U.S. patent application number 12/092791 was filed with the patent office on 2009-01-15 for methods and compositions comprising non-natural amino acids.
This patent application is currently assigned to AMBRX, INC.. Invention is credited to Ying Buechler, Anna-Maria A. Hays Putnam, Zhenwei Miao, Feng Tian.
Application Number | 20090018029 12/092791 |
Document ID | / |
Family ID | 38049316 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090018029 |
Kind Code |
A1 |
Miao; Zhenwei ; et
al. |
January 15, 2009 |
Methods and Compositions Comprising Non-Natural Amino Acids
Abstract
Disclosed herein are methods of detecting non-natural amino
acids and polypeptides that include at least one non-natural amino
acid. The non-natural amino acids, by themselves or as a part of a
polypeptide, can include a wide range of functionalities, including
but not limited to oxime, carbonyl, and/or hydroxylamine groups.
Also disclosed herein are non-natural amino acid polypeptides that
are further modified post-translationally, and methods for
detecting such polypeptides.
Inventors: |
Miao; Zhenwei; (San Diego,
CA) ; Tian; Feng; (San Diego, CA) ; Hays
Putnam; Anna-Maria A.; (San Diego, CA) ; Buechler;
Ying; (Carlsbad, CA) |
Correspondence
Address: |
ATTN: JOHN W. WALLEN, III;AMBRX, INC.
10975 NORTH TORREY PINES ROAD, SUITE 100
LA JOLLA
CA
92037
US
|
Assignee: |
AMBRX, INC.
La Jolla
CA
|
Family ID: |
38049316 |
Appl. No.: |
12/092791 |
Filed: |
November 16, 2006 |
PCT Filed: |
November 16, 2006 |
PCT NO: |
PCT/US06/44682 |
371 Date: |
August 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60737855 |
Nov 16, 2005 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/6.16;
435/7.1; 435/7.92; 436/501; 436/86; 506/18; 506/7; 530/344 |
Current CPC
Class: |
G01N 33/6812
20130101 |
Class at
Publication: |
506/9 ; 436/501;
436/86; 435/7.1; 506/7; 435/6; 435/7.92; 506/18; 530/344 |
International
Class: |
C40B 40/10 20060101
C40B040/10; G01N 33/53 20060101 G01N033/53; G01N 33/68 20060101
G01N033/68; C40B 30/00 20060101 C40B030/00; C07K 1/14 20060101
C07K001/14; C40B 30/04 20060101 C40B030/04; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of detecting a polypeptide comprising detecting a
non-naturally encoded amino acid side chain in the polypeptide.
2. The method of claim 1 wherein the polypeptide is detected using
a technique selected from the group consisting of immunoassays,
microarrays, microscopy, fluoroscent microscopy, electron
microscopy, electrophoresis, spectroscopy, chromogenic reactions,
radio-detection, radio-transmission, subatomic particle detection,
metal binding, chelating, structure or conformation changes,
enzymatic activity, specific binding, photography, magnetic field
measurement, sensors, electromagnetic energy detection, gene
expression, visible or invisible light detection, temperature
detection, and chemical detection.
3. The method of claim 2 wherein the immunoassays are selected from
the group consisting of Radioimmunoassay, Enzyme-linked
immunosorbent assay, Enzyme Multiplied Immunoassay, Microparticle
Enzyme Immunoassay, luminescent immunoassay, and fluorescent
immunoassay.
4. The method of claim 2 wherein the spectroscopy is selected from
the group consisting of SELDI, MALDI, fluorescence spectroscopy,
NMR, UV-Vis, and X-ray Crystallography.
5. The method of claim 2 wherein the electrophoresis is selected
from the group consisting of Gel Electrophoresis, Zone
Electrophoresis, Isoelectric Focusing Electrophoresis, Capillary
Electrophoresis, Capillary Zone Electrophoresis, Capillary Gel
Electrophoresis, Capillary Isotachophoresis, Capillary Isoelectric
Focusing Electrophoresis, and Capillary Electrochromatography.
6. The method of claim 1, wherein the polypeptide is ribosomally
synthesized.
7. The method of claim 1, wherein the non-natural amino acid
polypeptide is post-translationally modified.
8. A method of detecting a polypeptide comprising detecting a
non-naturally encoded amino acid side chain in the polypeptide that
has been post-translationally modified.
9. The method of claim 8 wherein the polypeptide is detected using
a technique selected from the group consisting of immunoassays,
microarrays, microscopy, fluorescent microscopy, electron
microscopy, electrophoresis, spectroscopy, chromogenic reactions,
radio-detection, radio-transmission, sub-atomic particle detection,
metal binding, chelating, structure or conformation changes,
enzymatic activity, specific binding, photography, magnetic field
measurement, sensors, electromagnetic energy detection, gene
expression, visible or invisible light detection, temperature
detection, and chemical detection.
10. The method of claim 9 wherein the immunoassays are selected
from the group consisting of Radioimmunoassay, Enzyme-linked
immunosorbent assay, Enzyme Multiplied Immunoassay, Microparticle
Enzyme Immunoassay, luminescent immunoassay, and fluorescent
immunoassay.
11. The method of claim 9 wherein the spectroscopy is selected from
the group consisting of SELDI, MALDI, fluorescence spectroscopy,
NMR, UV-Vis, and X-ray Crystallography.
12. The method of claim 9 wherein the electrophoresis is selected
from the group consisting of Gel Electrophoresis, Zone
Electrophoresis, Isoelectric Focusing Electrophoresis, Capillary
Electrophoresis, Capillary Zone Electrophoresis, Capillary Gel
Electrophoresis, Capillary Isotachophoresis, Capillary Isoelectric
Focusing Electrophoresis, and Capillary Electrochromatography.
13. The method of claim 8, wherein the polypeptide is ribosomally
synthesized.
14. A method of detecting a non-naturally encoded amino acid side
chain in said polypeptide, comprising contacting the non-naturally
encoded amino acid side chain with a molecule comprising a
functional group that specifically interacts with the non-naturally
encoded amino acid side chain.
15. A method for screening a library of molecules, comprising: a)
combining a polypeptide comprising a non-naturally encoded amino
acid with the library molecules under conditions to allow
interaction of the library molecules with the polypeptide
comprising a non-naturally encoded amino acid, b) identifying the
library, molecules which interact with the polypeptide comprising a
non-naturally encoded amino acid.
16. The method of claim 15, wherein the non-natural amino acid
polypeptide is ribosomally synthesized.
17. The method of claim 15, wherein the side chain of the
non-naturally encoded amino acid is post-translationally
modified.
18. The method of claim 17, wherein the non-natural amino acid
polypeptide is ribosomally synthesized.
19. The method of claim 15 wherein the library is chemical or
biological.
20. The method of claim 19 wherein the chemical library is organic
or inorganic.
21. The method of claim 19 wherein the biological library molecules
are selected from the group consisting of protein, peptide
polypeptide, DNA, RNA, virus, ribosomes, translation complexes,
bacteriophage, bacteria, and yeast.
22. The method of claim 15 wherein the interaction of the library
molecules with the polypeptide is specific binding of the library
molecules to the polypeptide.
23. The method of claim 15 wherein the interaction of the library
molecules with the polypeptide is covalent bond formation or
complex formation of the library molecules to the polypeptide.
24. The method of claim 15 further comprising the steps: c)
recovering a library molecule that interacts with the non-natural
amino acid polypeptide; and d) separating the non-natural amino
acid polypeptide from the library molecule, thereby obtaining
isolated library molecules.
25. A library of ribosomally made polypeptides comprising a
plurality of polypeptides having different amino acid sequences,
wherein each polypeptide comprises a non-natural amino acid.
26. The library of claim 25, wherein each polypeptide comprises the
same non-natural amino acid.
27. The library of claim 25, wherein each polypeptide comprises a
different non-natural amino acid.
28. The library of claim 25, wherein at least one polypeptide is
post-translationally modified.
29. The library of claim 27, wherein the polypeptides are identical
except for the non-naturally encoded amino acid.
30. The library of claim 25, wherein each polypeptide is homologous
to a polypeptide comprised of natural amino acids.
31. The library of claim 26, wherein each polypeptide is identical
except for the location of the non-natural amino acid.
32. A method of purifying a polypeptide having a non-naturally
encoded amino acid in the polypeptide chain, comprising contacting
the polypeptide with a substance that interacts with the
non-naturally encoded amino acid side chain in the polypeptide.
33. The method of claim 32 wherein the substance is a
chromatography matrix selected from the group consisting of liquid
chromatography, gas chromatography and supercritical fluid
chromatography.
34. The method of claim 33 wherein the liquid chromatography is
selected from the group consisting of partition chromatography,
adsorption chromatography, ion exchange chromatography, size
exclusion chromatography, thin layer chromatography and affinity
chromatography.
35. The method of claim 33 wherein the liquid chromatography is
selected from the group consisting of HPLC, column chromatography
and Batch treatment.
36. The method of claim 32 wherein the contacting of the
polypeptide with the substance occurs in a microfluidic device.
37. The method of claim 32 wherein the contacting of the
polypeptide with the substance occurs in a nanofluidic device.
38. The method of claim 34 wherein the partition chromatography is
selected from the group consisting of normal phase chromatography,
reversed phase chromatography and ion-pair chromatography.
39. The method of claim 34 wherein the thin layer chromatography is
selected from the group consisting of paper chromatography,
thin-layer chromatography and electrochromatography.
40. The method of claim 34 wherein the affinity chromatography is
selected from the group consisting of ligand chromatography, dye
chromatography, metal chelate chromatography, immunoaffinity
chromatography and hydrophobic interaction chromatography.
41. A method of purifying a polypeptide having a non-naturally
encoded amino acid in the polypeptide chain, comprising
precipitation of the polypeptide, wherein the non-naturally encoded
amino acid alters the solubility of the polypeptide when compared
to the solubility of the polypeptide without a non-naturally
encoded amino acid in the polypeptide chain.
42. The method of claim 41 wherein the precipitation is performed
by a compound selected from the group consisting of salts, acid,
base, and polymers.
43. The method of claim 41 wherein the purifying is by
immunoprecipitation.
44. The method of claim 32 wherein the substance is a magnetic
substance.
45. A method of purifying a ribosomally made polypeptide having a
non-naturally encoded amino acid in the polypeptide side chain,
comprising electrophoresis of the polypeptide, wherein the
non-naturally encoded amino acid alters the electrophoretic
mobility of the polypeptide when compared to the electrophoretic
mobility of the polypeptide without a non-naturally encoded amino
acid in the polypeptide chain.
46. The method of claim 45 where the electrophoresis is selected
from the group consisting of Gel Electrophoresis and Capillary
Electrophoresis.
47. The method of claim 46 wherein the Gel Electrophoresis, is
selected from the group consisting of Zone Electrophoresis and
Isoelectric Focusing Electrophoresis.
48. The method of claim 46 wherein the Capillary Electrophoresis is
selected from the group consisting of Capillary Zone
Electrophoresis, Capillary Gel Electrophoresis, Capillary
Isotachophoresis, Capillary Isoelectric Focusing Electrophoresis,
Capillary Electrochrormatography, Micellar electrokinetic capillary
chromatography, Isotachophoresis, and Transient
Isotachophoresis.
49. A method of purifying a ribosomally made polypeptide having a
non-naturally encoded amino acid in the polypeptide side chain,
comprising dialysis of the polypeptide, wherein the non-naturally
encoded amino acid alters the diffusion rate of the polypeptide
when compared to the diffusion rate of the polypeptide without a
non-naturally encoded amino acid in the polypeptide chain.
50. The method of claim 49 wherein the dialysis is
electrodialysis.
51. A method of purifying a non-natural amino acid polypeptide
comprising purifying the polypeptide by ultrafiltration.
52. The method of claim 32, wherein the polypeptide is expressed in
a microorganism.
53. The method of claim 32, wherein the polypeptide is made in a
microorganism selected from the group consisting of Escherichia
coli, Pseudomonas fluorescens, Bacillus subtilis, yeast, mammalian
cells and insect cells.
54. The method of claim 53 wherein the non-natural amino acid
polypeptide is a hybrid peptide that contain and affinity tag.
55. The method of claim 32, wherein the non-natural amino acid
polypeptide is post-translationally modified.
56. The method of claim 55, wherein the post-translational
modification is by an oxime exchange reaction.
57. The method of claim 32, wherein the non-natural amino acid is
posttranslationally modified to comprise an oxime group.
58. A method, comprising: a) substituting a non-naturally encoded
amino acid for a naturally encoded amino acid at a single
pre-selected site in a pre-selected polypeptide having at least one
known biological activity; and b) measuring a biological activity
of the pre-selected polypeptide comprising the non-naturally
encoded amino acid; and c) comparing the biological activity of the
pre-selected polypeptide of step b) with the pre-selected
polypeptide having a non-naturally encoded amino acid substituted
for a naturally encoded amino acid at a different position in the
pre-selected polypeptide chain or with the pre-selected polypeptide
without a substituted non-naturally encoded amino acid in the
polypeptide chain.
59. The method of claim 58 wherein the pre-selected positions in
the polypeptide chain for substitution with a non-naturally encoded
amino acid are sequential.
60. The method of claim 58 wherein the substituting of the
non-naturally encoded amino acid for a naturally encoded amino acid
at a single site in the pre-selected polypeptide chain is repeated
for every individual position in the polypeptide chain.
61. The method of claim 58 wherein the same non-naturally encoded
amino acid is substituted.
62. The method of claim 58 wherein a different non-naturally
encoded amino acid is substituted.
63. The method of claim 58 wherein more than one position of the
pre-selected polypeptide chain is substituted with a non-naturally
encoded amino acid.
64. A method for selecting a position for post-translational
modification of a pre-selected polypeptide comprising: a)
substituting a non-naturally encoded amino acid for a naturally
encoded amino acid at a single pre-selected site in a pre-selected
polypeptide having at least one known biological activity; and b)
measuring a biological activity of the pre-selected polypeptide
comprising the non-naturally encoded amino acid; and c) comparing
the biological activity of the pre-selected polypeptide of step b)
with the pre-selected polypeptide having a non-naturally encoded
amino acid substituted for a naturally encoded amino acid at a
different position in the pre-selected polypeptide chain or with
the pre-selected polypeptide without a substituted non-naturally
encoded amino acid in the polypeptide chain.
65. The method of claim 64 wherein the post-translational
modification is coupling to a water soluble polymer.
66. A composition comprising a polypeptide covalently linked to a
nucleic acid molecule at one or more specific amino acid positions
of said polypeptide, wherein said polypeptide and nucleic acid
molecule are covalently linked to an amino acid side chain of one
or more non-naturally encoded amino acids of said polypeptide.
Description
RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 60/737,855, entitled "Methods of Detecting
Non-Natural Amino Acid Polypeptides in vivo and in vitro" filed on
Nov. 16, 2005.
BACKGROUND OF THE INVENTION
[0002] The ability to incorporate non-genetically encoded amino
acids (i.e., "non-natural amino acids") into proteins permits the
introduction of chemical functional groups that could provide
valuable alternatives to the naturally-occurring functional groups,
such as the epsilon --NH2 of lysine, the sulfhydryl --SH of
cysteine, the imino group of histidine, etc. Certain chemical
functional groups are known to be inert to the functional groups
found in the 20 common, genetically-encoded amino acids but react
cleanly and efficiently to form stable linkages with functional
groups that can be incorporated onto non-natural amino acids.
[0003] Methods are now available to selectively introduce chemical
functional groups that are not found in proteins, that are
chemically inert to all of the functional groups found in the 20
common, genetically-encoded amino acids and that may be used to
react efficiently and selectively with reagents comprising certain
functional groups to form stable covalent linkages.
SUMMARY OF THE INVENTION
[0004] Described herein and incorporated by reference are methods,
compositions, techniques and strategies for making, purifying,
detecting, characterizing, and using non-natural amino acids,
non-natural amino acid polypeptides and modified non-natural amino
acid polypeptides.
[0005] This invention provides a method of detecting a polypeptide
that comprises detecting a non-naturally encoded amino acid side
chain in the polypeptide. In some embodiments, the polypeptide is
ribosomally synthesized. The invention also provides methods of
detecting a polypeptide that comprise detecting a non-naturally
encoded amino acid side chain in the polypeptide that has been
post-translationally modified. Also provided are methods of
detecting a non-naturally encoded amino acid side chain in said
polypeptide that comprise contacting the non-naturally encoded
amino acid side chain with a molecule comprising a functional group
that specifically interacts with the non-naturally encoded amino
acid side chain. Also provided are methods of purifying a
polypeptide having a non-naturally encoded amino acid in the
polypeptide chain. In some embodiments the method comprises
contacting the polypeptide with a substance that interacts with the
non-naturally encoded amino acid side chain in the polypeptide. In
other embodiments, the method of purifying a polypeptide having a
non-naturally encoded amino acid in the polypeptide chain comprises
precipitation of the polypeptide, wherein the non-naturally encoded
amino acid alters the solubility of the polypeptide when compared
to the solubility of the polypeptide without a non-naturally
encoded amino acid in the polypeptide chain. Methods of purifying a
ribosomally made polypeptide having a non-naturally encoded amino
acid in the polypeptide side chain comprises electrophoresis of the
polypeptide, wherein the non-naturally encoded amino acid alters
the electrophoretic mobility of the polypeptide when compared to
the electrophoretic mobility of the polypeptide without a
non-naturally encoded amino acid in the polypeptide chain are also
provided. In other embodiments, the method of purifying a
ribosomally made polypeptide having a non-naturally encoded amino
acid in the polypeptide side chain, comprises dialysis of the
polypeptide, wherein the non-naturally encoded amino acid alters
the diffusion rate of the polypeptide when compared to the
diffusion rate of the polypeptide without a non-naturally encoded
amino acid in the polypeptide chain.
[0006] The invention also provides a method for screening a library
of molecules, comprising: a) combining a polypeptide comprising a
non-naturally encoded amino acid with the library molecules under
conditions to allow interaction of the library molecules with the
polypeptide comprising a non-naturally encoded amino acid, and b)
identifying the library molecules which interact with the
polypeptide comprising a non-naturally encoded amino acid. In some
embodiments, a library of ribosomally made polypeptide comprising a
plurality of polypeptides having different amino acid sequences,
wherein each polypeptide comprises a non-natural amino acid is
screened.
[0007] The invention also provides methoda, comprising: a)
substituting a non-naturally encoded amino acid for a naturally
encoded amino acid at a single pre-selected site in a pre-selected
polypeptide having at least one known biological activity; and b)
measuring a biological activity of the pre-selected polypeptide
comprising the non-naturally encoded amino acid; and c) comparing
the biological activity of the pre-selected polypeptide of step b)
with the pre-selected polypeptide having a non-naturally encoded
amino acid substituted for a naturally encoded amino acid at a
different position in the pre-selected polypeptide chain or with
the pre-selected polypeptide without a substituted non-naturally
encoded amino acid in the polypeptide chain. In some embodiments, a
method for selecting a position for post-translational modification
of a pre-selected polypeptide comprises a) substituting a
non-naturally encoded amino acid for a naturally encoded amino acid
at a single pre-selected site in a pre-selected polypeptide having
at least one known biological activity; and b) measuring a
biological activity of the pre-selected polypeptide comprising the
non-naturally encoded amino acid; and c) comparing the biological
activity of the pre-selected polypeptide of step b) with the
pre-selected polypeptide having a non-naturally encoded amino acid
substituted for a naturally encoded amino acid at a different
position in the pre-selected polypeptide chain or with the
pre-selected polypeptide without a substituted non-naturally
encoded amino acid in the polypeptide chain.
[0008] It is to be understood that the methods and compositions
described herein and incorporated by reference are not limited to
the particular methodology, protocols, cell lines, constructs, and
reagents described herein and as such may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the methods and compositions described herein,
which will be limited only by the appended claims.
DEFINITIONS
[0009] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly indicates otherwise.
[0010] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the inventions described herein
belong. Although any methods, devices, and materials similar or
equivalent to those described herein can be used in the practice or
testing of the inventions described herein, the preferred methods,
devices and materials are now described.
[0011] All publications and patents mentioned herein are
incorporated herein by reference for the purpose of describing and
disclosing, for example, the constructs and methodologies that are
described in the publications, which might be used in connection
with the presently described inventions. The publications discussed
herein are provided solely for their disclosure prior to the filing
date of the present application. Nothing herein is to be construed
as an admission that the inventors described herein are not
entitled to antedate such disclosure by virtue of prior invention
or for any other reason.
[0012] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0013] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
di- and multivalent radicals, having the number of carbon atoms
designated (i.e. C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon radicals include, but are not
limited to, groups such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
those derivatives of alkyl defined in more detail below, such as
"heteroalkyl." Alkyl groups which are limited to hydrocarbon groups
are termed "homoalkyl".
[0014] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified, but not limited, by the structures
--CH.sub.2CH.sub.2-- and --CH.sub.2CH.sub.12CH.sub.2CH.sub.2--, and
further includes those groups described below as "heteroalkylene."
Typically, an alkyl (or alkylene) group will have from 1 to 24
carbon atoms, with those groups having 10 or fewer carbon atoms
being a particular embodiment of the methods and compositions
described herein. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer
carbon atoms.
[0015] The term "amino acid" refers to naturally occurring and
non-natural amino acids, as well as amino acid analogs and amino
acid mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally encoded amino acids are the 20
common amino acids (alanine, arginine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine) and pyrrolysine and
selenocysteine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, such as, homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (such as, norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid.
[0016] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0017] An "amino terminus modification group" refers to any
molecule that can be attached to the amino terminus of a
polypeptide. Similarly, a "carboxy terminus modification group"
refers to any molecule that can be attached to the carboxy terminus
of a polypeptide. Terminus modification groups include but are not
limited to various water soluble polymers, peptides or proteins
such as serum albumin, or other moieties that increase serum
half-life of peptides.
[0018] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic, hydrocarbon substituent which can be a
single ring or multiple rings (including but not limited to, from 1
to 3 rings) which are fused together or linked covalently. The term
"heteroaryl" refers to aryl groups (or rings) that contain from one
to four heteroatoms selected from N, O, and S, wherein the nitrogen
and sulfur atoms are optionally oxidized, and the nitrogen atom(s)
are optionally quaternized. A heteroaryl group can be attached to
the remainder of the molecule through a heteroatom. Non-limiting
examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below.
[0019] For brevity, the term "aryl" when used in combination with
other terms (including but not limited to, aryloxy, arylthioxy,
aralkyl) includes both aryl and heteroaryl rings as defined above.
Thus, the term "aralkyl" or "alkaryl" is meant to include those
radicals in which an aryl group is attached to an alkyl group
(including but not limited to, benzyl, phenethyl, pyridylmethyl and
the like) including those alkyl groups in which a carbon atom
(including but not limited to, a methylene group) has been replaced
by, for example, an oxygen atom (including but not limited to,
phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the
like).
[0020] A "bifunctional polymer" refers to a polymer comprising two
discrete functional groups that are capable of reacting
specifically with other moieties (including but not limited to,
amino acid side groups) to form covalent or non-covalent linkages.
A bifunctional linker having one functional group reactive with a
group on a particular biologically active component, and another
group reactive with a group on a second biological component, may
be used to form a conjugate that includes the first biologically
active component, the bifunctional linker and the second
biologically active component. Many procedures and linker molecules
for attachment of various compounds to peptides are known. See,
e.g., European Patent Application No. 188,256; U.S. Pat. Nos.
4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and
4,569,789 which are incorporated by reference herein. A
"multi-functional polymer" refers to a polymer comprising two or
more discrete functional groups that are capable of reacting
specifically with other moieties (including but not limited to,
amino acid side groups) to form covalent or non-covalent linkages.
A bi-functional polymer or multi-functional polymer may be any
desired length or molecular weight, and may be selected to provide
a particular desired spacing or conformation between one or more
molecules linked to the polypeptide and its binding partner or the
polypeptide.
[0021] The term "biologically active molecule", "biologically
active moiety" or "biologically active agent" when used herein
means any substance which can affect any physical or biochemical
properties of a biological system, pathway, molecule, or
interaction relating to an organism, including but not limited to
viruses, bacteria, bacteriophage, transposon, prion, insects,
fungi, plants, animals, and humans. In particular, as used herein,
biologically active molecules include but are not limited to any
substance intended for diagnosis, cure, mitigation, treatment, or
prevention of disease in humans or other animals, or to otherwise
enhance physical or mental well-being of humans or animals.
Examples of biologically active molecules include, but are not
limited to, peptides, proteins, enzymes, small molecule drugs, hard
drugs, soft drugs, carbohydrates, inorganic atoms or molecules,
dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins,
cells, viruses, liposomes, microparticles and micelles. Classes of
biologically active agents that are suitable for use with the
methods and compositions described herein include, but are not
limited to, drugs, prodrugs, radionuclides imaging agents,
polymers, antibiotics, fungicides, anti-viral agents,
anti-inflammatory agents, anti-tumor agents, cardiovascular agents,
anti-anxiety agents, hormones, growth factors, steroidal agents,
microbially derived toxins, and the like.
[0022] "Cofolding," as used herein, refers specifically to
refolding processes, reactions, or methods which employ at least
two polypeptides which interact with each other and result in the
transformation of unfolded or improperly folded polypeptides to
native, properly folded polypeptides.
[0023] A "comparison window," as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, including but not limited to, by the local homology
algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by
the homology alignment algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443, by the search for similarity method of Pearson
and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
manual alignment and visual inspection (see, e.g., Ausubel et al.,
Current Protocols in Molecular Biology (1995 supplement)).
[0024] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1997)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. The BLAST algorithm parameters W, T, and
X determine the sensitivity and speed of the alignment. The BLASTN
program (for nucleotide sequences) uses as defaults a wordlength
(W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of
both strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength of 3, and expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.
Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST
algorithm is typically performed with the "low complexity" filter
turned off.
[0025] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, less than about
0.01, or less than about 0.001.
[0026] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, "conservatively modified variants" refers
to those nucleic acids which encode identical or essentially
identical amino acid sequences, or where the nucleic acid does not
encode an amino acid sequence, to essentially identical sequences.
Because of the degeneracy of the genetic code, a large number of
functionally identical nucleic acids encode any given protein. For
instance, the codons GCA, GCC, GCG and GCU all encode the amino
acid alanine. Thus, at every position where an alanine is specified
by a codon, the codon can be altered to any of the corresponding
codons described without altering the encoded polypeptide. Such
nucleic acid variations are "silent variations," which are one
species of conservatively modified variations. Every nucleic acid
sequence herein which encodes a polypeptide also describes every
possible silent variation of the nucleic acid. One of skill will
recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine, and TGG, which is
ordinarily the only codon for tryptophan) can be modified to yield
a functionally identical molecule. Accordingly, each silent
variation of a nucleic acid which encodes a polypeptide is implicit
in each described sequence.
[0027] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are known to those of
ordinary skill in the art. Such conservatively modified variants
are in addition to and do not exclude polymorphic variants,
interspecies homologs, and alleles of the methods and compositions
described herein.
[0028] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
[0029] 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
[0030] (see, e.g., Creighton, Proteins: Structures and Molecular
Properties (W H Freeman & Co.; 2nd edition (December 1993)
[0031] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Thus, a cycloalkyl or heterocycloalkyl include saturated, partially
unsaturated and fully unsaturated ring linkages. Additionally, for
heterocycloalkyl, a heteroatom can occupy the position at which the
heterocycle is attached to the remainder of the molecule. Examples
of cycloalkyl include, but are not limited to, cyclopentyl,
cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the
like. Examples of heterocycloalkyl include, but are not limited to,
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like. Additionally, the term
encompasses bicyclic and tricyclic ring structures. Similarly, the
term "heterocycloalkylene" by itself or as part of another
substituent means a divalent radical derived from heterocycloalkyl,
and the term "cycloalkylene" by itself or as part of another
substituent means a divalent radical derived from cycloalkyl.
[0032] "Denaturing agent" or "denaturant," as used herein, is
defined as any compound or material which will cause a reversible
unfolding of a protein. The strength of a denaturing agent or
denaturant will be determined both by the properties and the
concentration of the particular denaturing agent or denaturant.
Suitable denaturing agents or denaturants may be chaotropes,
detergents, organic, water miscible solvents, phospholipids, or a
combination of two or more such agents. Suitable chaotropes
include, but are not limited to, urea, guanidine, and sodium
thiocyanate. Useful detergents may include, but are not limited to,
strong detergents such as sodium dodecyl sulfate, or
polyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl,
mild non-ionic detergents (e.g., digitonin), mild cationic
detergents such as
N->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic
detergents (e.g. sodium cholate or sodium deoxycholate) or
zwitterionic detergents including, but not limited to,
sulfobetaines (Zwittergent),
3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS),
and 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane
sulfonate (CHAPSO). Organic, water miscible solvents such as
acetonitrile, lower alkanols (especially C.sub.2-C.sub.4 alkanols
such as ethanol or isopropanol), or lower alkandiols (especially
C.sub.2-C.sub.4 alkandiols such as ethylene-glycol) may be used as
denaturants. Phospholipids useful in the methods and compositions
described herein may be naturally occurring phospholipids such as
phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,
and phosphatidylinositol or synthetic phospholipid derivatives or
variants such as dihexanoylphosphatidylcholine or
diheptanoylphosphatidylcholine.
[0033] The term "effective amount" as used herein refers to that
amount of the (modified) non-natural amino acid polypeptide being
administered which will relieve to some extent one or more of the
symptoms of the disease, condition or disorder being treated.
Compositions containing the (modified) non-natural amino acid
polypeptide described herein can be administered for prophylactic,
enhancing, and/or therapeutic treatments.
[0034] The terms "enhance" or "enhancing" means to increase or
prolong either in potency or duration a desired effect. Thus, in
regard to enhancing the effect of therapeutic agents, the term
"enhancing" refers to the ability to increase or prolong, either in
potency or duration, the effect of other therapeutic agents on a
system. An "enhancing-effective amount," as used herein, refers to
an amount adequate to enhance the effect of another therapeutic
agent in a desired system. When used in a patient, amounts
effective for this use will depend on the severity and course of
the disease, disorder or condition, previous therapy, the patient's
health status and response to the drugs, and the judgment of the
treating physician.
[0035] As used herein, the term "eukaryote" refers to organisms
belonging to the phylogenetic domain Eucarya such as animals
(including but not limited to, mammals, insects, reptiles, birds,
etc.), ciliates, plants (including but not limited to, monocots,
dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia,
protists, etc.
[0036] The terms "functional group", "active moiety", "activating
group", "leaving group", "reactive site", "chemically reactive
group" and "chemically reactive moiety" are used in the art and
herein to refer to distinct, definable portions or units of a
molecule. The terms are somewhat synonymous in the chemical arts
and are used herein to indicate the portions of molecules that
perform some function or activity and are reactive with other
molecules.
[0037] The term "halogen" includes fluorine, chlorine, iodine, and
bromine.
[0038] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and at
least one heteroatom selected from the group consisting of O, N, Si
and S, and wherein the nitrogen and sulfur atoms may optionally be
oxidized and the nitrogen heteroatom may optionally be quaternized.
The heteroatom(s) O, N and S and Si may be placed at any interior
position of the heteroalkyl group or at the position at which the
alkyl group is attached to the remainder of the molecule. Examples
include, but are not limited to, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CCH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified, but
not limited by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, the same or different heteroatoms can also
occupy either or both of the chain termini (including but not
limited to, alkyleneoxy, alkylenedioxy, alkyleneamino,
alkylenediamino, aminooxyalkylene, and the like). Still further,
for alkylene and heteroalkylene linking groups, no orientation of
the linking group is implied by the direction in which the formula
of the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--.
[0039] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same. Sequences are
"substantially identical" if they have a percentage of amino acid
residues or nucleotides that are the same (i.e., about 60%
identity, optionally about 65%, about 70%, about 75%, about 80%,
about 85%, about 90%, or about 95% identity over a specified
region), when compared and aligned for maximum correspondence over
a comparison window, or designated region as measured using one of
the following sequence comparison algorithms or by manual alignment
and visual inspection. This definition also refers to the
complement of a test sequence. The identity can exist over a region
that is at least about 50 amino acids or nucleotides in length, or
over a region that is 75-100 amino acids or nucleotides in length,
or, where not specified, across the entire sequence of a
polynucleotide or polypeptide.
[0040] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0041] The term "isolated," when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is free of at
least some of the cellular components with which it is associated
in the natural state, or that the nucleic acid or protein has been
concentrated to a level greater than the concentration of its in
vivo or in vitro production. It can be in a homogeneous state.
Isolated substances can be in either a dry or semi-dry state, or in
solution, including but not limited to an aqueous solution. It can
be a component of a pharmaceutical composition that comprises
additional pharmaceutically acceptable carriers and/or excipients.
Purity and homogeneity are typically determined using analytical
chemistry techniques such as polyacrylamide gel electrophoresis or
high performance liquid chromatography. A protein which is the
predominant species present in a preparation is substantially
purified. In particular, an isolated gene is separated from open
reading frames which flank the gene and encode a protein other than
the gene of interest. The term "purified" denotes that a nucleic
acid or protein gives rise to substantially one band in an
electrophoretic gel. Particularly, it may mean that the nucleic
acid or protein is at least 85% pure, at least 90% pure, at least
95% pure, at least 99% or greater pure.
[0042] The term "linkage" or "linker" is used herein to refer to
groups or bonds that normally are formed as the result of a
chemical reaction and typically are covalent linkages.
Hydrolytically stable linkages means that the linkages are
substantially stable in water and do not react with water at useful
pH values, including but not limited to, under physiological
conditions for an extended period of time, perhaps even
indefinitely. Hydrolytically unstable or degradable linkages mean
that the linkages are degradable in water or in aqueous solutions,
including for example, blood. Enzymatically unstable or degradable
linkages mean that the linkage can be degraded by one or more
enzymes. As understood in the art, PEG and related polymers may
include degradable linkages in the polymer backbone or in the
linker group between the polymer backbone and one or more of the
terminal functional groups of the polymer molecule. For example,
ester linkages formed by the reaction of PEG carboxylic acids or
activated PEG carboxylic acids with alcohol groups on a
biologically active agent generally hydrolyze under physiological
conditions to release the agent. Other hydrolytically degradable
linkages include but are not limited to carbonate linkages; imine
linkages resulted from reaction of an amine and an aldehyde;
phosphate ester linkages formed by reacting an alcohol with a
phosphate group; hydrazone linkages which are reaction product of a
hydrazide and an aldehyde; acetal linkages that are the reaction
product of an aldehyde and an alcohol; orthoester linkages that are
the reaction product of a formate and an alcohol; peptide linkages
formed by an amine group, including but not limited to at an end of
a polymer such as PEG, and a carboxyl group of a peptide; and
oligonucleotide linkages formed by a phosphoramidite group,
including but not limited to, at the end of a polymer, and a 5'
hydroxyl group of an oligonucleotide.
[0043] As used herein, the term "medium" or "media" includes any
culture medium, solution, solid, semi-solid, or rigid support that
may support or contain any host cell, including bacterial host
cells, yeast host cells, insect host cells, plant host cells,
eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic
host cells, E. coli, or Pseudomonas host cells, and cell contents.
Thus, the term may encompass medium in which the host cell has been
grown, e.g., medium into which the polypeptide has been secreted,
including medium either before or after a proliferation step. The
term also may encompass buffers or reagents that contain host cell
lysates, such as in the case where the polypeptide is produced
intracellularly and the host cells are lysed or disrupted to
release the polypeptide.
[0044] A "metabolite" of a (modified) non-natural amino acid
polypeptide disclosed herein is a derivative of that (modified)
non-natural amino acid polypeptide that is formed when the
(modified) non-natural amino acid polypeptide is metabolized. The
term "active metabolite" refers to a biologically active derivative
of a (modified) non-natural amino acid polypeptide that is formed
when the (modified) non-natural amino acid polypeptide is
metabolized. The term "metabolized" refers to the sum of the
processes (including, but not limited to, hydrolysis reactions and
reactions catalyzed by enzymes) by which a particular substance is
changed by an organism. Further information on metabolism may be
obtained from The Pharmacological Basis of Therapeutics, 9th
Edition, McGraw-Hill (1996). Metabolites of the (modified)
non-natural amino acid polypeptide disclosed herein can be
identified either by administration of (modified) non-natural amino
acid polypeptide to a host and analysis of tissue samples from the
host, or by incubation of (modified) non-natural amino acid
polypeptide with hepatic cells in vitro and analysis of the
resulting compounds.
[0045] The term "modified," as used herein refers to the presence
of a post-translational modification on a polypeptide. The form
"(modified)" term means that the polypeptides being discussed are
optionally modified, that is, the polypeptides under discussion can
be modified or unmodified.
[0046] As used herein, the term "modulated serum half-life" means
the positive or negative change in circulating half-life of a
(modified) polypeptide relative to its non-modified form. Serum
half-life is measured by taking blood samples at various time
points after administration of the polypeptide, and determining the
concentration of that molecule in each sample. Correlation of the
serum concentration with time allows calculation of the serum
half-life. Increased serum half-life desirably has at least about
two-fold, but a smaller increase may be useful, for example where
it enables a satisfactory dosing regimen or avoids a toxic effect.
In some embodiments, the increase is at least about three-fold, at
least about five-fold, or at least about ten-fold.
[0047] The term "modulated therapeutic half-life" as used herein
means the positive or negative change in the half-life of the
therapeutically effective amount of a (modified) polypeptide,
relative to its non-modified form. Therapeutic half-life is
measured by measuring pharmacokinetic and/or pharmacodynamic
properties of the molecule at various time points after
administration. Increased therapeutic half-life desirably enables a
particular beneficial dosing regimen, a particular beneficial total
dose, or avoids an undesired effect. In some embodiments, the
increased therapeutic half-life results from increased potency,
increased or decreased binding of the modified molecule to its
target, increased or decreased breakdown of the molecule by enzymes
such as proteases, or an increase or decrease in another parameter
or mechanism of action of the non-modified molecule.
[0048] As used herein, the term "non-eukaryote" refers to
non-eukaryotic organisms. For example, a non-eukaryotic organism
can belong to the Eubacteria (including but not limited to,
Escherichia coli, Thermus thermophilus, Bacillus
stearothermophilus, Pseudomonas fluorescens, Pseudomonas
aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or the
Archaea (including but not limited to, Methanococcus jannaschii,
Methanobacterium thermoautotrophicum, Halobacterium such as
Haloferax voleanii and Halobacterium species NRC-1, Archaeoglobus
fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum
pernix, etc.) phylogenetic domain.
[0049] A "non-natural amino acid" refers to an amino acid that is
not one of the 20 common amino acids or pyrrolysine or
selenocysteine; other terms that may be used synonymously with the
term "non-natural amino acid" is "non-naturally encoded amino
acid," "unnatural amino acid," "non-naturally-occurring amino
acid," and variously hyphenated and non-hyphenated versions
thereof. The term "non-natural amino acid" includes, but is not
limited to, amino acids that occur naturally by modification of a
naturally encoded amino acid (including but not limited to, the 20
common amino acids or pyrrolysine and selenocysteine) but are not
themselves incorporated into a growing polypeptide chain by the
translation complex. Examples of naturally-occurring amino acids
that are not naturally-encoded include, but are not limited to,
N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine,
and O-phosphotyrosine.
[0050] The term "nucleic acid" refers to deoxyribonucleotides,
deoxyribonucleosides, ribonucleosides or ribonucleotides and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogues of natural nucleotides which have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless
specifically limited otherwise, the term also refers
oligonucleotide analogs including PNA (peptidonucleic acid),
analogs of DNA used in antisense technology (phosphorothioates,
phosphoroamidates, and the like). Unless otherwise indicated, a
particular nucleic acid sequence also implicitly encompasses
conservatively modified variants thereof (including but not limited
to, degenerate codon substitutions) and complementary sequences as
well as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes
8:91-98 (1994)).
[0051] "Oxidizing agent," as used hereinwith respect to protein
refolding, is defined as any compound or material which is capable
of removing an electron from a compound being oxidized. Suitable
oxidizing agents include, but are not limited to, oxidized
glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized
erythreitol, and oxygen. A wide variety of oxidizing agents are
suitable for use in the methods and compositions described
herein.
[0052] As used herein, the term "polyalkylene glycol" refers to
polyethylene glycol, polypropylene glycol, polybutylene glycol, and
derivatives thereof. The term "polyalkylene glycol" encompasses
both linear and branched polymers and average molecular weights of
between 1 kDa and 100 kDa. Other exemplary embodiments are listed,
for example, in commercial supplier catalogs, such as Shearwater
Corporation's catalog "Polyethylene Glycol and Derivatives for
Biomedical Applications" (2001).
[0053] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. That is, a description directed to a polypeptide applies
equally to a description of a peptide and a description of a
protein, and vice versa. The terms apply to naturally occurring
amino acid polymers as well as amino acid polymers in which one or
more amino acid residues is a non-natural amino acid. As used
herein, the terms encompass amino acid chains of any length,
including full length proteins, wherein the amino acid residues are
linked by covalent peptide bonds.
[0054] The term "post-translationally modified" refers to any
modification of a natural or non-natural amino acid that occurs to
such an amino acid after it has been incorporated into a
polypeptide chain. The term encompasses, by way of example only,
co-translational in vivo, modifications, co-translational in vitro
modifications (such as in a cell-free translation system),
post-translational in vivo modifications, and post-translational in
vitro modifications.
[0055] A "prodrug" refers to an agent that is converted into the
parent drug in vivo. Prodrugs are often useful because, in some
situations, they may be easier to administer than the parent drug.
They may, for instance, be bioavailable by oral administration
whereas the parent is not. The prodrug may also have improved
solubility in pharmaceutical compositions over the parent drug.
[0056] In prophylactic applications, compositions containing the
(modified) non-natural amino acid polypeptide are administered to a
patient susceptible to or otherwise at risk of a particular
disease, disorder or condition. Such an amount is defined to be a
"prophylactically effective amount." In this use, the precise
amounts also depend on the patient's state of health, weight, and
the like. It is considered well within the skill of the art for one
to determine such prophylactically effective amounts by routine
experimentation (e.g., a dose escalation clinical trial).
[0057] The term "protected" refers to the presence of a "protecting
group" or moiety that prevents reaction of the chemically reactive
functional group under certain reaction conditions. The protecting
group will vary depending on the type of chemically reactive group
being protected. For example, if the chemically reactive group is
an amine or a hydrazide, the protecting group can be selected from
the group of tert-butyloxycarbonyl (t-Boc) and
9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group
is a thiol, the protecting group can be orthopyridyldisulfide. If
the chemically reactive group is a carboxylic acid, such as
butanoic or propionic acid, or a hydroxyl group, the protecting
group can be benzyl or an alkyl group such as methyl, ethyl, or
tert-butyl. Other protecting groups known in the art may also be
used in or with the methods and compositions described herein,
including photolabile groups such as Nvoc and MeNvoc.
[0058] By way of example only, blocking/protecting groups may be
selected from:
##STR00001##
[0059] Other protecting groups are described in Greene and Wuts,
Protective Groups in Organic Synthesis, 3rd Ed., John Wiley &
Sons, New York, N.Y., 1999, which is incorporated herein by
reference in its entirety.
[0060] A "recombinant host cell" or "host cell" refers to a cell
that includes an exogenous polynucleotide, regardless of the method
used for insertion, for example, direct uptake, transduction,
f-mating, or other methods known in the art to create recombinant
host cells. The exogenous polynucleotide may be maintained as a
nonintegrated vector, for example, a plasmid, or alternatively, may
be integrated into the host genome.
[0061] "Reducing agent," as used herein with respect to protein
refolding, is defined as any compound or material which maintains
sulfhydryl groups in the reduced state and reduces intra- or
intermolecular disulfide bonds. Suitable reducing agents include,
but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol,
dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and
reduced glutathione. A wide variety of reducing agents are suitable
for use in the methods and compositions described herein.
[0062] "Refolding," as used herein describes any process, reaction
or method which transforms disulfide bond containing polypeptides
from an improperly folded or unfolded state to a native or properly
folded conformation with respect to disulfide bonds.
[0063] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(including but not limited to, total cellular or library DNA or
RNA).
[0064] The phrase "stringent hybridization conditions" refers to
conditions of low ionic strength and high temperature as is known
in the art. Typically, under stringent conditions a probe will
hybridize to its target subsequence in a complex mixture of nucleic
acid (including but not limited to, total cellular or library DNA
or RNA) but does not hybridize to other sequences in the complex
mixture. Stringent conditions are sequence-dependent and will be
different in different circumstances. Longer sequences hybridize
specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Laboratory
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes
(including but not limited to, 10 to 50 nucleotides) and at least
about 60.degree. C. for long probes (including but not limited to,
greater than 50 nucleotides). Stringent conditions may also be
achieved with the addition of destabilizing agents such as
formamide. For selective or specific hybridization, a positive
signal may be at least two times background, optionally 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C. Such washes can be performed for 5, 15, 30,
60, 120, or more minutes.
[0065] The term "subject" as used herein, refers to an animal, in
some embodiments a mammal, and in other embodiments a human, who is
the object of treatment, observation or experiment.
[0066] The term "substantially purified" refers to a polypeptide
that may be substantially or essentially free of components that
normally accompany or interact with the protein as found in its
naturally occurring environment, i.e. a native cell, or host cell
in the case of recombinantly produced polypeptide. A polypeptide
that may be substantially free of cellular material includes
preparations of protein having less than about 30%, less than about
25%, less than about 20%, less than about 15%, less than about 10%,
less than about 5%, less than about 4%, less than about 3%, less
than about 2%, or less than about 1% (by dry weight) of
contaminating protein. When the polypeptide or variant thereof is
recombinantly produced by the host cells, the protein may be
present at about 30%, about 25%, about 20%, about 15%, about 10%,
about 5%, about 4%, about 3%, about 2%, or about 1% or less of the
dry weight of the cells. When the polypeptide or variant thereof is
recombinantly produced by the host cells, the protein may be
present in the culture medium at about 5 g/L, about 4 g/L, about 3
g/L, about 2 g/L, about 1 g/L, about 750 mg/L, about 500 mg/L,
about 250 mg/L, about 10 mg/L, about 50 mg/L, about 10 mg/L, or
about 1 mg/L or less of the dry weight of the cells. Thus,
"substantially purified" polypeptide as produced by the methods
described herein may have a purity level of at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, specifically, a purity level of at least
about 75%, 80%, 85%, and more specifically, a purity level of at
least about 90%, a purity level of at least about 95%, a purity
level of at least about 99% or greater as determined by appropriate
methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary
electrophoresis.
[0067] The term "substituents" includes but is not limited to
"non-interfering substituents." "Non-interfering substituents" are
those groups that yield stable compounds. Suitable non-interfering
substituents or radicals include, but are not limited to, halo,
C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10
alkynyl, C.sub.1-C.sub.10 alkoxy, C.sub.5-C.sub.12 aralkyl,
C.sub.3-C.sub.12 cycloalkyl, C.sub.4-C.sub.12 cycloalkenyl, phenyl,
substituted phenyl, toluoyl, xylenyl, biphenyl, C.sub.2-C.sub.12
alkoxyalkyl, C.sub.5-C.sub.12 alkoxyaryl, C.sub.5-C.sub.12
aryloxyalkyl, C.sub.7-C.sub.12 oxyaryl, C.sub.1-C.sub.6
alkylsulfinyl, C.sub.1-C.sub.10 alkylsulfonyl,
--(CH.sub.2).sub.m--O--(C.sub.1-C.sub.10 alkyl) wherein m is from 1
to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl,
heterocyclic radical, substituted heterocyclic radical, nitroalkyl,
--NO.sub.2, --CN, --NRC(O)--(C.sub.1-C.sub.10 alkyl),
--C(O)--(C.sub.1-C.sub.10 alkyl), C.sub.2-C.sub.10 alkthioalkyl,
--C(O)O--(C.sub.1-C.sub.10 alkyl), --OH, --SO.sub.2, .dbd.S,
--COOH, --NR.sub.2, carbonyl, --C(O)--(C.sub.1-C.sub.10 alkyl)-CF3,
--C(O)--CF3, --C(O)NR2, --(C.sub.1-C.sub.10
aryl)-S--(C.sub.6-C.sub.10 aryl), --C(O)--(C.sub.6-C.sub.10 aryl),
--(CH.sub.2).sub.m--O--(CH.sub.2).sub.m--O--(C.sub.1-C.sub.10
alkyl) wherein each m is from 1 to 8, --C(O)NR.sub.2,
--C(S)NR.sub.2, --SO.sub.2NR.sub.2, --NRC(O)NR.sub.2,
--NRC(S)NR.sub.2, salts thereof, and the like. Each R group in the
preceding list is independently selected from the group consisting
of H, alkyl or substituted alkyl, aryl or substituted aryl, or
alkaryl. Where substituent groups are specified by their
conventional chemical formulas, written from left to right, they
equally encompass the chemically identical substituents that would
result from writing the structure from right to left, for example,
--CH.sub.2O-- is equivalent to --OCH.sub.2--.
[0068] Substituents for alkyl and heteroalkyl radicals (including
those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to:
--OR, .dbd.O, .dbd.NR, .dbd.N--OR, --NR.sub.2, --SR, -halogen,
--SiR.sub.3, --OC(O)R, --C(O)R, --CO.sub.2R, --CONR.sub.2,
--OC(O)NR.sub.2, --NRC(O)R, --NR--C(O)NR.sub.2, --NR(O).sub.2R,
--NR--C(NR.sub.2).dbd.NR, --S(O)R, --S(O).sub.2R,
--S(O).sub.2NR.sub.2, --NRSO.sub.2R, --CN and --NO.sub.2 in a
number ranging from zero to (2m'+1), where m' is the total number
of carbon atoms in such a radical. Each R group in the preceding
list is independently selected from the group consisting of
hydrogen, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, including but not limited to, aryl substituted
with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or
thioalkoxy groups, or aralkyl groups. When two R groups are
attached to the same nitrogen atom, they can be combined with the
nitrogen atom to form a 5-, 6-, or 7-membered ring. For example,
--NR.sub.2 is meant to include, but not be limited to,
1-pyrrolidinyl and 4-morpholinyl. From the above discussion of
substituents, one of skill in the art will understand that the term
"alkyl" is meant to include groups including carbon atoms bound to
groups other than hydrogen groups, such as haloalkyl (including but
not limited to, --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl
(including but not limited to, --C(O)CH.sub.3, --C(O)CF.sub.3,
--C(O)CH.sub.2OCH.sub.3, and the like).
[0069] Similar to the substituents described for the alkyl radical,
substituents for aryl and heteroaryl groups are varied and are
selected from, but are not limited to --OR, .dbd.O, .dbd.NR,
.dbd.N--OR, --NR.sub.2, --SR, -halogen, --SiR.sub.3, --OC(O)R,
--C(O)R, --CO.sub.2R, --CONR.sub.2, --OC(O)NR.sub.2, --NRC(O)R,
--NR--C(O)NR.sub.2, --NR(O).sub.2R, --NR--C(NR.sub.2).dbd.NR,
--S(O)R, --S(O).sub.2R, --S(O).sub.2NR.sub.2, --NRSO.sub.2R, --CN,
--NO.sub.2, --R, --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where each R group in the preceding
list is independently selected from hydrogen, alkyl, heteroalkyl,
aryl and heteroaryl.
[0070] In therapeutic applications, compositions containing the
(modified) non-natural amino acid polypeptide are administered to a
patient already suffering from a disease, condition or disorder, in
an amount sufficient to cure or at least partially arrest the
symptoms of the disease, disorder or condition. Such an amount is
defined to be a "therapeutically effective amount," and will depend
on the severity and course of the disease, disorder or condition,
previous therapy, the patient's health status and response to the
drugs, and the judgment of the treating physician. It is considered
well within the skill of the art for one to determine such
therapeutically effective amounts by routine experimentation (e.g.,
a dose escalation clinical trial).
[0071] As used herein, the term "test ligand" refers to an agent,
which can be a compound, molecule or complex, which is being tested
for its ability to bind to a non-natural amino acid polypeptide,
such as a protein or -protein complex in its native form is known
to be associated with or causative of a disease or condition in a
living organism, such as a vertebrate, particularly a mammal and
even more particularly a human. Since binding of a ligand to its
non-natural amino acid polypeptide must occur for the ligand to
have a direct effect on the non-natural amino acid polypeptide,
binding as indicated by the present assay method is a strong
indication of the therapeutic potential of a ligand identified as
described herein.
[0072] A test ligand which can be assessed by the present method
can be virtually any agent, including, but not limited to, metals,
polypeptides, proteins, lipids, polysaccharides, polynucleotides
and small organic molecules. A test ligand which is shown to bind a
non-natural amino acid polypeptide is referred to as a ligand.
Complex mixtures of substances, including but not limited to,
natural product extracts, which include more than one test ligand
can be tested and if there is a positive response (i.e., if binding
to the non-natural amino acid polypeptide occurs), the ligand which
bound the non-natural amino acid polypeptide can be purified from
the mixture prior to further assessment of its therapeutic
potential.
[0073] The term "treating" is used to refer to either prophylactic
and/or therapeutic treatments.
[0074] As used herein, the term "water soluble polymer" refers to
any polymer that is soluble in aqueous solvents. Linkage of water
soluble polymers to a polypeptide can result in changes including,
but not limited to, increased or modulated serum half-life, or
increased or modulated therapeutic half-life relative to the
unmodified form, modulated immunogenicity, modulated physical
association characteristics such as aggregation and multimer
formation, altered receptor binding, altered binding to one or more
binding partners, and altered receptor dimerization or
multimerization. The water soluble polymer may or may not have its
own biological activity and may be utilized as a linker for
attaching the polypeptide to other substances, including but not
limited to one or more polypeptides, or one or more biologically
active molecules. Suitable polymers include, but are not limited
to, polyethylene glycol, polyethylene glycol propionaldehyde, mono
C1-C10 alkoxy or aryloxy derivatives thereof (described in U.S.
Pat. No. 5,252,714 which is incorporated by reference herein),
monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl
alcohol, polyamino acids, divinylether maleic anhydride,
N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives
including dextran sulfate, polypropylene glycol, polypropylene
oxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,
heparin fragments, polysaccharides, oligosaccharides, glycans,
cellulose and cellulose derivatives, including but not limited to
methylcellulose and carboxymethyl cellulose, starch and starch
derivatives, polypeptides, polyalkylene glycol and derivatives
thereof, copolymers of polyalkylene glycols and derivatives
thereof, polyvinyl ethyl ethers, and
alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or
mixtures thereof. Examples of such water soluble polymers include
but are not limited to polyethylene glycol and serum albumin.
[0075] Unless otherwise indicated, conventional methods of mass
spectroscopy, NMR, HPLC, protein chemistry, biochemistry,
recombinant DNA techniques and pharmacology, within the skill of
the art are employed.
[0076] Compounds (including, but not limited to non-natural amino
acids, (modified) non-natural amino acid polypeptides and reagents
for producing either of the aforementioned compounds) presented
herein include isotopically-labelled compounds, which are identical
to those recited in the various formulas and structures presented
herein, but for the fact that one or more atoms are replaced by an
atom having an atomic mass or mass number different from the atomic
mass or mass number usually found in nature. Examples of isotopes
that can be incorporated into the present compounds include
isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and
chlorine, such as .sup.2H, .sup.3H. .sup.13C, .sup.14C, .sup.15N,
.sup.18O, .sup.17O, .sup.35S, .sup.18F, .sup.36Cl, respectively.
Certain isotopically-labelled compounds described herein, for
example those into which radioactive isotopes such as .sup.3H and
.sup.14C are incorporated, are useful in drug and/or substrate
tissue distribution assays. Further, substitution with isotopes
such as deuterium, i.e., .sup.2H, can afford certain therapeutic
advantages resulting from greater metabolic stability, for example
increased in vivo half-life or reduced dosage requirements.
[0077] Some of the compounds herein (including, but not limited to
non-natural amino acids, (modified) non-natural amino acid
polypeptides and reagents for producing either of the
aforementioned compounds) have asymmetric carbon atoms and can
therefore exist as enantiomers or diastereomers. Diasteromeric
mixtures can be separated into their individual diastereomers on
the basis of their physical chemical differences by methods known,
for example, by chromatography and/or fractional crystallization.
Enantiomers can be separated by converting the enantiomeric mixture
into a diastereomeric mixture by reaction with an appropriate
optically active compound (e.g., alcohol), separating the
diastereomers and converting (e.g., hydrolyzing) the individual
diastereomers to the corresponding pure enantiomers. All such
isomers, including diastereomers, enantiomers, and mixtures thereof
are considered as part of the compositions described herein.
[0078] In additional or further embodiments, the compounds
described herein (including, but not limited to non-natural amino
acids, (modified) non-natural amino acid polypeptides and reagents
for producing either of the aforementioned compounds) are used in
the form of pro-drugs. In additional or further embodiments, the
compounds described herein (including, but not limited to
non-natural amino acids, (modified) non-natural amino acid
polypeptides and reagents for producing either of the
aforementioned compounds) are metabolized upon administration to an
organism in need to produce a metabolite that is then used to
produce a desired effect, including a desired therapeutic effect.
In further or additional embodiments are active metabolites of
non-natural amino acids and (modified) non-natural amino acid
polypeptides.
[0079] The methods and formulations described herein include the
use of N-oxides, crystalline forms (also known as polymorphs), or
pharmaceutically acceptable salts of non-natural amino acids and
(modified) non-natural amino acid polypeptides. In some situations,
non-natural amino acids and (modified) non-natural amino acid
polypeptides may exist as tautomers. All tautomers are included
within the scope of the non-natural amino acids and (modified)
non-natural amino acid polypeptides presented herein. In addition,
the non-natural amino acids and (modified) non-natural amino acid
polypeptides described herein can exist in unsolvated as well as
solvated forms with pharmaceutically acceptable solvents such as
water, ethanol, and the like. The solvated forms of the non-natural
amino acids and (modified) non-natural amino acid polypeptides
presented herein are also considered to be disclosed herein.
[0080] Those skilled in the art will recognize that some of the
compounds herein (including, but not limited to non-natural amino
acids, (modified) non-natural amino acid polypeptides and reagents
for producing either of the aforementioned compounds) can exist in
several tautomeric forms. All such tautomeric forms are considered
as part of the compositions described herein. Also, for example all
enol-keto forms of any compounds (including, but not limited to
non-natural amino acids, (modified) non-natural amino acid
polypeptides and reagents for producing either of the
aforementioned compounds) herein are considered as part of the
compositions described herein.
[0081] Some of the compounds herein (including, but not limited to
non-natural amino acids, (modified) non-natural amino acid
polypeptides and reagents for producing either of the
aforementioned compounds) are acidic and may form a salt with a
pharmaceutically acceptable cation. Some of the compounds herein
(including, but not limited to non-natural amino acids, (modified)
non-natural amino acid polypeptides and reagents for producing
either of the aforementioned compounds) can be basic and
accordingly, may form a salt with a pharmaceutically acceptable
anion. All such salts, including di-salts are within the scope of
the compositions described herein and they can be prepared by
conventional methods. For example, salts can be prepared by
contacting the acidic and basic entities, in either an aqueous,
non-aqueous or partially aqueous medium. The salts are recovered by
using at least one of the following techniques: filtration,
precipitation with a non-solvent followed by filtration,
evaporation of the solvent, or, in the case of aqueous solutions,
lyophilization.
[0082] Salts, for example, include: (1) acid addition salts, formed
with inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like; or
formed with organic acids such as acetic acid, propionic acid,
hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic
acid, lactic acid, malonic acid, succinic acid, malic acid, maleic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic
acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
2-naphthalenesulfonic acid,
4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid),
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, lauryl sulfuric acid, gluconic acid, glutamic acid,
hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid,
and the like; (2) salts formed when an acidic proton present in the
parent compound either is replaced by a metal ion, e.g., an alkali
metal ion, an alkaline earth ion, or an aluminum ion; or
coordinates with an organic base. Acceptable organic bases include
ethanolamine, diethanolamine, triethanolamine, tromethamine,
N-methylglucamine, and the like. Acceptable inorganic bases include
aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium
carbonate, sodium hydroxide, and the like.
[0083] It should be understood that a reference to a salt includes
the solvent addition forms or crystal forms thereof, particularly
solvates or polymorphs. Solvates contain either stoichiometric or
non-stoichiometric amounts of a solvent, and are often formed
during the process of crystallization. Hydrates are formed when the
solvent is water, or alcoholates are formed when the solvent is
alcohol. Polymorphs include the different crystal packing
arrangements of the same elemental composition of a compound.
Polymorphs usually have different X-ray diffraction patterns,
infrared spectra, melting points, density, hardness, crystal shape,
optical and electrical properties, stability, and solubility.
Various factors such as the recrystallization solvent, rate of
crystallization, and storage temperature may cause a single crystal
form to dominate.
INCORPORATION BY REFERENCE
[0084] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference in their
entirety for all purposes to the same extent as if each individual
publication, patent, or patent application was specifically and
individually indicated to be incorporated by reference for all
purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0086] FIG. 1 presents a schematic representation of the
relationship of certain aspects of the methods, compositions,
strategies and techniques described herein.
[0087] FIG. 1a presents various protein detection techniques.
[0088] FIG. 2 presents an illustrative, non-limiting example of
reactions where an amino acid functionality (A), translationally
incorporated (or otherwise incorporated) into a polypeptide, reacts
with reactant (B) to yield a modified polypeptide.
[0089] FIG. 3 presents an illustrative, non-limiting example of
formation of oxime-containing non-natural amino acid components by
reaction of carbonyl-containing non-natural amino acid components
with hydroxylamine-containing reagents.
[0090] FIG. 4 presents an illustrative, non-limiting example of
formation of oxime-containing non-natural amino acid components by
reaction of hydroxylamine-containing non-natural amino acid
components with carbonyl-containing reagents.
[0091] FIG. 5 presents an illustrative, non-limiting example of
formation of oxime-containing non-natural amino acid components by
oxime-containing non-natural amino acid components with
carbonyl-containing reagents.
[0092] FIG. 6 presents an illustrative, non-limiting example of
formation of oxime-containing non-natural amino acid components by
reactions of dicarbonyl-containing non-natural amino acid
components with hydroxylamine-containing reagents.
[0093] FIG. 7 presents an illustrative, non-limiting example of
formation of oxime-containing non-natural amino acid components by
reactions of hydroxylamine-containing non-natural amino acid
components with dicarbonyl-containing reagents.
[0094] FIG. 8 presents an illustrative, non-limiting example of
formation of oxime-containing non-natural amino acid components by
oxime exchange reactions of oxime-containing non-natural amino acid
components with carbonyl or dicarbonyl-containing reagents.
[0095] FIG. 9 presents non-limiting examples of molecules that are
site specifically attached to proteins through oxime formation
between carbonyl of non-natural amino acid incorporated into a
polypeptide and the hydroxylamine of the molecule.
[0096] FIG. 10 shows an example of a purification method for a
non-natural amino acid polypeptide utilizing a resin that reacts
with the non-natural amino acid.
[0097] FIG. 11 shows an example of a method in which the
purification of a non-natural amino acid polypeptide and
conjugation of the polypeptide is performed in "one pot".
[0098] FIG. 12 shows an example of resin selection and
functionalization.
[0099] FIG. 13 shows an example of affinity purification of a
non-natural amino acid polypeptide using hydroxylamine resin.
[0100] FIG. 14 shows an example of purification of a non-natural
amino acid polypeptide using an aldehyde resin.
[0101] FIG. 15 shows an example of purification of native proteins
from a non-natural amino acid precursor that is converted to
tyrosine after cleavage.
[0102] FIG. 16 shows non-limiting examples of non-natural amino
acids.
[0103] FIG. 17 shows SDS-PAGE analysis of hGH-single strand DNA
conjugate 1) Reaction mixture of the conjugation reaction; 2)
Purified hGH-ssDNA conjugate by HIC column.
[0104] FIG. 18 shows protein-ssDNA conjugate hybridization.
[0105] FIG. 19 shows native 14% glycine gel analysis of hGH-ssDNA
conjugate hybridization; hGH-ssDNA conjugate (5 .mu.l) with: 1) 0
.mu.l; 2) 2 .mu.l; 3) 4 .mu.l; 4) 6 .mu.l; 5) 8 .mu.l; 6) 10 .mu.l,
of 1 .mu.M FTam28d3; and 7) 2 .mu.l; 8) 4 .mu.l; 9) 8 .mu.l, of 10
.mu.M FTam28-d3.
[0106] FIG. 20 shows native gel analysis of 5 .mu.l of hGH-ssDNA
mixed with 1) 0 .mu.l; 2) 1 .mu.l; 3) 4 .mu.l, of 100 .mu.M
FTam28-d3; and hGH mixed with 4) 1 .mu.l; 5) 0 .mu.l, of 100 .mu.M
FTam28-d3.
[0107] FIG. 21 shows assemblies of 1-D hGH structure using DNA as a
template.
DETAILED DESCRIPTION OF THE INVENTION
[0108] I. Introduction
[0109] Recently, an entirely new technology in the protein sciences
has been reported, which promises to overcome many of the
limitations associated with site-specific modifications of
proteins. Specifically, new components have been added to the
protein biosynthetic machinery of the prokaryote Escherichia coli
(E. coli) (e.g., L. Wang, et al., (2001), Science 292:498-500) and
the eukaryote Saccharomyces cerevisiae (S. cerevisiae) (e.g., J.
Chin et al., Science 301:964-7 (2003)), which has enabled the
incorporation of non-natural amino acids to proteins in vivo. A
number of new amino acids with novel chemical, physical or
biological properties, including photoaffinity labels and
photoisomerizable amino acids, keto amino acids, and glycosylated
amino acids have been incorporated efficiently and with high
fidelity into proteins in E. coli and in yeast in response to the
amber codon, TAG, using this methodology. See, e.g., J. W. Chin et
al., (2002), Journal of the American Chemical Society 124:9026-9027
(incorporated by reference in its entirety); J. W. Chin, & P.
G. Schultz, (2002), ChemBioChem 3(11):1135-1137 (incorporated by
reference in its entirety); J. W. Chin, et al., (2002), PNAS United
States of America 99:11020-11024 (incorporated by reference in its
entirety); and, L. Wang, & P. G. Schultz, (2002), Chem. Comm.,
1:1-11 (incorporated by reference in its entirety). These studies
have demonstrated that it is possible to selectively and routinely
introduce chemical functional groups that are not found in
proteins, that are chemically inert to all of the functional groups
found in the 20 common, genetically-encoded amino acids and that
may be used to react efficiently and selectively to form stable
covalent linkages.
[0110] II. Overview
[0111] FIG. 1 is an overview of the compositions, methods and
techniques that are described herein. At one level, incorporated by
reference from U.S. Patent Application Nos. 60/638,418, 60/638,527,
60/639,195, 60/696,210, 60/696,302, and 60/696,068 in their
entirety are the tools (methods, compositions, techniques) for
creating and using a polypeptide comprising at least one
non-natural amino acid or modified non-natural amino acid. Such
non-natural amino acid polypeptides may contain further
functionality, including but not limited to, a label; a dye; a
polymer; a water-soluble polymer; a derivative of polyethylene
glycol; a photocrosslinker; a cytotoxic compound; a drug; an
affinity label; a photoaffinity label; a reactive compound; a
resin; a second protein or polypeptide or polypeptide analog; an
antibody or antibody fragment; a metal chelator; a cofactor; a
fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an
antisense polynucleotide; a saccharide, a water-soluble dendrimer,
a cyclodextrin, an inhibitory ribonucleic acid; a biomaterial; a
nanoparticle; a spin label; a fluorophore, a metal-containing
moiety; a radioactive moiety; a novel functional group; a group
that covalently or noncovalently interacts with other molecules; a
photocaged moiety; an actinic radiation excitable moiety; a
photoisomerizable moiety; biotin; a derivative of biotin; a biotin
analogue; a moiety incorporating a heavy atom; a chemically
cleavable group; a photocleavable group; an elongated side chain; a
carbon-linked sugar; a redox-active agent; an amino thioacid; a
toxic moiety; an isotopically labeled moiety; a biophysical probe;
a phosphorescent group; a chemiluminescent group; an electron dense
group; a magnetic group; an intercalating group; a chromophore; an
energy transfer agent; a biologically active agent; a detectable
label; a small molecule; a quantum dot; a nanotransmitter; and any
combination of the above.
[0112] As shown in FIG. 1, in one aspect are methods for selecting
and designing a polypeptide to be modified using the methods,
compositions and techniques are further described in U.S. Patent
Application Nos. 60/638,418, 60/638,527, 60/639,195, 60/696,210,
60/696,302, and 60/696,068 which are incorporated by reference in
their entirety. The new polypeptide may be designed de novo,
including by way of example only, as part of high-throughput
screening process (in which case numerous polypeptides may be
designed, synthesized, characterized and/or tested) or based on the
interests of the researcher. The new polypeptide may also be
designed based on the structure of a known or partially
characterized polypeptide. By way of example only, the Growth
Hormone Gene Superfamily (see infra) has been the subject of
intense study by the scientific community; a new polypeptide may be
designed based on the structure of a member or members of this gene
superfamily. The principles for selecting which amino acid(s) to
substitute and/or modify are described separately herein. The
choice of which modification to employ is also described herein,
and can be used to meet the need of the experimenter or end user.
Modifications include, by way of example only, manipulating the
therapeutic effectiveness of the polypeptide, improving the safety
profile of the polypeptide, adjusting the pharmacokinetics of the
polypeptide, providing additional functionality to the polypeptide,
incorporating a tag, label or detectable signal into the
polypeptide, easing the isolation properties of the polypeptide,
and any combination of the aforementioned modifications.
[0113] Thus, polypeptides comprising at least one non-natural amino
acid or modified non-natural amino acid are further provided and
described in U.S. Patent Application Nos. 60/638,418, 60/638,527,
60/639,195, 60/696,210, 60/696,302, and 60/696,068 which are
incorporated by reference in their entirety. A very wide variety of
non-naturally encoded amino acids are suitable for use in the
present invention. Any number of non-naturally encoded amino acids
can be introduced into a polypeptide. In general, the introduced
non-naturally encoded amino acids are substantially chemically
inert toward the 20 common, genetically-encoded amino acids (i.e.,
alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, and valine). In some embodiments, the non-naturally
encoded amino acids include side chain functional groups that react
efficiently and selectively with functional groups not found in the
20 common amino acids (including but not limited to, azido, ketone,
aldehyde and aminooxy groups) to form stable conjugates. Because
the non-naturally encoded amino acids of the invention typically
differ from the natural amino acids only in the structure of the
side chain, the non-naturally encoded amino acids form amide bonds
with other amino acids, including but not limited to, natural or
non-naturally encoded, in the same manner in which they are formed
in naturally occurring polypeptides. However, the non-naturally
encoded amino acids have side chain groups that distinguish them
from the natural amino acids. For example, the side chain (R group)
optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-,
hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl,
ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho,
phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester,
thioacid, hydroxylamine, amino group, or the like or any
combination thereof.
[0114] Other non-naturally occurring amino acids of interest that
may be suitable for use in the present invention include, but are
not limited to, amino acids comprising a photoactivatable
cross-linker, spin-labeled amino acids, fluorescent amino acids,
metal binding amino acids, metal-containing amino acids,
radioactive amino acids, amino acids with novel functional groups,
amino acids that covalently or noncovalently interact with other
molecules, photocaged and/or photoisomerizable amino acids, amino
acids comprising biotin or a biotin analogue, glycosylated amino
acids such as a sugar substituted serine, other carbohydrate
modified amino acids, keto-containing amino acids, amino acids
comprising polyethylene glycol or polyether, heavy atom substituted
amino acids, chemically cleavable and/or photocleavable amino
acids, amino acids with an elongated side chains as compared to
natural amino acids, including but not limited to, polyethers or
long chain hydrocarbons, including but not limited to, greater than
about 5 or greater than about 10 carbons, carbon-linked
sugar-containing amino acids, redox-active amino acids, amino
thioacid containing amino acids, and amino acids comprising one or
more toxic moiety.
[0115] A number of non-natural amino acids for incorporation into
polypeptides are found in WO 2002/085923 entitled "In vivo
incorporation of unnatural amino acids" which is incorporated by
reference herein in its entirety. Methods and compositions for the
in vivo incorporation of non-naturally encoded amino acids are
described in U.S. Patent Application Publication 2003/0082575 (Ser.
No. 10/126,927) which is incorporated by reference herein in its
entirety. Methods for selecting an orthogonal tRNA-tRNA synthetase
pair for use in in vivo translation system of an organism are also
described in U.S. Patent Application Publications 2003/0082575
(Ser. No. 10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which
are incorporated by reference herein in their entirety. PCT
Publication No. WO 04/035743 entitled "Site Specific Incorporation
of Keto Amino Acids into Proteins," which is incorporated by
reference herein in its entirety, describes orthogonal RS and tRNA
pairs for the incorporation of keto amino acids. PCT Publication
No. WO 04/094593 entitled "Expanding the Eukaryotic Genetic Code,"
which is incorporated by reference herein in its entirety,
describes orthogonal RS and tRNA pairs for the incorporation of
non-naturally encoded amino acids in eukaryotic host cells.
Non-naturally encoded amino acids have side chain groups that
distinguish them from the natural amino acids. The side chain may
comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-,
hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol,
seleno-, sulfonyl-, borate, boronate, phospho, phosphono,
phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid,
hydroxylamine, amino group, or the like or any combination
thereof.
[0116] In certain embodiments, polypeptides with at least one
non-natural amino acid or modified non-natural amino acid group
include at least one post-translational modification at some
position on the polypeptide. In some embodiments the
post-translational modification occurs via the cellular machinery
(e.g., glycosylation, acetylation, acylation, lipid-modification,
palmitoylation, palmitate addition, phosphorylation,
glycolipid-linkage modification, and the like), in many instances,
such cellular-machinery-based post-translational modifications
occur at the naturally occurring amino acid sites on the
polypeptide, however, in certain embodiments, the
cellular-machinery-based post-translational modifications occur on
the non-natural amino acid site(s) on the polypeptide.
[0117] In other embodiments the post-translational modification
does not utilize the cellular machinery, but is instead providing
by attachment of a molecule (including but not limited to, a label;
a dye; a polymer; a water-soluble polymer; a derivative of
polyethylene glycol; a photocrosslinker; a cytotoxic compound; a
drug; an affinity label; a photoaffinity label; a reactive
compound; a resin; a second protein or polypeptide or polypeptide
analog; an antibody or antibody fragment; a metal chelator; a
cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a
RNA; an antisense polynucleotide; a saccharide, a water-soluble
dendrimer, a cyclodextrin, an inhibitory ribonucleic acid; a
biomaterial; a nanoparticle; a spin label; a fluorophore, a
metal-containing moiety; a radioactive moiety; a novel functional
group; a group that covalently or noncovalently interacts with
other molecules; a photocaged moiety; an actinic radiation
excitable moiety; a photoisomerizable moiety; biotin; a derivative
of biotin; a biotin analogue; a moiety incorporating a heavy atom;
a chemically cleavable group; a photocleavable group; an elongated
side chain; a carbon-linked sugar; a redox-active agent; an amino
thioacid; a toxic moiety; an isotopically labeled moiety; a
biophysical probe; a phosphorescent group; a chemiluminescent
group; an electron dense group; a magnetic group; an intercalating
group; a chromophore; an energy transfer agent; a biologically
active agent; a detectable label; a small molecule, a quantum dot;
a nanotransmitter; and any combination of the above) comprising a
second reactive group to the at least one non-natural amino acid
comprising a first reactive group (including but not limited to,
non-natural amino acid containing a ketone, aldehyde, acetal,
hemiacetal, oxime, or hydroxylamine functional group) utilizing
chemistry methodology that is known to one of ordinary skill in the
art to be suitable for the particular reactive groups. In certain
embodiments, the post-translational modification is made in vivo in
a eukaryotic cell or in a non-eukaryotic cell. In certain
embodiments, the post-translational modification is made in vitro.
Also included with this aspect are methods for producing,
purifying, characterizing and using such polypeptides containing at
least one such post-translationally modified non-natural amino
acids.
[0118] Also included within the scope of the methods, compositions,
strategies and techniques further described in U.S. Patent
Application Nos. 60/638,418, 60/638,527, 60/639,195, 60/696,210,
60/696,302, and 60/696,068 which are incorporated by reference in
their entirety are reagents capable of reacting with a non-natural
amino acid that is part of a polypeptide so as to produce any of
the aforementioned post-translational modifications. In general,
the resulting post-translationally modified non-natural amino acid
will contain at least one non-natural amino acid which may undergo
subsequent modification reactions. Also included with this aspect
are methods for producing, purifying, characterizing and using such
reagents that are capable of any such post-translational
modifications of such non-natural amino acid(s).
[0119] In certain embodiments, the protein includes at least one
post-translational modification that is made in vivo by one host
cell, where the post-translational modification is not normally
made by another host cell type. In certain embodiments, the protein
includes at least one post-translational modification that is made
in vivo by a eukaryotic cell, where the post-translational
modification is not normally made by a non-eukaryotic cell.
Examples of post-translational modifications include, but are not
limited to, glycosylation, acetylation, acylation,
lipid-modification, palmitoylation, palmitate addition,
phosphorylation, glycolipid-linkage modification, and the like. In
one embodiment, the post-translational modification comprises
attachment of an oligosaccharide to an asparagine by a
GlcNAc-asparagine linkage (including but not limited to, where the
oligosaccharide comprises (GlcNAc-Man).sub.2-Man-GlcNAc-GlcNAc, and
the like). In another embodiment, the post-translational
modification comprises attachment of an oligosaccharide (including
but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or
threonine by a GalNAc-serine, a GalNAc-threonine, a GlcNAc-serine,
or a GlcNAc-threonine linkage. In certain embodiments, a protein or
polypeptide can comprise a secretion or localization sequence, an
epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or
the like. Examples of secretion signal sequences include, but are
not limited to, a prokaryotic secretion signal sequence, a
eukaryotic secretion signal sequence, a eukaryotic secretion signal
sequence 5'-optimized for bacterial expression, a novel secretion
signal sequence, pectate lyase secretion signal sequence, Omp A
secretion signal sequence, and a phage secretion signal sequence.
Examples of secretion signal sequences, include, but are not
limited to, STII (prokaryotic), Fd GIII and M13 (phage), Bgl2
(yeast), and the signal sequence bla derived from a transposon.
Also included with this aspect are methods for producing,
purifying, characterizing and using such polypeptides containing at
least one such post-translational modification.
[0120] The protein or polypeptide of interest can contain at least
one, at least two, at least three, at least four, at least five, at
least six, at least seven, at least eight, at least nine, or ten or
more non-natural amino acids. The non-natural amino acids can be
the same or different, for example, there can be 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or more different sites in the protein that comprise 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more different non-natural amino
acids. In certain embodiments, at least one, but fewer than all, of
a particular amino acid present in a naturally occurring version of
the protein is substituted with an non-natural amino acid.
[0121] The methods and compositions provided and described herein
include polypeptides comprising at least one non-natural amino
acid. Introduction of at least one non-natural amino acid into a
polypeptide can allow for the application of conjugation
chemistries that involve specific chemical reactions, including,
but not limited to, with one or more non-natural amino acids while
not reacting with the commonly occurring 20 amino acids. Once
incorporated, the amino acid side chains can then be modified by
utilizing chemistry methodologies known to those of ordinary skill
in the art to be suitable for the particular functional groups or
substituents present in the naturally encoded amino acid.
[0122] The non-natural amino acid methods and compositions
described herein provides conjugates of substances having a wide
variety of functional groups, substituents or moieties, with other
substances including but not limited to a label; a dye; a polymer;
a water-soluble polymer; a derivative of polyethylene glycol; a
photocrosslinker; a cytotoxic compound; a drug; an affinity label;
a photoaffinity label; a reactive compound; a resin; a second
protein or polypeptide or polypeptide analog; an antibody or
antibody fragment; a metal chelator; a cofactor; a fatty acid; a
carbohydrate; a polynucleotide; a DNA; a RNA; an antisense
polynucleotide; a saccharide; a water-soluble dendrimer; a
cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a
nanoparticle; a spin label; a fluorophore, a metal-containing
moiety; a radioactive moiety; a novel functional group; a group
that covalently or noncovalently interacts with other molecules; a
photocaged moiety; an actinic radiation excitable moiety; a
photoisomerizable moiety; biotin; a derivative of biotin; a biotin
analogue; a moiety incorporating a heavy atom; a chemically
cleavable group; a photocleavable group; an elongated side chain; a
carbon-linked sugar; a redox-active agent; an amino thioacid; a
toxic moiety; an isotopically labeled moiety; a biophysical probe;
a phosphorescent group; a chemiluminescent group; an electron dense
group; a magnetic group; an intercalating group; a chromophore; an
energy transfer agent; a biologically active agent; a detectable
label; a small molecule; a quantum dot; a nanotransmitter; and any
combination of the above. Conjugation of a non-natural amino acid
polypeptide with a molecule, including but not limited to, biotin
may enable purification of the conjugate.
[0123] In another aspect of the compositions, methods, techniques
and strategies further described in U.S. Patent Application Nos.
60/638,418, 60/638,527, 60/639,195, 60/696,210, 60/696,302, and
60/696,068 which are incorporated by reference in their entirety
are methods for studying or using any of the aforementioned
(modified) non-natural amino acid polypeptides. Included within
this aspect, by way of example only, are therapeutic, diagnostic,
assay-based, industrial, cosmetic, plant biology, environmental,
energy-production, and/or military uses which would benefit from a
polypeptide comprising a (modified) non-natural amino acid
polypeptides or protein.
[0124] The invention provides a method for detecting the
aforementioned (modified) non-natural amino acid polypeptides or a
fragment thereof. Such non-natural amino acid polypeptides or a
fragment thereof can be obtained by combining the non-natural amino
acid polypeptides or a fragment thereof with a library of molecules
under conditions suitable to allow specific interactions. The
invention also provides a method for detecting the aforementioned
(modified) non-natural amino acid polypeptides or a fragment
thereof where non-natural amino acid polypeptides or a fragment
thereof are obtained by combining the non-natural amino acid
polypeptides or a fragment thereof with the library of proteins or
a portion thereof under conditions suitable to allow specific
interaction. Such interactions include but are not limited to
acetylation, carboxylation, acylation, phosphorylation,
dephosphorylation, ubiquitination, glycosylation, lipid
modification, ADP-ribosylation, bioavailability and half-life. Such
libraries include alpha-1 antitrypsin, angiostatin, antihemolytic
factor, antibody, apolipoprotein, apoprotein, atrial natriuretic
factor, atrial natriuretic polypeptide, atrial peptide, C-X-C
chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10,
GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine,
CC chemokine, monocyte chemoattractant protein-1, monocyte
chemoattractant protein-2, monocyte chemoattractant protein-3,
monocyte inflammatory protein-1 alpha, monocyte inflammatory
protein-i beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065,
T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony
stimulating factor (CSF), complement factor 5a, complement
inhibitor, complement receptor 1, cytokine, epithelial neutrophil
activating peptide-78, MIP-16, MCP-1, epidermal growth factor
(EGF), epithelial neutrophil activating peptide, erythropoietin
(EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII,
Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin,
four-helical bundle protein, G-CSF, glp-1, GM-CSF,
glucocerebrosidase, gonadotropin, growth factor, growth factor
receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth
factor (hGF), hirudin, human growth hormone (hGH), human serum
albumin, ICAM-1. ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin,
insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN),
IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
keratinocyte growth factor (KGF), lanreotide, lactoferrin, leukemia
inhibitory factor, luciferase, neurturin, neutrophil inhibitory
factor (NIF), oncostatin M, osteogenic protein, oncogene product,
paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone,
pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A,
pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin,
SCF, small biosynthetic protein, soluble complement receptor I,
soluble I-CAM 1, soluble interleukin receptor, soluble TNF
receptor, somatomedin, somatostatin, somatotropin, streptokinase,
superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2,
SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase,
toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen
activator, tumor growth factor (TGF), tumor necrosis factor, tumor
necrosis factor alpha, tumor necrosis factor beta, tumor necrosis
factor receptor (TNFR), urotensin-II, VLA-4 protein, VCAM-1
protein, vascular endothelial growth factor (VEGF), urokinase, mos,
ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen
receptor, progesterone receptor, testosterone receptor, aldosterone
receptor, LDL receptor, and corticosterone.
[0125] III. Location of Non-Natural Amino Acids in Polypeptides
[0126] The non-natural amino acid polypeptides or a fragment
thereof disclosed herein, include incorporation of one or more
non-natural amino acids into a polypeptide. One or more non-natural
amino acids may be incorporated at a particular position which does
not disrupt activity of the polypeptide. This can be achieved by
making "conservative" substitutions, including but not limited to,
substituting hydrophobic amino acids with hydrophobic amino acids,
bulky amino acids for bulky amino acids, hydrophilic amino acids
for hydrophilic amino acids and/or inserting the non-natural amino
acid in a location that is not required for activity.
[0127] A variety of biochemical and structural approaches can be
employed to select the desired sites for substitution with a
non-natural amino acid within the polypeptide. Any position of the
polypeptide chain is suitable for selection to incorporate a
non-natural amino acid, and selection may be based on rational
design or by random selection for any or no particular desired
purpose. Selection of desired sites may be for producing a
non-natural amino acid polypeptide (which may be further modified
or remain unmodified) having any desired property or activity,
including but not limited to agonists, super-agonists, inverse
agonists, antagonists, receptor binding modulators, receptor
activity modulators, modulators of binding to one or more binding
partners, binding partner activity modulators, binding partner
conformation modulators, dimer or multimer formation, no change to
activity or property compared to the native molecule, or
manipulating any physical or chemical property of the polypeptide
such as solubility, aggregation, or stability. For example,
locations in the polypeptide required for biological activity of a
polypeptide can be identified using point mutation analysis,
alanine scanning or homolog scanning methods known in the art.
Methods similar to those described in Cunningham, B. and Wells, J.,
Science, 244:1081-1085 (1989) and Cunningham, B., et al. Science
243: 1330-1336 (1989) may be used to identify residues that are
critical for bioactivity and/or may be used to identify antibody
and receptor epitopes. U.S. Pat. Nos. 5,580,723; 5,834,250;
6,013,478; 6,428,954; and 6,451,561, which are incorporated by
reference herein, describe methods for the systematic analysis of
the structure and function of polypeptides by identifying active
domains which influence the activity of the polypeptide with a
target substance. Residues other than those identified as critical
to biological activity by alanine or homolog scanning mutagenesis
may be good candidates for substitution with a non-natural amino
acid depending on the desired activity sought for the polypeptide.
Alternatively, the sites identified as critical to biological
activity may also be good candidates for substitution with a
non-natural amino acid, again depending on the desired activity
sought for the polypeptide. Another alternative would be to simply
make serial substitutions in each position on the polypeptide chain
with a non-natural amino acid and observe the effect on the
activities of the polypeptide. It is readily apparent to those of
ordinary skill in the art that any means, technique, or method for
selecting a position for substitution with a non-natural amino acid
into any polypeptide is suitable for use in the present
invention.
[0128] The structure and activity of naturally-occurring mutants of
a polypeptide that contain deletions can also be examined to
determine regions of the protein that are likely to be tolerant of
substitution with a non-natural amino acid. Once residues that are
likely to be intolerant to substitution with non-natural amino
acids have been eliminated, the impact of proposed substitutions at
each of the remaining positions can be examined from the
three-dimensional structure of the relevant polypeptide, and any
associated ligands or binding proteins. X-ray crystallographic and
NMR structures of many polypeptides are available in the Protein
Data Bank (PDB, www.rcsb.org), a centralized database containing
three-dimensional structural data of large molecules of proteins
and nucleic acids. Thus, those of ordinary skill in the art can
readily identify amino acid positions that can be substituted with
non-natural amino acids.
[0129] Exemplary sites of incorporation of a non-natural amino acid
include, but are not limited to, those that are excluded from
potential receptor binding regions, regions for binding to one or
more binding partners, may be fully or partially solvent exposed,
have minimal or no hydrogen-bonding interactions with nearby
residues, may be minimally exposed to nearby reactive residues, may
be on one or more of the exposed faces of the polypeptide, may be
in regions that are highly flexible or structurally rigid as
predicted by the three-dimensional, secondary, tertiary, or
quaternary structure of the polypeptide, bound or unbound to its
associated receptor, ligand or binding proteins., or coupled or not
coupled to another polypeptide or other biologically active
molecule, or may modulate the conformation of the polypeptide
itself or a dimer or multimer comprising one or more polypeptide,
by altering the flexibility or rigidity of the complete structure
as desired.
[0130] A wide variety of non-natural amino acids can be substituted
for, or incorporated into, a given position in a polypeptide. In
general, a particular non-natural amino acid may be selected for
incorporation based on an examination of the three dimensional
crystal structure of a polypeptide with its associated ligand,
receptor and/or binding proteins, secondary, tertiary or quaternary
structure, a preference for conservative substitutions (i.e.,
aryl-based non-natural amino acids, such as p-acetylphenylalanine
or O-propargyltyrosine substituting for Phe, Tyr or Trp), and the
specific conjugation chemistry that one desires to introduce into
the polypeptide protein.
[0131] The method further includes incorporating into the protein
the non-natural amino acid, where the non-natural amino acid
comprises a first reactive group; and contacting the protein with a
molecule (including but not limited to a label; a dye; a polymer; a
water-soluble polymer; a derivative of polyethylene glycol; a
photocrosslinker; a cytotoxic compound; a drug; an affinity label;
a photoaffinity label; a reactive compound; a resin; a second
protein or polypeptide or polypeptide analog; an antibody or
antibody fragment; a metal chelator; a cofactor; a fatty acid; a
carbohydrate; a polynucleotide; a DNA; a RNA; an antisense
polynucleotide; a saccharide; a water-soluble dendrimer; a
cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a
nanoparticle; a spin label; a fluorophore, a metal-containing
moiety; a radioactive moiety; a novel functional group; a group
that covalently or noncovalently interacts with other molecules; a
photocaged moiety; an actinic radiation excitable moiety; a
photoisomerizable moiety; biotin; a derivative of biotin; a biotin
analogue; a moiety incorporating a heavy atom; a chemically
cleavable group; a photocleavable group; an elongated side chain; a
carbon-linked sugar; a redox-active agent; an amino thioacid; a
toxic moiety; an isotopically labeled moiety; a biophysical probe;
a phosphorescent group; a chemiluminescent group; an electron dense
group; a magnetic group; an intercalating group; a chromophore; an
energy transfer agent; a biologically active agent; a detectable
label; a small molecule; a quantum dot; a nanotransmitter; and any
combination of the above) that comprises a second reactive
group.
[0132] In some cases, the non-natural amino acid substitution(s) or
incorporation(s) will be combined with other additions,
substitutions, or deletions within the polypeptide to affect other
biological traits. In some cases, the other additions,
substitutions or deletions may increase the stability (including
but not limited to, resistance to proteolytic degradation) of the
polypeptide or increase affinity of the polypeptide for its
appropriate receptor, ligand and/or binding proteins. In some
cases, the other additions, substitutions or deletions may increase
the solubility (including but not limited to, when expressed in E.
coli or other host cells) of the polypeptide. In some cases, sites
are selected for substitution with a naturally encoded or
non-natural amino acid in addition to another site for
incorporation of a non-natural amino acid for the purpose of
increasing the polypeptide solubility following expression in E.
coli recombinant host cells. In some cases, the polypeptides
comprise another addition, substitution, or deletion that modulates
affinity for the associated ligand, binding proteins, and/or
receptor, modulates (including but not limited to, increases or
decreases) receptor dimerization, stabilizes receptor dimers,
modulates circulating half-life, modulates release or
bio-availability, facilitates purification, or improves or alters a
particular route of administration. Similarly, polypeptide can
comprise chemical or enzyme cleavage sequences, protease cleavage
sequences, reactive groups, antibody-binding domains (including but
not limited to, FLAG or poly-His) or other affinity based sequences
(including but not limited to, FLAG, poly-His, GST, etc.) or linked
molecules (including but not limited to, biotin) that improve
detection (including but not limited to, GFP), purification,
transport through tissues or cell membranes, prodrug release or
activation, size reduction, or other traits of the polypeptide.
[0133] IV. Growth Hormone Supergene Family as Exemplar
[0134] The methods, compositions, strategies and techniques
described herein are not limited to a particular type, class or
family of polypeptides or proteins. By way of example only, the
polypeptide can be homologous to a therapeutic protein selected
from the group consisting of: alpha-1 antitrypsin, angiostatin,
antihemolytic factor, antibody, antibody fragments, apolipoprotein,
apoprotein, atrial natriuretic factor, atrial natriuretic
polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2,
ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG,
calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte
chemoattractant protein-1, monocyte chemoattractant protein-2,
monocyte chemoattractant protein-3, monocyte inflammatory protein-1
alpha, monocyte inflammatory protein-i beta, RANTES, 1309, R83915,
R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit
ligand, collagen, colony stimulating factor (CSF), complement
factor 5a, complement inhibitor, complement receptor 1, cytokine,
epithelial neutrophil activating peptide-78, MIP-16, MCP-1,
epidermal growth factor (EGF), epithelial neutrophil activating
peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor
VII, Factor VIII, Factor X, fibroblast growth factor (FGF),
fibrinogen, fibronectin, four-helical bundle protein, G-CSF, glp-1,
GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth
factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte
growth factor (hGF), hirudin, human growth hormone (hGH), human
serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor,
insulin, insulin-like growth factor (IGF), IGF-I, IGF-II,
interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL),
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1,
IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia
inhibitory factor, luciferase, neurturin, neutrophil inhibitory
factor (NIF), oncostatin M, osteogenic protein, oncogene product,
paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone,
pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A,
pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin,
SCF, small biosynthetic protein, soluble complement receptor 1,
soluble I-CAM 1, soluble interleukin receptor, soluble TNF
receptor, somatomedin, somatostatin, somatotropin, streptokinase,
superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2,
SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase,
toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen
activator, tumor growth factor (TGF), tumor necrosis factor, tumor
necrosis factor alpha, tumor necrosis factor beta, tumor necrosis
factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular
endothelial growth factor (VEGF), urokinase, mos, ras, raf, met,
p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone
receptor, testosterone receptor, aldosterone receptor, LDL
receptor, and corticosterone.
[0135] Antibody fragments herein include antibodies that are
smaller components that exist within full-length antibodies, and
antibodies that have been engineered. Antibody fragments include
but are not limited to Fv, Fc, Fab, and (Fab') 2, single chain Fv
(scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid
antibodies, CDR1, CDR2, CDR3, combinations of CDR's, variable
regions, framework regions, constant regions, and the like (Maynard
& Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson,
1998, Curr. Opin. Biotechnol. 9:395-402). Another functional
substructure is a single chain Fv (scFv), comprised of the variable
regions of the immunoglobulin heavy and light chain, covalently
connected by a peptide linker (S-z Hu et al., 1996, Cancer
Research, 56, 3055-3061). These small (Mr 25,000) proteins
generally retain specificity and affinity for antigen in a single
polypeptide and can provide a convenient building block for larger,
antigen-specific molecules. Polypeptides also include the antibody
heavy chain, light chain, variable region, alternative scaffold
non-antibody molecules, and bispecific antibodies, as well as other
antigen-binding polypeptides or fragments thereof.
[0136] Thus, the following description of the growth hormone
supergene family is provided for illustrative purposes and by way
of example only and not as a limit on the scope of the methods,
compositions, strategies and techniques described herein. Further,
reference to GH polypeptides in this application is intended to use
the generic term as an example of any member of the GH supergene
family. Thus, it is understood that the modifications and
chemistries described herein with reference to GH polypeptides or
protein can be equally applied to any member of the GH supergene
family, including those specifically listed herein or incorporated
by reference.
[0137] The following proteins include those encoded by genes of the
growth hormone (GH) supergene family (Bazan, F., Immunology Today
11: 350-354 (1990); Bazan, J. F. Science 257: 410-413 (1992); Mott,
H. R. and Campbell, I. D., Current Opinion in Structural Biology 5:
114-121 (1995); Silvennoinen, O. and Ihie, J. N., SIGNALLING BY THE
HEMATOPOIETIC CYTOKINE RECEPTORS (1996)): growth hormone,
prolactin, placental lactogen, erythropoietin (EPO), thrombopoietin
(TPO), interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9,
IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M,
ciliary neurotrophic factor, leukemia inhibitory factor, alpha
interferon, beta interferon, gamma interferon, omega interferon,
tau interferon, granulocyte-colony stimulating factor (G-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF),
macrophage colony stimulating factor (M-CSF) and cardiotrophin-1
(CT-1) ("the GH supergene family"). It is anticipated that
additional members of this gene family will be identified in the
future through gene cloning and sequencing. Members of the GH
supergene family have similar secondary and tertiary structures,
despite the fact that they generally have limited amino acid or DNA
sequence identity. The shared structural features allow new members
of the gene family to be readily identified and the non-natural
amino acid methods and compositions described herein and
incorporated by reference similarly applied.
[0138] Structures of a number of cytokines, including G-CSF (Zink
et al., FEBS Lett. 314:435 (1992); Zink et al., Biochemistry
33:8453 (1994); Hill et al., Proc. Natl. Acad. Sci. USA 90:5167
(1993)), GM-CSF (Diederichs, K., et al. Science 154: 1779-1782
(1991); Walter et al., J. Mol. Biol. 224:1075-1085 (1992)), IL-2
(Bazan, J. F. and McKay, D. B. Science 257: 410-413 (1992)), IL-4
(Redfield et al., Biochemistry 30: 11029-11035 (1991); Powers et
al., Science 256:1673-1677 (1992)), and IL-5 (Milburn et al.,
Nature 363: 172-176 (1993)) have been determined by X-ray
diffraction and NMR studies and show striking conservation with the
GH structure, despite a lack of significant primary sequence
homology. IFN is considered to be a member of this family based
upon modeling and other studies (Lee et al., J. Interferon Cytokine
Res. 15:341 (1995); Murgolo et al., Proteins 17:62 (1993);
Radhakrishnan et al., Structure 4:1453 (1996); Klavs et al., J.
Mol. Biol. 274:661 (1997)). EPO is considered to be a member of
this family based upon modeling and mutagenesis studies (Boissel et
al., J. Biol. Chem. 268: 15983-15993 (1993); Wen et al., J. Biol.
Chem. 269: 22839-22846 (1994)). A large number of additional
cytokines and growth factors including ciliary neurotrophic factor
(CNTF), leukemia inhibitory factor (LIF), thrombopoietin (TPO),
oncostatin M, macrophage colony stimulating factor (M-CSF), IL-3,
IL-6, IL-7, IL-9, IL-12, IL-13, IL-15, and granulocyte-colony
stimulating factor (G-CSF), as well as the IFN's such as alpha,
beta, omega, tau, epsilon, and gamma interferon belong to this
family (reviewed in Mott and Campbell, Current Opinion in
Structural Biology 5: 114-121 (1995); Silvennoinen and Ihle (1996)
SIGNALLING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS). All of the
above cytokines and growth factors are now considered to comprise
one large gene family.
[0139] In addition to sharing similar secondary and tertiary
structures, members of this family share the property that they
must oligomerize cell surface receptors to activate intracellular
signaling pathways. Some GH family members, including but not
limited to; GH and EPO, bind a single type of receptor and cause it
to form homodimers. Other family members, including but not limited
to, IL-2, IL4. and IL-6, bind more than one type of receptor and
cause the receptors to form heterodimers or higher order aggregates
(Davis et al., (1993) Science 260: 1805-1808; Paonessa et al.,
1995) EMBO J. 14: 1942-1951; Mott and Campbell, Current Opinion in
Structural Biology 5: 114-121 (1995)). Mutagenesis studies have
shown that, like GH, these other cytokines and growth factors
contain multiple receptor binding sites, typically two, and bind
their cognate receptors sequentially (Mott and Campbell, Current
Opinion in Structural Biology 5: 114-121 (1995); Matthews et al.,
(1996) Proc. Natl. Acad. Sci. USA 93: 9471-9476). Like GH, the
primary receptor binding sites for these other family members occur
primarily in the four alpha helices and the A-B loop. The specific
amino acids in the helical bundles that participate in receptor
binding differ amongst the family members. Most of the cell surface
receptors that interact with members of the GH supergene family are
structurally related and comprise a second large multi-gene family.
See, e.g. U.S. Pat. No. 6,608,183, which is incorporated by
reference herein.
[0140] A general conclusion reached from mutational studies of
various members of the GH supergene family is that the loops
joining the alpha helices generally tend to not be involved in
receptor binding. In particular the short B-C loop appears to be
non-essential for receptor binding in most, if not all, family
members. For this reason, the B-C loop may be substituted with
non-natural amino acids as described herein in members of the GH
supergene family. The A-B loop, the C-D loop (and D-E loop of
interferon/IL-10-like members of the GH superfamily) may also be
substituted with a non-natural amino acid. Amino acids proximal to
helix A and distal to the final helix also tend not to be involved
in receptor binding and also may be sites for introducing
non-natural amino acids. In some embodiments, a non-natural amino
acid is substituted at any position within a loop structure
including but not limited to the first 1, 2, 3, 4, 5, 6, 7, or more
amino acids of the A-B, B-C, C-D or D-E loop. In some embodiments,
a non-natural amino acid is substituted within the last 1, 2, 3, 4,
5, 6, 7, or more amino acids of the A-B, B-C, C-D or D-E loop.
[0141] Certain members of the GH family, including but not limited
to, EPO, IL-2, IL-3, IL-4, IL-6, IFN, GM-CSF, TPO, IL-10, IL-12
p35, IL-13, IL-15 and beta interferon contain N-linked and/or
O-linked sugars. The glycosylation sites in the proteins occur
almost exclusively in the loop regions and not in the alpha helical
bundles. Because the loop regions generally are not involved in
receptor binding and because they are sites for the covalent
attachment of sugar groups, they may be useful sites for
introducing non-natural amino acid substitutions into the proteins.
Amino acids that comprise the N- and O-linked glycosylation sites
in the proteins may be sites for non-natural amino acid
substitutions because these amino acids are surface-exposed.
Therefore, the natural protein can tolerate bulky sugar groups
attached to the proteins at these sites and the glycosylation sites
tend to be located away from the receptor binding sites.
[0142] Additional members of the GH gene family are likely to be
discovered in the future. New members of the GH supergene family
can be identified through computer-aided secondary and tertiary
structure analyses of the predicted protein sequences. Members of
the GH supergene family typically possess four or five amphipathic
helices joined by non-helical amino acids (the loop regions). The
proteins may contain a hydrophobic signal sequence at their
N-terminus to promote secretion from the cell. Such later
discovered members of the GH supergene family also are included
within the methods and compositions described herein. International
Patent Application entitled "Modified Four Helical Bundle
Polypeptides and Their Uses" (WO 05/074650 on Aug. 18, 2005), which
is incorporated by reference herein in its entirety, provides
methods for site selection and incorporation of non-natural amino
acids into polypeptides.
[0143] V. Non-Natural Amino Acids
[0144] A very wide variety of non-natural amino acids are suitable
for use in the methods and compositions described herein as long as
the non-natural amino acid has at least one of the following four
properties: (1) at least one functional group on the sidechain of
the non-natural amino acid with at least one characteristic and/or
activity and/or reactivity orthogonal to the chemical reactivity of
the 20 common, genetically-encoded amino acids (i.e., alanine,
arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic
acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
and valine), or at least orthogonal to the chemical reactivity of
the naturally occurring amino acids present in the polypeptide that
includes the non-natural amino acid; (2) the introduced non-natural
amino acid is substantially chemically inert toward the 20 common,
genetically-encoded amino acids; (3) the non-natural amino acid can
be stably incorporated into a polypeptide; the stability may be
commensurate with the naturally-occurring amino acids or under
typical physiological conditions, and such incorporation may occur
via an in vivo system; and (4) the non-natural amino acid includes
an oxime functional group or a functional group that can be
transformed into an oxime group by reacting with a reagent, and may
be reacted under conditions that do not destroy the biological
properties of the polypeptide that includes the non-natural amino
acid (unless of course such a destruction of biological properties
is the purpose of the modification/transformation), or preferably
where the transformation can occur under aqueous conditions at a pH
between about 2 and about 10 or a pH between about 4 and about 8,
and the reactive site on the non-natural amino acid may be an
electrophilic site. Illustrative, non-limiting examples of amino
acids that satisfy these four properties for non-natural amino
acids that can be used with the compositions and methods further
described in U.S. Patent Application Nos. 60/638,418, 60/638,527,
60/639,195, 60/696,210, 60/696,302, and 60/696,068 which are
incorporated by reference in their entirety. Any number of
non-natural amino acids can be introduced into the polypeptide.
Non-natural amino acids may also include protected or masked oximes
or protected or masked groups that can be transformed into an oxime
group after deprotection of the protected group or unmasking of the
masked group.
[0145] Non-natural amino acids of interest that may be suitable for
use in the methods and compositions described herein include, but
are not limited to, amino acids comprising a photoactivatable
cross-linker, spin-labeled amino acids, fluorescent amino acids,
metal binding amino acids, metal-containing amino acids,
radioactive amino acids, amino acids with novel functional groups,
amino acids that covalently or noncovalently interact with other
molecules, photocaged and/or photoisomerizable amino acids, amino
acids comprising biotin or a biotin analogue, glycosylated amino
acids such as a sugar substituted serine, other carbohydrate
modified amino acids, keto-containing amino acids, amino acids
comprising polyethylene glycol or polyether, heavy atom substituted
amino acids, chemically cleavable and/or photocleavable amino
acids, amino acids with an elongated side chains as compared to
natural amino acids, including but not limited to, polyethers or
long chain hydrocarbons, including but not limited to, greater than
about 5 or greater than about 10 carbons, carbon-linked
sugar-containing amino acids, redox-active amino acids, amino
thioacid containing amino acids, and amino acids comprising one or
more toxic moiety.
[0146] In some embodiments, non-natural amino acids comprise a
saccharide moiety. Examples of such amino acids include
N-acetyl-L-glucosaminyl-L-serine,
N-acetyl-L-galactosaminyl-L-serine,
N-acetyl-L-glucosaminyl-L-threonine,
N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.
Examples of such amino acids also include examples where the
naturally-occurring N- or O-linkage between the amino acid and the
saccharide is replaced by a covalent linkage not commonly found in
nature--including but not limited to, an alkene, an oxime, a
thioether, an amide and the like. Examples of such amino acids also
include saccharides that are not commonly found in
naturally-occurring proteins such as 2-deoxy-glucose,
2-deoxygalactose and the like.
[0147] The chemical moieties via non-natural amino acids that can
be incorporated into proteins offer a variety of advantages and
manipulations of the protein. For example, the unique reactivity of
a carbonyl functional group (including a keto functional group)
allows selective modification of proteins with any of a number of
hydrazine- or hydroxylamine-containing reagents in vitro and in
vivo. A heavy atom non-natural amino acid, for example, can be
useful for phasing X-ray structure data. The site-specific
introduction of heavy atoms using non-natural amino acids also
provides selectivity and flexibility in choosing positions for
heavy atoms. Photoreactive non-natural amino acids (including but
not limited to, amino acids with benzophenone and arylazides
(including but not limited to, phenylazide) side chains), for
example, allow for efficient in vivo and in vitro photocrosslinking
of protein. Examples of photoreactive non-natural amino acids
include, but are not limited to, p-azido-phenylalanine and
p-benzoyl-phenylalanine. The protein with the photoreactive
non-natural amino acids can then be crosslinked at will by
excitation of the photoreactive group-providing temporal control.
In one example, the methyl group of a non-natural amino can be
substituted with an isotopically labeled, including but not limited
to, methyl group, as a probe of local structure and dynamics,
including but not limited to, with the use of nuclear magnetic
resonance and vibrational spectroscopy.
[0148] Many non-naturally encoded amino acids are commercially
available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),
Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or
Peptech (Burlington, Mass., USA). Those that are not commercially
available are optionally synthesized. For organic synthesis
techniques, see, e.g., Organic Chemistry by Fessendon and
Fessendon, (1982, Second Edition, Willard Grant Press, Boston
Mass.); Advanced Organic Chemistry by March (Third Edition, 1985,
Wiley and Sons, New York); and Advanced Organic Chemistry by Carey
and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New
York). Many non-natural amino acids are based on natural amino
acids, such as tyrosine, glutamine, phenylalanine, and the
like.
[0149] A. Cellular Uptake of Non-Natural Amino Acids
[0150] Non-natural amino acid uptake by a eukaryotic cell is one
issue that is typically considered when designing and selecting
non-natural amino acids, including but not limited to, for
incorporation into a protein. For example, the high charge density
of .alpha.-amino acids suggests that these compounds are unlikely
to be cell permeable. Natural amino acids are taken up into the
eukaryotic cell via a collection of protein-based transport
systems. A rapid screen can be done which assesses which
non-natural amino acids, if any, are taken up by cells. See, e.g.,
the toxicity assays in, e.g., U.S. Patent Publication No. US
2004/0198637 entitled "Protein Arrays," which is incorporated by
reference; and Liu, D. R. & Schultz, P. G. (1999) Progress
toward the evolution of an organism with an expanded genetic code.
PNAS United States 96:4780-4785. Although uptake is easily analyzed
with various assays, an alternative to designing non-natural amino
acids that are amenable to cellular uptake pathways is to provide
biosynthetic pathways to create amino acids in vivo.
[0151] B. Biosynthesis of Non-Natural Amino Acids
[0152] Many biosynthetic pathways already exist in cells for the
production of amino acids and other compounds. While a biosynthetic
method for a particular non-natural amino acid may not exist in
nature, including but not limited to, in a eukaryotic cell, the
methods and compositions described herein include such methods. For
example, biosynthetic pathways for non-natural amino acids are
optionally generated in host cell by adding new enzymes or
modifying existing host cell pathways. Additional new enzymes are
optionally naturally occurring enzymes or artificially evolved
enzymes. For example, the biosynthesis of p-aminophenylalanine (as
presented in an example in WO 2002/085923 entitled "In vivo
incorporation of unnatural amino acids") relies on the addition of
a combination of known enzymes from other organisms. The genes for
these enzymes can be introduced into a eukaryotic cell by
transforming the cell with a plasmid comprising the genes. The
genes, when expressed in the cell, provide an enzymatic pathway to
synthesize the desired compound. Examples of the types of enzymes
that are optionally added are provided in the examples below.
Additional enzymes sequences are found, for example, in Genbank.
Artificially evolved enzymes are also optionally added into a cell
in the same manner. In this manner, the cellular machinery and
resources of a cell are manipulated to produce non-natural amino
acids.
[0153] A variety of methods are available for producing novel
enzymes for use in biosynthetic pathways or for evolution of
existing pathways. For example, recursive recombination, including
but not limited to, as developed by Maxygen, Inc. (available on the
World Wide Web at www.maxygen.com), is optionally used to develop
novel enzymes and pathways. See, e.g., Stemmer (1994), Rapid
evolution of a protein in vitro by DNA shuffling, Nature
370(4):389-391; and, Stemmer, (1994), DNA shuffling by random
fragmentation and reassembly: In vitro recombination for molecular
evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751. Similarly
DesignPath.TM., developed by Genencor (available on the World Wide
Web at genencor.com) is optionally used for metabolic pathway
engineering, including but not limited to, to engineer a pathway to
create O-methyl-L-tyrosine in a cell. This technology reconstructs
existing pathways in host organisms using a combination of new
genes, including but not limited to, identified through functional
genomics, and molecular evolution and design. Diversa Corporation
(available on the world wide web at diversa.com) also provides
technology for rapidly screening libraries of genes and gene
pathways, including but not limited to, to create new pathways.
[0154] Typically, the non-natural amino acid produced with an
engineered biosynthetic pathway is produced in a concentration
sufficient for efficient protein biosynthesis, including but not
limited to, a natural cellular amount, but not to such a degree as
to affect the concentration of the other amino acids or exhaust
cellular resources. Typical concentrations produced in vivo in this
manner are about 10 mM to about 0.05 mM. Once a cell is transformed
with a plasmid comprising the genes used to produce enzymes desired
for a specific pathway and a non-natural amino acid is generated,
in vivo selections are optionally used to further optimize the
production of the non-natural amino acid for both ribosomal protein
synthesis and cell growth.
[0155] VI. Polypeptides with Non-Natural Amino Acids
[0156] The compositions and methods further described in U.S.
Patent Application Nos. 60/638,418, 60/638,527, 60/639,195,
60/696,210, 60/696,302, and 60/696,068; U.S. Patent Application
Publications 2003/0082575 (Ser. No. 10/126,927) and 2003/0108885
(Ser. No. 10/126,931); WO 04/035743 entitled "Site Specific
Incorporation of Keto Amino Acids into Proteins," and PCT
Publication No. WO 04/094593 entitled "Expanding the Eukaryotic
Genetic Code," which are incorporated by reference in their
entirety provide for the incorporation of at least one non-natural
amino acid into a polypeptide. The non-natural amino acid may be
present at any location on the polypeptide, including any terminal
position or any internal position of the polypeptide. The
non-natural amino acid polypeptides described herein may be
produced biosynthetically or non-biosyntheticially. By
biosynthetically is meant any method utilizing a translation system
(cellular or non-cellular), including use of at least one of the
following components: a polynucleotide, a codon, a tRNA, and a
ribosome. By non-biosynthetically is meant any method not utilizing
a translation system: this approach can be further divided into
methods utilizing solid state peptide synthetic methods, solid
phase peptide synthetic methods, methods that utilize at least one
enzyme, and methods that do not utilize at least one enzyme; of
course any of this sub-divisions may overlap and many methods may
utilize a combination of these sub-divisions.
[0157] The methods, compositions, strategies and techniques
described herein are not limited to a particular type, class or
family of polypeptides or proteins. Indeed, virtually any
polypeptides may include but are not limited to at least one
non-natural amino acids further described in U.S. Patent
Application Nos. 60/638,418, 60/638,527, 60/639,195, 60/696,210,
60/696,302, and 60/696,068; U.S. Patent Application Publications
2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No.
10/126,931), WO 04/035743 entitled "Site Specific Incorporation of
Keto Amino Acids into Proteins," PCT Publication No. WO 04/094593
entitled "Expanding the Eukaryotic Genetic Code," and PCT
Publication WO 05/074650 entitled "Modified Four Helical Bundle
Polypeptides and Their Uses," which are incorporated by reference
herein. The non-natural amino acid polypeptides may be further
modified as described in U.S. Patent Application Nos. 60/638,418,
60/638,527, 60/639,195, 60/696,210, 60/696,302, and 60/696,068;
U.S. Patent Application Publications 2003/0082575 (Ser. No.
10/126,927) and 2003/0108885 (Ser. No. 10/126,931), WO 04/035743
entitled "Site Specific Incorporation of Keto Amino Acids into
Proteins," PCT Publication No. WO 04/094593 entitled "Expanding the
Eukaryotic Genetic Code," and PCT Publication WO 05/074650 entitled
"Modified Four Helical Bundle Polypeptides and Their Uses," which
are incorporated by reference herein or the non-natural amino acid
polypeptide may be used without further modification. In one
aspect, a composition includes at least one protein with at least
one, including but not limited to, at least two, at least three, at
least four, at least five, at least six, at least seven, at least
eight, at least nine, or at least ten or more non-natural amino
acids. The polypeptides may comprise one or more natural amino acid
substitutions.
[0158] Although embodiments of the non-natural amino acid
polypeptides further described in U.S. Patent Application Nos.
60/638,418, 60/638,527, 60/639,195, 60/696,210, 60/696,302, and
60/696,068 which are incorporated by reference may be chemically
synthesized via solid phase peptide synthesis methods (e.g., on a
solid resin), by solution phase peptide synthesis methods, and/or
without the aid of enzymes, other embodiments of the non-natural
amino acid polypeptides described herein allow synthesis via a cell
membrane, cellular extract, or lysate system or via an in vivo
system, i.e., using the cellular machinery of a prokarote or
eukaryote cell.
[0159] VII. Compositions and Methods Comprising Nucleic Acids and
Oligonucleotides
[0160] A. General Recombinant Nucleic Acid Methods for Use
[0161] U.S. Patent Application Nos. 60/638,418, 60/638,527,
60/639,195, 60/696,210, 60/696,302, and 60/696,068; and PCT
Publication WO 05/074650 entitled "Modified Four Helical Bundle
Polypeptides and Their Uses," which are incorporated by reference
herein, discuss nucleic acids encoding a polypeptide of interest
(including by way of example a GH polypeptide), and how it may be
isolated, cloned and often altered using recombinant methods. Such
embodiments are used, including but not limited to, for protein
expression or during the generation of variants, derivatives,
expression cassettes, or other sequences derived from a
polypeptide. In some embodiments, the sequences encoding the
polypeptides are operably linked to a heterologous promoter.
[0162] A nucleotide sequence encoding a polypeptide comprising a
non-natural amino acid may be synthesized on the basis of the amino
acid sequence of the parent polypeptide, and then changing the
nucleotide sequence so as to effect introduction (i.e.,
incorporation or substitution) or removal (i.e., deletion or
substitution) of the relevant amino acid residue(s). The nucleotide
sequence may be conveniently modified by site-directed mutagenesis
in accordance with conventional methods. Alternatively, the
nucleotide sequence may be prepared by chemical synthesis,
including but not limited to, by using an oligonucleotide
synthesizer, wherein oligonucleotides are designed based on the
amino acid sequence of the desired polypeptide, and preferably
selecting those codons that are favored in the host cell in which
the recombinant polypeptide will be produced. For example, several
small oligonucleotides coding for portions of the desired
polypeptide may be synthesized and assembled by PCR, ligation or
ligation chain reaction. See, e.g., Barany, et al., Proc. Natl.
Acad. Sci. 88: 189-193 (1991); U.S. Pat. No. 6,521,427 which are
incorporated by reference herein.
[0163] B. Selector Codons
[0164] Selector codons encompassed within the methods and
compositions further described in U.S. Patent Application Nos.
60/638,418, 60/638,527, 60/639,195, 60/696,210, 60/696,302, and
60/696,068; and PCT Publication WO 05/074650 entitled "Modified
Four Helical Bundle Polypeptides and Their Uses," which are
incorporated by reference in their entirety expand the genetic
codon framework of protein biosynthetic machinery. For example, a
selector codon includes, but is not limited to, a unique three base
codon, a nonsense codon, such as a stop codon, including but not
limited to, an amber codon (UAG), or an opal codon (UGA), an ochre
codon, a unnatural codon, a four or more base codon, a rare codon,
or the like. There is a wide range in the number of selector codons
that can be introduced into a desired gene, including but not
limited to, one or more, two or more, three or more, 4, 5, 6, 7, 8,
9, 10 or more in a single polynucleotide encoding at least a
portion of a polypeptide of interest.
[0165] In some cases, it involves the use of a selector codon that
is a stop codon for the incorporation of one or more non-natural
amino acids in vivo. The incorporation of non-natural amino acids
in vivo can be done without significant perturbation of the
eukaryotic host cell. Selector codons also comprise extended
codons, including but not limited to, four or more base codons,
such as, four, five, six or more base codons. For a given system, a
selector codon can also include one of the natural three base
codons, where the endogenous system does not use (or rarely uses)
the natural base codon. Selector codons optionally include
unnatural base pairs. These unnatural base pairs further expand the
existing genetic alphabet. For in vivo usage, the unnatural
nucleoside is membrane permeable and is phosphorylated to form the
corresponding triphosphate. In addition, the increased genetic
information is stable and not destroyed by cellular enzymes. A
translational bypassing system can also be used to incorporate a
non-natural amino acid in a desired polypeptide. In certain
embodiments, the protein or polypeptide of interest (or portion
thereof) is encoded by a nucleic acid. Typically, the nucleic acid
comprises at least one selector codon, at least two selector
codons, at least three selector codons, at least four selector
codons, at least five selector codons, at least six selector
codons, at least seven selector codons, at least eight selector
codons, at least nine selector codons, ten or more selector
codons.
[0166] VIII. In Vivo Generation of Polypeptides Comprising
Non-Natural Amino Acids
[0167] The polypeptides can be generated in vivo using modified
tRNA and tRNA synthetases to add to or substitute amino acids that
are not encoded in naturally-occurring systems. All the methods for
generating, screening methods and organisms used for in vivo
generation of polypeptides comprising non-natural amino acids which
are further described in U.S. Patent Application Nos. 60/638,418,
60/638,527, 60/639,195, 60/696,210, 60/696,302, and 60/696,068;
U.S. Patent Application Publications 2003/0082575 (Ser. No.
10/126,927) and 2003/0108885 (Ser. No. 10/126,931), PCT Publication
No. WO 04/094593 entitled "Expanding the Eukaryotic Genetic Code,"
and PCT Publication WO 05/074650 entitled "Modified Four Helical
Bundle Polypeptides and Their Uses," which are incorporated by
reference in their entirety.
[0168] Methods for generating tRNAs and tRNA synthetases which use
amino acids that are not encoded in naturally-occurring systems are
described in, e.g., U.S. Patent Application Publications
2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No.
10/126,931) which are incorporated by reference herein. These
methods involve generating a translational machinery that functions
independently of the synthetases and tRNAs endogenous to the
translation system (and are therefore sometimes referred to as
"orthogonal"). In further or additional embodiments, the
translation system comprises an orthogonal tRNA (O-tRNA) and an
orthogonal aminoacyl tRNA synthetase (O-RS). A wide variety of
orthogonal tRNAs and aminoacyl tRNA synthetases have been described
in the art for inserting particular synthetic amino acids into
polypeptides, and are generally suitable for in the methods to
produce the non-natural amino acid polypeptides.
[0169] Use of O-tRNA/aminoacyl-tRNA synthetases involves selection
of a specific codon which encodes the non-natural amino acid. While
any codon can be used, it is generally desirable to select a codon
that is rarely or never used in the cell in which the
O-tRNA/aminoacyl-tRNA synthetase is expressed. Specific selector
codon(s) can be introduced into appropriate positions in the
polynucleotide coding sequence using mutagenesis methods known in
the art (including but not limited to, site-specific mutagenesis,
cassette mutagenesis, restriction selection mutagenesis, etc.).
[0170] A. Expression in Non-Eukaryotes and Eukaryotes
[0171] To obtain high level expression of a cloned polynucleotide,
one typically subclones polynucleotides encoding a desired
polypeptide into an expression vector that contains a strong
promoter to direct transcription, a transcription/translation
terminator, and if for a nucleic acid encoding a protein, a
ribosome binding site for translational initiation. Suitable
bacterial promoters are well known in the art and described, e.g.,
in Sambrook et al. and Ausubel et al. Bacterial expression systems
and eukaryotic host cell or non-eukaryotic host cell systems
further described in U.S. Patent Application Nos. 60/638,418,
60/638,527, 60/639,195, 60/696,210, 60/696,302, and 60/696,068;
U.S. Patent Application Publications 2003/0082575 (Ser. No.
10/126,927) and 2003/0108885 (Ser. No. 10/126,931), PCT Publication
No. WO 04/094593 entitled "Expanding the Eukaryotic Genetic Code,"
and PCT Publication WO 05/074650 entitled "Modified Four Helical
Bundle Polypeptides and Their Uses," which are incorporated by
reference in their entirety may be used to biosynthesize proteins
that comprise non-natural amino acids in large useful
quantities.
[0172] 1. Expression Systems, Culture, and Isolation
[0173] The desired polypeptide may be expressed in any number of
suitable expression systems including, for example, yeast, insect
cells, mammalian cells, Pseudomonas cells, and bacteria. A
description of exemplary expression systems is further described in
U.S. Patent Application Nos. 60/638,418, 60/638,527, 60/639,195,
60/696,210, 60/696,302, and 60/696,068; U.S. Patent Application
Publications 2003/0082575 (Ser. No. 10/126,927) and 2003/0108885
(Ser. No. 10/126,931), PCT Publication No. WO 04/094593 entitled
"Expanding the Eukaryotic Genetic Code," and PCT Publication WO
05/074650 entitled "Modified Four Helical Bundle Polypeptides and
Their Uses," which are incorporated by reference in their
entirety
[0174] 2. Purification of Non-Natural Amino Acid Polypeptides
[0175] General Purification Methods Any one of a variety of
isolation steps may be performed on the cell lysate, extract,
culture medium, inclusion bodies, periplasmic space of the host
cells, cytoplasm of the host cells, or other material comprising
the desired polypeptide or mixtures resulting from any isolation
steps including, but not limited to, affinity chromatography, ion
exchange chromatography, hydrophobic interaction chromatography,
gel filtration chromatography, high performance liquid
chromatography ("HPLC"), reversed phase-HPLC ("RP-HPLC"), expanded
bed adsorption, or any combination and/or repetition thereof and in
any appropriate order. General purification methods, equipment,
preferred embodiments and other purification techniques are further
described in U.S. Patent Application Nos. 60/638,418, 60/638,527,
60/639,195, 60/696,210, 60/696,302, and 60/696,068; and WO
05/074650 entitled "Modified Four Helical Bundle Polypeptides and
Their Uses which are incorporated by reference in their
entirety.
[0176] B. In Vivo Post-Translational Modifications
[0177] By producing proteins or polypeptides of interest with at
least one non-natural amino acid in eukaryotic cells, proteins or
polypeptides include eukaryotic post-translational modifications.
In certain embodiments, a protein includes at least one non-natural
amino acid and at least one post-translational modification that is
made in vivo by a eukaryotic cell, where the post-translational
modification is not made by a prokaryotic cell. For example, the
post-translation modification is further described in U.S. Patent
Application Nos. 60/638,418, 60/638,527, 60/639,195, 60/696,210,
60/696,302, and 60/696,068; and WO 05/074650 entitled "Modified
Four Helical Bundle Polypeptides and Their Uses which are
incorporated by reference in their entirety.
[0178] One advantage of a non-natural amino acid is that it
presents additional chemical moieties that can be used to add
additional molecules. These modifications can be made in vivo in a
eukaryotic or non-eukaryotic cell, or in vitro. Thus, in certain
embodiments, the post-translational modification is through the
non-natural amino acid.
[0179] IX. Expression in Alternate Systems
[0180] Several strategies have been employed to introduce unnatural
amino acids into proteins in non-recombinant host cells,
mutagenized host cells, or in cell-free systems. These systems are
also suitable for use in making the non-natural amino acid
polypeptides. Derivatization of amino acids with reactive
side-chains such as Lys, Cys and Tyr resulted in the conversion of
lysine to N.sup.2-acetyl-lysine. Chemical synthesis also provides a
straightforward method to incorporate unnatural amino acids. With
the recent development of enzymatic ligation and native chemical
ligation of peptide fragments, it is possible to make larger
proteins. See, e.g., P. E. Dawson and S. B. H. Kent, Annu. Rev.
Biochem, 69:923 (2000). Chemical peptide ligation and native
chemical ligation are described in U.S. Pat. No. 6,184,344, U.S.
Patent Publication No. 2004/0138412, U.S. Patent Publication No.
2003/0208046, WO 02/098902, and WO 03/042235, which are
incorporated by reference herein. A general in vitro biosynthetic
method in which a suppressor tRNA chemically acylated with the
desired unnatural amino acid is added to an in vitro extract
capable of supporting protein biosynthesis, has been used to
site-specifically incorporate over 100 unnatural amino acids into a
variety of proteins of virtually any size. See, e.g., V. W.
Cornish, D. Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl.,
1995, 34:621 (1995); C. J. Noren, S. J. Anthony-Cahill, M. C.
Griffith, P. G. Schultz, A general method for site-specific
incorporation of unnatural amino acids into proteins, Science
244:182-188 (1989); and, J. D. Bain, C. G. Glabe, T. A. Dix, A. R.
Chamberlin, E. S. Diala, Biosynthetic site-specific incorporation
of a non-natural amino acid into a polypeptide, J. Am. Chem. Soc.
111:8013-8014 (1989). A broad range of functional groups has been
introduced into proteins for studies of protein stability, protein
folding, enzyme mechanism, and signal transduction.
[0181] An in vivo method, termed selective pressure incorporation,
was developed to exploit the promiscuity of wild-type synthetases.
See, e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M.
Dong, L. Moroder and R. Huber, FASEB J., 13:41 (1999). An
auxotrophic strain, in which the relevant metabolic pathway
supplying the cell with a particular natural amino acid is switched
off, is grown in minimal media containing limited concentrations of
the natural amino acid, while transcription of the target gene is
repressed. At the onset of a stationary growth phase, the natural
amino acid is depleted and replaced with the unnatural amino acid
analog. Induction of expression of the recombinant protein results
in the accumulation of a protein containing the unnatural analog.
For example, using this strategy, o, m and p-fluorophenylalanines
have been incorporated into proteins, and exhibit two
characteristic shoulders in the UV spectrum which can be easily
identified, see, e.g., C. Minks, R. Huber, L. Moroder and N.
Budisa, Anal. Biochem., 284:29 (2000); trifluoromethionine has been
used to replace methionine in bacteriophage T4 lysozyme to study
its interaction with chitooligosaccharide ligands by .sup.19F NMR,
see, e.g., H. Duewel, E. Daub, V. Robinson and J. F. Honek,
Biochemistry, 36:3404 (1997); and trifluoroleucine has been
incorporated in place of leucine, resulting in increased thermal
and chemical stability of a leucine-zipper protein. See, e.g., Y.
Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F. DeGrado and D.
A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001). Moreover,
selenomethionine and telluromethionine are incorporated into
various recombinant proteins to facilitate the solution of phases
in X-ray crystallography. See, e.g., W. A. Hendrickson, J. R.
Horton and D. M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K.
Lewinski, M. Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M.
Hatada, Nat. Struct. Biol., 1:283 (1994); N. Budisa, B. Steipe, P.
Demange, C. Eckerskorn, J. Kellermann and R. Huber, Eur. J.
Biochem., 230:788 (1995); and, N. Budisa, W. Karnbrock, S.
Steinbacher, A. Humm, L. Prade, T. Neuefeind, L. Moroder and R.
Huber, J. Mol. Biol., 270:616 (1997). Methionine analogs with
alkene or alkyne functionalities have also been incorporated
efficiently, allowing for additional modification of proteins by
chemical means. See, e.g., J. C. van Hest and D. A. Tirrell, FEBS
Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.
Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and
D. A. Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No.
6,586,207; U.S. Patent Publication 2002/0042097, which are
incorporated by reference herein.
[0182] The success of this method depends on the recognition of the
unnatural amino acid analogs by aminoacyl-tRNA synthetases, which,
in general, require high selectivity to insure the fidelity of
protein translation. One way to expand the scope of this method is
to relax the substrate specificity of aminoacyl-tRNA synthetases,
which has been achieved in a limited number of cases. For example,
replacement of Ala.sup.294 by Gly in Escherichia coli
phenylalanyl-tRNA synthetase (PheRS) increases the size of
substrate binding pocket, and results in the acylation of tRNAPhe
by p-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H.
Hennecke, Biochemistry, 33:7107 (1994). An Escherichia coli strain
harboring this mutant PheRS allows the incorporation of
p-Cl-phenylalanine or p-Br-phenylalanine in place of phenylalanine.
See, e.g., M. Ibba and H. Hennecke, FEBS Lett., 364:272 (1995);
and, N. Sharma, R. Furter, P. Kast and D. A. Tirrell, FEBS Lett.,
467:37 (2000). Similarly, a point mutation Phe130Ser near the amino
acid binding site of Escherichia coli tyrosyl-tRNA synthetase was
shown to allow azatyrosine to be incorporated more efficiently than
tyrosine. See, F. Hamano-Takaku, T. Iwama, S. Saito-Yano, K.
Takaku, Y. Monden, M. Kitabatake, D. Soll and S. Nishimura, J.
Biol. Chem., 275:40324 (2000).
[0183] Another strategy to incorporate unnatural amino acids into
proteins in vivo is to modify synthetases that have proofreading
mechanisms. These synthetases cannot discriminate and therefore
activate amino acids that are structurally similar to the cognate
natural amino acids. This error is corrected at a separate site,
which deacylates the mischarged amino acid from the tRNA to
maintain the fidelity of protein translation. If the proofreading
activity of the synthetase is disabled, structural analogs that are
misactivated may escape the editing function and be incorporated.
This approach has been demonstrated recently with the valyl-tRNA
synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A. Nangle, T.
L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P. Marliere,
Science, 292:501 (2001). ValRS can misaminoacylate tRNAVal with
Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids are
subsequently hydrolyzed by the editing domain. After random
mutagenesis of the Escherichia coli chromosome, a mutant
Escherichia coli strain was selected that has a mutation in the
editing site of ValRS. This edit-defective ValRS incorrectly
charges tRNAVal with Cys. Because Abu sterically resembles Cys
(--SH group of Cys is replaced with --CH3 in Abu), the mutant ValRS
also incorporates Abu into proteins when this mutant Escherichia
coli strain is grown in the presence of Abu. Mass spectrometric
analysis shows that about 24% of valines are replaced by Abu at
each valine position in the native protein.
[0184] Solid-phase synthesis and semisynthetic methods have also
allowed for the synthesis of a number of proteins containing novel
amino acids. For example, see the following publications and
references cited within, which are as follows: Crick, F. H. C.,
Barrett, L. Brenner, S. Watts-Tobin, R. General nature of the
genetic code for proteins. Nature, 192:1227-1232 (1961); Hofmann,
K., Bohn, H. Studies on polypeptides. XXXVI. The effect of
pyrazole-imidazole replacements on the S-protein activating potency
of an S-peptide fragment, J. Am. Chem, 88(24):5914-5919 (1966);
Kaiser, E. T. Synthetic approaches to biologically active peptides
and proteins including enyzmes, Acc Chem Res, 22:47-54 (1989);
Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptide segment coupling
catalyzed by the semisynthetic enzyme thiosubtilisin, J Am Chem
Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H. Constructing
proteins by dovetailing unprotected synthetic peptides:
backbone-engineered HIV protease, Science, 256(5054):221-225
(1992); Chaiken, I. M. Semisynthetic peptides and proteins, CRC
Crit Rev Biochem, 11(3):255-301 (1981); Offord, R. E. Protein
engineering by chemical means? Protein Eng., 1(3):151-157 (1987);
and, Jackson, D. Y., Burnier, J., Quan, C., Stanley, M., Tom, J.,
Wells, J. A. A Designed Peptide Ligase for Total Synthesis of
Ribonuclease A with Unnatural Catalytic Residues, Science,
266(5183):243 (1994).
[0185] Chemical modification has been used to introduce a variety
of unnatural side chains, including cofactors, spin labels and
oligonucleotides into proteins in vitro. See, e.g., Corey, D. R.,
Schultz, P. G. Generation of a hybrid sequence-specific
single-stranded deoxyribonuclease, Science, 238(4832):1401-1403
(1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E. The chemical
modification of enzymatic specificity, Annu Rev Biochem, 54:565-595
(1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation of enyzme
active sites, Science, 226(4674):505-511 (1984); Neet, K. E., Nanci
A, Koshland, D. E. Properties of thiol-subtilisin, J Biol. Chem,
243(24):6392-6401 (1968); Polgar, L. et M. L. Bender. A new enzyme
containing a synthetically formed active site. Thiol-subtilisin. J.
Am Chem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.
Schultz, P. G. Introduction of nucleophiles and spectroscopic
probes into antibody combining sites, Science, 242(4881):1038-1040
(1988).
[0186] Alternatively, biosynthetic methods that employ chemically
modified aminoacyl-tRNAs have been used to incorporate several
biophysical probes into proteins synthesized in vitro. See the
following publications and references cited within: Brunner, J. New
Photolabeling and crosslinking methods, Annu. Rev Biochem,
62:483-514 (1993); and, Krieg, U. C., Walter, P., Hohnson, A. E.
Photocrosslinking of the signal sequence of nascent preprolactin of
the 54-kilodalton polypeptide of the signal recognition particle,
Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).
[0187] Previously, it has been shown that unnatural amino acids can
be site-specifically incorporated into proteins in vitro by the
addition of chemically aminoacylated suppressor tRNAs to protein
synthesis reactions programmed with a gene containing a desired
amber nonsense mutation. Using these approaches, one can substitute
a number of the common twenty amino acids with close structural
homologues, e.g., fluorophenylalanine for phenylalanine, using
strains auxotropic for a particular amino acid. See, e.g., Noren,
C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G. A general
method for site-specific incorporation of unnatural amino acids
into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,
Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,
Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific
Incorporation of a non-natural amino acid into a polypeptide, J. Am
Chem Soc, 111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51
(1999); Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C.
J., Schultz, P. G. Biosynthetic method for introducing unnatural
amino acids site-specifically into proteins, Methods in Enz., vol.
202, 301-336 (1992); and, Mendel, D., Cornish, V. W. & Schultz,
P. G. Site-Directed Mutagenesis with an Expanded Genetic Code, Annu
Rev Biophys. Biomol Struct. 24, 435-62 (1995).
[0188] For example, a suppressor tRNA was prepared that recognized
the stop codon UAG and was chemically aminoacylated with an
unnatural amino acid. Conventional site-directed mutagenesis was
used to introduce the stop codon TAG, at the site of interest in
the protein gene. See, e.g., Sayers, J. R., Schmidt, W. Eckstein,
F. 5'-3' Exonucleases in phosphorothioate-based
olignoucleotide-directed mutagensis, Nucleic Acids Res,
16(3):791-802 (1988). When the acylated suppressor tRNA and the
mutant gene were combined in an in vitro transcription/translation
system, the unnatural amino acid was incorporated in response to
the UAG codon which gave a protein containing that amino acid at
the specified position. Experiments using [.sup.3H]-Phe and
experiments with .alpha.-hydroxy acids demonstrated that only the
desired amino acid is incorporated at the position specified by the
UAG codon and that this amino acid is not incorporated at any other
site in the protein. See, e.g., Noren, et al, supra; Kobayashi et
al., (2003) Nature Structural Biology 10(6):425-432; and, Ellman,
J. A., Mendel, D., Schultz, P. G. Site-specific incorporation of
novel backbone structures into proteins, Science, 255(5041):197-200
(1992).
[0189] A tRNA may be aminoacylated with a desired amino acid by any
method or technique, including but not limited to, chemical or
enzymatic aminoacylation.
[0190] Aminoacylation may be accomplished by aminoacyl tRNA
synthetases or by other enzymatic molecules, including but not
limited to, ribozymes. The term "ribozyme" is interchangeable with
"catalytic RNA." Cech and coworkers (Cech, 1987, Science,
236:1532-1539; McCorkle et al., 1987, Concepts Biochem. 64:221-226)
demonstrated the presence of naturally occurring RNAs that can act
as catalysts (ribozymes). However, although these natural RNA
catalysts have only been shown to act on ribonucleic acid
substrates for cleavage and splicing, the recent development of
artificial evolution of ribozymes has expanded the repertoire of
catalysis to various chemical reactions. Studies have identified
RNA molecules that can catalyze aminoacyl-RNA bonds on their own
(2')3'-termini (Illangakekare et al., 1995 Science 267:643-647),
and an RNA molecule which can transfer an amino acid from one RNA
molecule to another (Lohse et al., 1996, Nature 381:442-444).
[0191] U.S. Patent Application Publication 2003/0228593, which is
incorporated by reference herein, describes methods to construct
ribozymes and their use in aminoacylation of tRNAs with naturally
encoded and non-naturally encoded amino acids.
Substrate-immobilized forms of enzymatic molecules that can
aminoacylate tRNAs, including but not limited to, ribozymes, may
enable efficient affinity purification of the aminoacylated
products. Examples of suitable substrates include agarose,
sepharose, and magnetic beads. The production and use of a
substrate-immobilized form of ribozyme for aminoacylation is
described in Chemistry and Biology 2003, 10:1077-1084 and U.S.
Patent Application Publication 2003/0228593, which is incorporated
by reference herein.
[0192] Chemical aminoacylation methods include, but are not limited
to, those introduced by Hecht and coworkers (Hecht, S. M. Ace.
Chem. Res. 1992, 25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.;
Chang, P.; Hecht, S. M. Biochemistry 1988, 27, 7254; Hecht, S. M.;
Alford, B. L.; Kuroda, Y.; Kitano, S. J. Biol. Chem. 1978, 253,
4517) and by Schultz, Chamberlin, Dougherty and others (Cornish, V.
W.; Mendel, D.; Schultz, P. G. Angew. Chem. Int. Ed. Engl. 1995,
34, 621; Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am.
Chem. Soc. 1991, 113, 2722; Noren, C. J.; Anthony-Cahill, S. J.;
Griffith, M. C.; Schultz, P. G. Science 1989, 244, 182; Bain, J.
D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J. Am. Chem. Soc.
1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356, 537;
Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,
4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M.
W. et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol.
Chem. 1996, 271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999,
121, 34), to avoid the use of synthetases in aminoacylation. Such
methods or other chemical aminoacylation methods may be used to
aminoacylate tRNA molecules of the invention.
[0193] Methods for generating catalytic RNA may involve generating
separate pools of randomized ribozyme sequences, performing
directed evolution on the pools, screening the pools for desirable
aminoacylation activity, and selecting sequences of those ribozymes
exhibiting desired aminoacylation activity.
[0194] Ribozymes can comprise motifs and/or regions that facilitate
acylation activity, such as a GGU motif and a U-rich region. For
example, it has been reported that U-rich regions can facilitate
recognition of an amino acid substrate, and a GGU-motif can form
base pairs with the 3' termini of a tRNA. In combination, the GGU
and motif and U-rich region facilitate simultaneous recognition of
both the amino acid and tRNA simultaneously, and thereby facilitate
aminoacylation of the 3' terminus of the tRNA.
[0195] Ribozymes can be generated by in vitro selection using a
partially randomized r24mini conjugated with tRNA.sup.Asn.sub.CCCG,
followed by systematic engineering of a consensus sequence found in
the active clones. An exemplary ribozyme obtained by this method is
termed "Fx3 ribozyme" and is described in U.S. Pub. App. No.
2003/0228593, the contents of which is incorporated by reference
herein, acts as a versatile catalyst for the synthesis of various
aminoacyl-tRNAs charged with cognate non-natural amino acids.
[0196] Aminoacylate tRNAs ribozymes can be immobilized on a
substrate so as to enable efficient affinity purification of the
aminoacylated tRNAs. Examples of suitable substrates include, but
are not limited to, agarose, sepharose, and magnetic beads.
Ribozymes can be immobilized on resins by taking advantage of the
chemical structure of RNA, such as the 3'-cis-diol on the ribose of
RNA can be oxidized with periodate to yield the corresponding
dialdehyde to facilitate immobilization of the RNA on the resin.
Various types of resins can be used including inexpensive hydrazide
resins wherein reductive amination makes the interaction between
the resin and the ribozyme an irreversible linkage. Synthesis of
aminoacyl-tRNAs can be significantly facilitated by this on-column
aminoacylation technique. Kourouklis et al. Methods 2005; 36:239-4
describe a column-based aminoacylation system.
[0197] Isolation of the aminoacylated tRNAs can be accomplished in
a variety of ways. One suitable method is to elute the
aminoacylated tRNAs from a column with a buffer such as a sodium
acetate solution with 10 mM EDTA, a buffer containing 50 mM
N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid), 12.5 mM
KCl, pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH
7.0).
[0198] The aminoacylated tRNAs can be added to translation
reactions in order to incorporate the amino acid with which the
tRNA was aminoacylated in a position of choice in a polypeptide
made by the translation reaction. Examples of translation systems
in which the aminoacylated tRNAs of the present invention may be
used include, but are not limited to cell lysates. Cell lysates
provide reaction components necessary for in vitro translation of a
polypeptide from an input mRNA. Examples of such reaction
components include but are not limited to ribosomal proteins, rRNA,
amino acids, tRNAs, GTP, ATP, translation initiation and elongation
factors and additional factors associated with translation.
Additionally, translation systems may be batch translations or
compartmentalized translation. Batch translation systems combine
reaction components in a single compartment while compartmentalized
translation systems separate the translation reaction components
from reaction products that can inhibit the translation efficiency.
Such translation systems are available commercially.
[0199] Further, a coupled transcription/translation system may be
used. Coupled transcription/translation systems allow for both
transcription of an input DNA into a corresponding mRNA, which is
in turn translated by the reaction components. An example of a
commercially available coupled transcription/translation is the
Rapid Translation System (RTS, Roche Inc.). The system includes a
mixture containing E. coli lysate for providing translational
components such as ribosomes and translation factors. Additionally,
an RNA polymerase is included for the transcription of the input
DNA into an mRNA template for use in translation. RTS can use
compartmentalization of the reaction components by way of a
membrane interposed between reaction compartments, including a
supply/waste compartment and a transcription/translation
compartment.
[0200] Aminoacylation of tRNA may be performed by other agents,
including but not limited to, transferases, polymerases, catalytic
antibodies, multi-functional proteins, and the like.
[0201] Stephan in Scientist 2005 Oct. 10; pages 30-33 describes
additional methods to incorporate non-naturally encoded amino acids
into proteins. Lu et al. in Mol Cell. 2001 October; 8(4):759-69
describe a method in which a protein is chemically ligated to a
synthetic peptide containing unnatural amino acids (expressed
protein ligation).
[0202] Microinjection techniques have also been use incorporate
unnatural amino acids into proteins. See, e.g., M. W. Nowak, P. C.
Kearney, J. R. Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman,
W. G. Zhong, J. Thorson, J. N. Abelson, N. Davidson, P. G. Schultz,
D. A. Dougherty and H. A. Lester, Science, 268:439 (1995); and, D.
A. Dougherty, Curr. Opin. Chem. Biol., 4:645 (2000). A Xenopus
oocyte was coinjected with two RNA species made in vitro: an mRNA
encoding the target protein with a UAG stop codon at the amino acid
position of interest and an amber suppressor tRNA aminoacylated
with the desired unnatural amino acid. The translational machinery
of the oocyte then inserts the unnatural amino acid at the position
specified by UAG. This method has allowed in vivo
structure-function studies of integral membrane proteins, which are
generally not amenable to in vitro expression systems. Examples
include the incorporation of a fluorescent amino acid into
tachykinin neurokinin-2 receptor to measure distances by
fluorescence resonance energy transfer, see, e.g., G. Turcatti, K.
Nemeth, M. D. Edgerton, U. Meseth, F. Talabot, M. Peitsch, J.
Knowles, H. Vogel and A. Chollet, J. Biol. Chem., 271:19991 (1996);
the incorporation of biotinylated amino acids to identify
surface-exposed residues in ion channels, see, e.g., J. P.
Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739
(1997); the use of caged tyrosine analogs to monitor conformational
changes in an ion channel in real time, see, e.g., J. C. Miller, S.
K. Silverman, P. M. England, D. A. Dougherty and H. A. Lester,
Neuron, 20:619 (1998); and, the use of alpha hydroxy amino acids to
change ion channel backbones for probing their gating mechanisms.
See, e.g., P. M. England, Y. Zhang, D. A. Dougherty and H. A.
Lester, Cell, 96:89 (1999); and, T. Lu, A. Y. Ting, J. Mainland, L.
Y. Jan, P. G. Schultz and J. Yang, Nat. Neurosci., 4:239
(2001).
[0203] The ability to incorporate unnatural amino acids directly
into proteins in vivo offers the advantages of high yields of
mutant proteins, technical ease, the potential to study the mutant
proteins in cells or possibly in living organisms and the use of
these mutant proteins in therapeutic treatments. The ability to
include unnatural amino acids with various sizes, acidities,
nucleophilicities, hydrophobicities, and other properties into
proteins can greatly expand our ability to rationally and
systematically manipulate the structures of proteins, both to probe
protein function and create new proteins or organisms with novel
properties. However, the process is difficult, because the complex
nature of tRNA-synthetase interactions that are required to achieve
a high degree of fidelity in protein translation.
[0204] In one attempt to site-specifically incorporate para-F-Phe,
a yeast amber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase
pair was used in a p-F-Phe resistant, Phe auxotrophic Escherichia
coli strain. See, e.g., R. Furter, Protein Sci., 7:419 (1998). It
may also be possible to obtain expression of a polynucleotide using
a cell-free (in-vitro) translational system. Translation systems
may be cellular or cell-free, and may be prokaryotic or eukaryotic.
Cellular translation systems include, but are not limited to, whole
cell preparations such as permeabilized cells or cell cultures
wherein a desired nucleic acid sequence can be transcribed to mRNA
and the mRNA translated. Cell-free translation systems are
commercially available and many different types and systems are
well-known. Examples of cell-free systems include, but are not
limited to, prokaryotic lysates such as Escherichia coli lysates,
and eukaryotic lysates such as wheat germ extracts, insect cell
lysates, rabbit reticulocyte lysates, rabbit oocyte lysates and
human cell lysates. Eukaryotic extracts or lysates may be preferred
when the resulting protein is glycosylated, phosphorylated or
otherwise modified because many such modifications are only
possible in eukaryotic systems. Some of these extracts and lysates
are available commercially (Promega; Madison, Wis.; Stratagene; La
Jolla, Calif.; Amersham; Arlington Heights, Ill.; GIBCO/BRL; Grand
Island, N.Y.). Membranous extracts, such as the canine pancreatic
extracts containing microsomal membranes, are also available which
are useful for translating secretory proteins. In these systems,
which can include either mRNA as a template (in-vitro translation)
or DNA as a template (combined in-vitro transcription and
translation), the in vitro synthesis is directed by the ribosomes.
Considerable effort has been applied to the development of
cell-free protein expression systems. See, e.g., Kim, D. M. and J.
R. Swartz, Biotechnology and Bioengineering, 74 :309-316 (2001);
Kim, D. M. and J. R. Swartz, Biotechnology Letters, 22, 1537-1542,
(2000); Kim, D. M., and J. R. Swartz, Biotechnology Progress, 16,
385-390, (2000); Kim, D. M., and J. R. Swartz, Biotechnology and
Bioengineering, 66, 180-188, (1999); and Patnaik, R. and J. R.
Swartz, Biotechniques 24, 862-868, (1998); U.S. Pat. No. 6,337,191;
U.S. Patent Publication No. 2002/0081660; WO 00/55353; WO 90/05785,
which are incorporated by reference herein. Another approach that
may be applied to the expression of non-natural amino acid
polypeptides includes the mRNA-peptide fusion technique. See, e.g.,
R. Roberts and J. Szostak, Proc. Natl Acad. Sci. (USA)
94:12297-12302 (1997); A. Frankel, et al., Chemistry & Biology
10:1043-1050 (2003). In this approach, an mRNA template linked to
puromycin is translated into peptide on the ribosome. If one or
more tRNA molecules has been modified, non-natural amino acids can
be incorporated into the peptide as well. After the last mRNA codon
has been read, puromycin captures the C-terminus of the peptide. If
the resulting mRNA-peptide conjugate is found to have interesting
properties in an in vitro assay, its identity can be easily
revealed from the mRNA sequence. In this way, one may screen
libraries of non-natural amino acid polypeptides to identify
polypeptides having desired properties. More recently, in vitro
ribosome translations with purified components have been reported
that permit the synthesis of peptides substituted with
non-naturally encoded amino acids. See, e.g., A. Forster et al.,
Proc. Natl. Acad. Sci. (USA) 100:6353 (2003).
[0205] Reconstituted translation systems may also be used. Mixtures
of purified translation factors have also been used successfully to
translate mRNA into protein as well as combinations of lysates or
lysates supplemented with purified translation factors such as
initiation factor-1 (IF-1), IF-2, IF-3 (.alpha. or .beta.),
elongation factor T (EF-Tu), or termination factors. Cell-free
systems may also be coupled transcription/translation systems
wherein DNA is introduced to the system, transcribed into mRNA and
the mRNA translated as described in Current Protocols in Molecular
Biology (F. M. Ausubel et al. editors, Wiley Interscience, 1993),
which is hereby specifically incorporated by reference. RNA
transcribed in eukaryotic transcription system may be in the form
of heteronuclear RNA (hnRNA) or 5'-end caps (7-methyl guanosine)
and 3'-end poly A tailed mature mRNA, which can be an advantage in
certain translation systems. For example, capped mRNAs are
translated with high efficiency in the reticulocyte lysate
system.
Post-Translational Modifications of Non-Natural Amino Acid
Components of a Polypeptide
[0206] Methods, compositions, techniques and strategies have been
developed to site-specifically incorporate non-natural amino acids
during the in vivo translation of proteins. By incorporating a
non-natural amino acid with a sidechain chemistry that is
orthogonal to those of the naturally-occurring amino acids, this
technology makes possible the site-specific derivatization of
recombinant proteins. As a result, a major advantage of the
methods, compositions, techniques and strategies is that
derivatized proteins can now be prepared as defined homogeneous
products.
[0207] The non-natural amino acid polypeptides described above are
useful for, including but not limited to, novel therapeutics,
diagnostics, catalytic enzymes, industrial enzymes, binding
proteins and including but not limited to, the study of protein
structure and function. See, e.g., Dougherty, (2000) Unnatural
Amino Acids as Probes of Protein Structure and Function, Current
Opinion in Chemical Biology, 4:645-652. Other uses for the
non-natural amino acid polypeptides described above include, by way
of example only, assay-based, cosmetic, plant biology,
environmental, energy-production, and/or military uses. However,
the non-natural amino acid polypeptides described above can undergo
further modifications so as to incorporate new or modified
functionalities, including manipulating the therapeutic
effectiveness of the polypeptide, improving the safety profile of
the polypeptide, adjusting the pharmacokinetics, pharmacologics
and/or pharmacodynamics of the polypeptide (e.g., increasing water
solubility, bioavailability, increasing serum half-life, increasing
therapeutic half-life, modulating immunogenicity, modulating
biological activity, or extending the circulation time), providing
additional functionality to the polypeptide, incorporating a tag,
label or detectable signal into the polypeptide, easing the
isolation properties of the polypeptide, and any combination of the
aforementioned modifications.
[0208] The methods, compositions, strategies and techniques
described herein are not limited to a particular type, class or
family of polypeptides or proteins. Indeed, virtually any
polypeptides may include at least one non-natural amino acid. A
composition may include at least one protein with at least one,
including but not limited to, at least two, at least three, at
least four, at least five, at least six, at least seven, at least
eight, at least nine, or at least ten or more non-natural amino
acids that have been post-translationally modified. The
post-translationally-modified non-natural amino acids can be the
same or different, including but not limited to, there can be 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 or more different sites in the protein
that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different
post-translationally-modified non-natural amino acids. A
composition may include a protein with at least one, but fewer than
all, of a particular amino acid present in the protein as
substituted with the post-translationally-modified non-natural
amino acid. For a given protein with more than one
post-translationally-modified non-natural amino acids, the
post-translationally-modified non-natural amino acids can be
identical or different (including but not limited to, the protein
can include two or more different types of
post-translationally-modified non-natural amino acids, or can
include two of the same post-translationally-modified non-natural
amino acid). For a given protein with more than two
post-translationally-modified non-natural amino acids, the
post-translationally-modified non-natural amino acids can be the
same, different or a combination of a multiple
post-translationally-modified non-natural amino acid of the same
kind with at least one different post-translationally-modified
non-natural amino acid.
[0209] For example, the post-translational modification can be
through a nucleophilic-electrophilic reaction. Most reactions
currently used for the selective modification of proteins involve
covalent bond formation between nucleophilic and electrophilic
reaction partners, including but not limited to the reaction of
.alpha.-haloketones with histidine or cysteine side chains.
Selectivity in these cases is determined by the number and
accessibility of the nucleophilic residues in the protein. In
proteins of the invention, other more selective reactions can be
used such as the reaction of an unnatural keto-amino acid with
hydrazides or aminooxy compounds, in vitro and in vivo. See, e.g.,
Cornish, et al., (1996) J. Am. Chem. Soc., 118:8150-8151; Mahal, et
al., (1997) Science, 276:1125-1128; Wang, et al., (2001) Science
292:498-500; Chin, et al., (2002) J. Am. Chem. Soc. 124:9026-9027;
Chin, et al., (2002) Proc. Natl. Acad. Sci., 99:11020-11024; Wang,
et al., (2003) Proc. Natl. Acad. Sci., 100:56-61; Zhang, et al.,
(2003) Biochemistry, 42:6735-6746; and, Chin, et al., (2003)
Science, 301:964-7. This allows the selective labeling of virtually
any protein with a host of reagents including fluorophores,
crosslinking agents, saccharide derivatives and cytotoxic
molecules. See also, U.S. Pat. No. 6,927,042 entitled "Glycoprotein
synthesis," which is incorporated by reference herein.
[0210] A. Modifications of Non-Natural Amino Acid Components
[0211] The various modifications of non-natural amino acid
components (which includes non-natural amino acids, as well as the
non-natural amino acid portion of a polypeptide or other polymer)
include, but are not limited to, [0212] (i) reactions of
carbonyl-containing non-natural amino acid components with
hydroxylamine-containing reagents to form oxime-containing
non-natural amino acid components; [0213] (ii) reactions of
hydroxylamine-containing non-natural amino acid components with
carbonyl-containing reagents to form oxime-containing non-natural
amino acid components; [0214] (iii) reactions of oxime-containing
non-natural amino acid components, formed by reaction of carbonyls
and hydroxylamines as in (i) and (ii), with different
carbonyl-containing reagents to form new oxime-containing
non-natural amino acid components via an oxime exchange reaction;
[0215] (iv) reactions of dicarbonyl-containing non-natural amino
acid components with hydroxylamine-containing reagents to form
oxime-containing non-natural amino acid components; [0216] (v)
reactions of hydroxylamine-containing non-natural amino acid
components with dicarbonyl-containing reagents to form
oxime-containing non-natural amino acid components; [0217] (vi)
reactions of oxime-containing non-natural amino acid components,
formed by reaction of dicarbonyls and hydroxylamines as in (iv) and
(v), with a different dicarbonyl-containing reagents to form new
oxime-containing non-natural amino acid components via an oxime
exchange reaction;
[0218] Such reactions are depicted in FIG. 2 wherein the amino acid
functionality (A), translationally incorporated (or otherwise
incorporated) into a polypeptide, reacts with reactant (B) to yield
a modified polypeptide. Such reactions may further occur with the
amino acid functionality (A) on a polymer (including, by way of
example, a polynucleotide, a polynucleoside, a polysaccharide, or
combinations thereof), wherein reaction with reactant (B) yields a
modified polymer. For convenience, the modifications described in
this section and other parts herein use "polypeptide" or
"polypeptides," by way of example, to illustrate the various
modifications. However, the modifications described herein apply
equally well to nonnatural amino acids incorporated into other
molecules, including, but not limited to, polynucleotide(s),
polynucleoside(s), polysaccharide(s), synthetic polymer(s), or
combinations thereof.
[0219] The term "components", as used herein, refers to nonnatural
amino acids, nonnatural amino acid polypeptides, polymers which
contain nonnatural amino acids, nucleic acid sequences which
contain selector codons, nonnatural amino acid polypeptides linked
to polymers, nonnatural amino acid polypeptides linked to polymers
which contain nonnatural amino acids, nonnatural amino acid
polypeptides linked to nucleic acid sequences, nonnatural amino
acid polypeptides linked to nucleic acid sequences; each of which
may independently be a part of, or incorporated into, a
polypeptide, a nonnatural amino acid polypeptide, nucleic acid
sequence, or a polymer.
[0220] Description of these various reaction schemes have been
disclosed in U.S. Provisional Patent Application Nos. 60/638,418,
60/638,527, 60/639,195, 60/696,210, 60/696,302, and 60/696,068,
each of which is herein incorporated by reference in its entirety.
The disclosures provided within each of the above provisional
patent applications apply fully to the methods, compositions,
techniques and strategies for making, detecting, purifying,
characterizing, and using non-natural amino acids, non-natural
amino acid polypeptides and modified non-natural amino acid
polypeptides described herein to the same extent as if such
disclosures were fully presented herein.
Reactions of Carbonyl-Containing Non-Natural Amino Acid Components
with Hydroxylamine-Containing Reagents to Form Oxime-Containing
Non-Natural Amino Acid Components
[0221] Non-natural amino acids with electrophile-containing
sidechains including, but not limited to carbonyl groups such as
aldehydes, esters, thioesters and ketones, can be incorporated into
polypeptides. The incorporation of such non-natural amino acids
with such electrophilic sidechains into polypeptides makes possible
site-specific derivatization of this sidechain via nucleophilic
attack of the carbonyl group. When the attacking nucleophile is a
hydroxylamine, an oxime-derivatized polypeptide will be generated.
The methods for derivatizing and/or further modifying may be
conducted with a polypeptide that has been purified prior to the
derivatization step or after the derivatization step. Further, the
derivatization step can occur under mildly acidic to slightly basic
conditions, including by way of example, between a pH of about 2 to
about 10, or between a pH of about 2 to about 8, or between a pH of
about 4 to about 8.
[0222] Modification of carbonyl sidechains, of non-natural amino
acids incorporated into polypeptides, with hydroxylamine-containing
reagents or other functional groups with similar chemical
reactivity affords modified polypeptides containing oxime linkages.
The reactions and the resulting structures of such modified
polypeptides are shown in FIG. 3.
[0223] Certain embodiments described herein are polypeptides
containing non-natural amino acids with sidechains comprising an
oxime group. In other embodiments such oxime groups may be further
modified, such as, by way of example only, formation of masked
oxime groups (which can be readily converted into oxime groups),
protected oxime groups (which upon deprotection can be readily
converted into oxime groups available for other chemical
reactions), or new oxime groups via oxime exchange reactions.
[0224] Non-limiting examples of such modified polypeptide oxime
linkages are shown below:
##STR00002##
Reactions of Hydroxylamine-Containing Non-Natural Amino Acid
Components with Carbonyl-Containing Reagents to Form
Oxime-Containing Non-Natural Amino Acid Components
[0225] The incorporation of non-natural amino acids containing
hydroxylamine groups into polypeptides allows for reaction with a
variety of electrophilic groups including, but not limited to,
carbonyl group such as ketones, esters, thioesters and aldehydes.
The nucleophilicity of the hydroxylamine group permits it to react
efficiently and selectively with a variety of molecules that
contain carbonyl functionality, or other functional groups with
similar chemical reactivity, under mild conditions in aqueous
solution to form the corresponding oxime linkage. This
site-specific derivatization and/or further modifying of such
sidechains via nucleophilic attack of the carbonyl group may be
conducted with a polypeptide that has been purified prior to the
derivatization step or after the derivatization step. Further, the
derivatization step can occur under mildly acidic to slightly basic
conditions, including by way of example, between a pH of about 2 to
about 10, a pH of about 2 to about 8, or between a pH of about 4 to
about 8.
[0226] Modification of hydroxylamine groups of nonnatural amino
acids incorporated into polypeptides with carbonyl-containing
reagents affords modified polypeptides containing oxime linkages.
The reactions and the resulting structures of such modified
polypeptides are shown in FIG. 4.
[0227] Certain embodiments described herein are polypeptides
containing non-natural amino acids with sidechains comprising an
oxime group. In other embodiments such oxime groups may be further
modified, such as, by way of example only, formation of masked
oxime groups (which can be readily converted into oxime groups),
protected oxime groups (which upon deprotection can be readily
converted into oxime groups available for other chemical
reactions), or new oxime groups via oxime exchange reactions.
[0228] Non-limiting examples of such modified polypeptide oxime
linkages are shown below:
##STR00003##
Reactions of Oxime-Containing Non-Natural Amino Acid Components
Formed by Reaction of Carbonyls and Hydroxylamines, with Different
Carbonyl-Containing Reagents to Form New Oxime-Containing
Non-Natural Amino Acid Components via an Oxime Exchange
Reaction
[0229] Non-natural amino acids containing an oxime group allow for
reaction with a variety of reagents that contain certain reactive
carbonyl groups (including but not limited to, aldehydes, esters,
thioesters and ketones) to form new non-natural amino acids (which
can be incorporated into a polypeptide) comprising a new oxime
group. Such an oxime exchange reaction allows for the further
functionalization of non-natural amino acid polypeptides.
[0230] Modification of oxime sidechains, of nonnatural amino acids
incorporated into polypeptides, with carbonyl-containing reagents,
or other functional groups with similar chemical reactivity,
affords modified polypeptides containing new oxime linkages. The
reactions and the resulting structures of such modified
polypeptides are shown in FIG. 5.
[0231] Certain embodiments described herein are polypeptides
containing non-natural amino acids with sidechains comprising an
oxime group. In other embodiments such oxime groups may be further
modified, such as, by way of example only, formation of masked
oxime groups (which can be readily converted into oxime groups),
protected oxime groups (which upon deprotection can be readily
converted into oxime groups available for other chemical
reactions), or new oxime groups via oxime exchange reactions.
Reactions of Dicarbonyl-Containing Non-Natural Amino Acid
Components with Hydroxylamine-Containing Reagents to form
Oximes
[0232] Non-natural amino acids with electrophile-containing
sidechains including, but not limited to dicarbonyl groups such as
a diketone group, a ketoaldehyde group, a ketoacid group, a
ketoester group, and a ketothioester group), a dicarbonyl-like
group (which has reactivity similar to a dicarbonyl group and is
structurally similar to a carbonyl group), a masked dicarbonyl
group (which can be readily converted into a dicarbonyl group), or
a protected dicarbonyl group (which has reactivity similar to a
dicarbonyl group upon deprotection), can be incorporated into
polypeptides. The incorporation of such unnatural amino acid with
such electrophilic sidechains into polypeptides makes possible
site-specific derivatization of this sidechain via nucleophilic
attack of the carbonyl group. When the attacking nucleophile is a
hydroxylamine, an oxime-derivatized polypeptide will be generated.
The methods for derivatizing and/or further modifying may be
conducted with a polypeptide that has been purified prior to the
derivatization step or after the derivatization step. Further, the
derivatization step can occur under mildly acidic to slightly basic
conditions, including by way of example, between a pH of about 2 to
about 10, a pH of about 2 to about 8, or between a pH of about 4 to
about 8.
[0233] Modification of dicarbonyl sidechains, of nonnatural amino
acids incorporated into polypeptides, with hydroxylamine-containing
reagents, or other functional groups with similar chemical
reactivity, affords modified polypeptides containing oxime
linkages. The reactions and the resulting structures of such
modified polypeptides are shown in FIG. 6.
[0234] Certain embodiments described herein are polypeptides
containing non-natural amino acids with sidechains comprising an
oxime group. In other embodiments such oxime groups may be further
modified, such as, by way of example only, formation of masked
oxime groups (which can be readily converted into oxime groups),
protected oxime groups (which upon deprotection can be readily
converted into oxime groups available for other chemical
reactions), or new oxime groups via oxime exchange reactions.
[0235] Non-limiting examples of such modified polypeptide oxime
linkages are shown below:
##STR00004##
Reactions of Hydroxyalamine-Containing Non-Natural Amino Acid
Components with Dicarbonyl-Containing Reagents to Form Oximes
[0236] The incorporation of non-natural amino acids containing
hydroxylamine groups into polypeptides allows for reaction with a
variety of electrophilic groups including, but not limited to,
dicarbonyl group such as a diketone group, a ketoaldehyde group, a
ketoacid group, a ketoester group, and a ketothioester group, a
dicarbonyl-like group (which has reactivity similar to a dicarbonyl
group and is structurally similar to a carbonyl group), a masked
dicarbonyl group (which can be readily converted into a dicarbonyl
group), or a protected dicarbonyl group (which has reactivity
similar to a dicarbonyl group upon deprotection). The
nucleophilicity of the hydroxylamine group permits it to react
efficiently and selectively with a variety of molecules that
contain such dicarbonyl functionality, or other functional groups
with similar chemical reactivity, under mild conditions in aqueous
solution to form the corresponding oxime linkage. This
site-specific derivatization and/or further modifying of such
sidechains via nucleophilic attack of the dicarbonyl group may be
conducted with a polypeptide that has been purified prior to the
derivatization step or after the derivatization step. Further, the
derivatization step can occur under mildly acidic to slightly basic
conditions, including by way of example, between a pH of about 2 to
about 10, a pH of about 2 to about 8, or between a pH of about 4 to
about 8.
[0237] Modification of hydroxylamine groups, of nonnatural amino
acids incorporated into polypeptides, with dicarbonyl-containing
reagents affords modified polypeptides containing oxime linkages.
The reactions and the resulting structures of such modified
polypeptides are shown in FIG. 7.
[0238] Certain embodiments described herein are polypeptides
containing non-natural amino acids with sidechains comprising an
oxime group. In other embodiments such oxime groups may be further
modified, such as, by way of example only, formation of masked
oxime groups (which can be readily converted into oxime groups),
protected oxime groups (which upon deprotection can be readily
converted into oxime groups available for other chemical
reactions), or new oxime groups via oxime exchange reactions.
[0239] Non-limiting examples of such modified polypeptide oxime
linkages are shown below:
##STR00005##
Reactions of Oxime-Containing Non-Natural Amino Acid Components
Formed by Reaction of Dicarbonyls and Hydroxylamines, with Carbonyl
or Different Dicarbonyl-Containing Reagents to Form New Oximes via
an Oxime Exchange Reaction
[0240] Non-natural amino acids containing an oxime group allow for
reaction with a variety of reagents that contain certain reactive
dicarbonyl groups, including, but not limited to, diketone groups,
ketoaldehyde groups, ketoacid groups, ketoester groups,
ketothioester groups, dicarbonyl-like groups (which has reactivity
similar to a dicarbonyl group and is structurally similar to a
carbonyl group), masked dicarbonyl groups (which can be readily
converted into a dicarbonyl group), or protected dicarbonyl groups
(which has reactivity similar to a dicarbonyl group upon
deprotection) to form new non-natural amino acids (which can be
incorporated into a polypeptide) comprising a new oxime group. Such
an oxime exchange reaction allows for the further functionalization
of non-natural amino acid polypeptides.
[0241] Modification of oxime sidechains, of nonnatural amino acids
incorporated into polypeptides, with dicarbonyl-containing
reagents, or other functional groups with similar chemical
reactivity, affords modified polypeptides containing new oxime
linkages. The reactions and the resulting structures of such
modified polypeptides are shown in FIG. 8.
[0242] Certain embodiments described herein are polypeptides
containing non-natural amino acids with sidechains comprising an
oxime group. In other embodiments such oxime groups may be further
modified, such as, by way of example only, formation of masked
oxime groups (which can be readily converted into oxime groups),
protected oxime groups (which upon deprotection can be readily
converted into oxime groups available for other chemical
reactions), or new oxime groups via oxime exchange reactions.
[0243] B. Enhancing Affinity for Serum Albumin
[0244] Various molecules can also be fused to the non-natural amino
acid polypeptides described herein to modulate the half-life in
serum. In some cases, molecules are linked or fused to the
(modified) non-natural amino acid polypeptides described herein to
enhance affinity for endogenous serum albumin in an animal.
[0245] For example, in some cases, a recombinant fusion of a
polypeptide and an albumin binding sequence is made. In other
cases, the (modified) non-natural amino acid polypeptides described
herein are acylated with fatty acids. In other cases, the
(modified) non-natural amino acid polypeptides described herein are
fused directly with serum albumin (including but not limited to,
human serum albumin). Those of skill in the art will recognize that
a wide variety of other molecules can also be linked to non-natural
amino acid polypeptides, modified or unmodified, as described
herein, to modulate binding to serum albumin or other serum
components. Further discussion regarding the enhancement affinity
for serum albumin is described in U.S. Patent Application Nos.
60/638,418, 60/638,527, 60/639,195, 60/696,210, 60/696,302, and
60/696,068; PCT Publication WO 05/074650 entitled "Modified Four
Helical Bundle Polypeptides and Their Uses," which are incorporated
by reference in their entirety.
[0246] C. Glycosylation of Non-Natural Amino Acid Polypeptides
Described Herein
[0247] The methods and compositions described herein include
polypeptides incorporating one or more non-natural amino acids
bearing saccharide residues. The saccharide residues may be either
natural (including but not limited to, N-acetylglucosamine) or
non-natural (including but not limited to, 3-fluorogalactose). The
saccharides may be linked to the non-natural amino acids either by
an N- or O-linked glycosidic linkage (including but not limited to,
N-acetylgalactose-L-serine) or a non-natural linkage (including but
not limited to, an oxime or the corresponding C- or S-linked
glycoside).
[0248] The saccharide (including but not limited to, glycosyl)
moieties can be added to the non-natural amino acid polypeptides
either in vivo or in vitro. In some cases, a polypeptide comprising
a carbonyl-containing non-natural amino acid is modified with a
saccharide derivatized with an aminooxy group to generate the
corresponding glycosylated polypeptide linked via an oxime linkage.
Once attached to the non-natural amino acid, the saccharide may be
further elaborated by treatment with glycosyltransferases and other
enzymes to generate an oligosaccharide bound to the non-natural
amino acid polypeptide. See, e.g., H. Liu, et al. J. Am. Chem. Soc.
125: 1702-1703 (2003).
[0249] D. Use of Linking Groups and Applications, Including
Polypeptide Dimers and Multimers
[0250] In addition to adding functionality directly to the
non-natural amino acid polypeptide, the non-natural amino acid
portion of the polypeptide may first be modified with a
multifunctional (e.g., bi-, tri, tetra-) linker molecule that then
subsequently is further modified. That is, at least one end of the
multifunctional linker molecule reacts with at least one
non-natural amino acid in a polypeptide and at least one other end
of the multifunctional linker is available for further
functionalization. If all ends of the multifunctional linker are
identical, then (depending upon the stoichiometric conditions)
homomultimers of the non-natural amino acid polypeptide may be
formed. If the ends of the multifunctional linker have distinct
chemical reactivities, then at least one end of the multifunctional
linker group will be bound to the non-natural amino acid
polypeptide and the other end can subsequently react with a
different functionality, including by way of example only: a label;
a dye; a polymer; a water-soluble polymer; a derivative of
polyethylene glycol; a photocrosslinker; a cytotoxic compound; a
drug; an affinity label; a photoaffinity label; a reactive
compound; a resin; a second protein or polypeptide or polypeptide
analog; an antibody or antibody fragment; a metal chelator; a
cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a
RNA; an antisense polynucleotide; a saccharide; a water-soluble
dendrimer; a cyclodextrin; an inhibitory ribonucleic acid; a
biomaterial; a nanoparticle; a spin label; a fluorophore, a
metal-containing moiety; a radioactive moiety; a novel functional
group; a group that covalently or noncovalently interacts with
other molecules; a photocaged moiety; an actinic excitable moiety;
a photoisomerizable moiety; biotin; a biotin analogue; a moiety
incorporating a heavy atom; a chemically cleavable group; a
photocleavable group; an elongated side chain; a carbon-linked
sugar; a redox-active agent; an amino thioacid; a toxic moiety; an
isotopically labeled moiety; a biophysical probe; a phosphorescent
group; a chemiluminescent group; an electron dense group; a
magnetic group; an intercalating group; a chromophore; an energy
transfer agent; a biologically active agent; a detectable label; a
small molecule; a quantum dot; a nanotransmitter; and any
combination of the above.
[0251] Further use of linking groups and applications, including
polypeptide dimers and multimers are further described in U.S.
Patent Application Nos. 60/638,418, 60/638,527, 60/639,195,
60/696,210, 60/696,302, and 60/696,068; PCT Publication WO
05/074650 entitled "Modified Four Helical Bundle Polypeptides and
Their Uses," which are incorporated by reference in their
entirety.
[0252] E. Example of Adding Functionality: Easing the Isolation
Properties of a Polypeptide
[0253] A naturally-occurring or non-natural amino acid polypeptide
may be difficult to isolate from a sample for a number of reasons,
including but not limited to the solubility or binding
characteristics of the polypeptide. For example, in the preparation
of a polypeptide for therapeutic use, such a polypeptide may be
isolated from a recombinant system that has been engineered to
overproduce the polypeptide. However, because of the solubility or
binding characteristics of the polypeptide, achieving a desired
level of purity often proves difficult. The methods, compositions,
techniques and strategies further described in U.S. Patent
Application Nos. 60/638,418, 60/638,527, 60/639,195, 60/696,210,
60/696,302, and 60/696,068; PCT Publication WO 05/074650 entitled
"Modified Four Helical Bundle Polypeptides and Their Uses," which
are incorporated by reference in their entirety provide a solution
to this situation.
[0254] F. Example of Adding Functionality: Detecting the Presence
of a Polypeptide
[0255] A naturally-occurring or non-natural amino acid polypeptide
may be difficult to detect in a sample (including an in vivo sample
and an in vitro sample) for a number of reasons, including but not
limited to the lack of a reagent or label that can readily bind to
the polypeptide. The methods, compositions, techniques and
strategies further described in U.S. Patent Application Nos.
60/638,418, 60/638,527, 60/639,195, 60/696,210, 60/696,302, and
60/696,068; PCT Publication WO 05/074650 entitled "Modified Four
Helical Bundle Polypeptides and Their Uses," which are incorporated
by reference in their entirety provide a solution to this
situation.
[0256] G. Example of Adding Functionality: Improving the
Therapeutic Properties of a Polypeptide
[0257] A naturally-occurring or non-natural amino acid polypeptide
will be able to provide a certain therapeutic benefit to a patient
with a particular disorder, disease or condition. Such a
therapeutic benefit will depend upon a number of factors, including
by way of example only: the safety profile of the polypeptide, and
the pharmacokinetics, pharmacologics and/or pharmacodynamics of the
polypeptide (e.g., water solubility, bioavailability, serum
half-life, therapeutic half-life, immunogenicity, biological
activity, or circulation time). In addition, it may be advantageous
to provide additional functionality to the polypeptide, such as an
attached cytotoxic compound or drug, or it may be desirable to
attach additional polypeptides to form the homo- and
heteromultimers described herein. Such modifications preferably do
not destroy the activity and/or tertiary structure of the original
polypeptide. The methods, compositions, techniques and strategies
further described in U.S. Patent Application Nos. 60/638,418,
60/638,527, 60/639,195, 60/696,210, 60/696,302, and 60/696,068; PCT
Publication WO 05/074650 entitled "Modified Four Helical Bundle
Polypeptides and Their Uses," which are incorporated by reference
in their entirety provide solutions to these issues.
[0258] X. Therapeutic Uses of Modified Polypeptides
[0259] The (modified) non-natural amino acid polypeptides described
herein, including homo- and hetero-multimers thereof find multiple
uses, including but not limited to: therapeutic, diagnostic,
assay-based, industrial, cosmetic, plant biology, environmental,
energy-production, and/or military uses. As a non-limiting
illustration, the following therapeutic uses of (modified)
non-natural amino acid polypeptides are provided.
[0260] The (modified) non-natural amino acid polypeptides described
herein are useful for treating a wide range of disorders.
Administration of the (modified) non-natural amino acid polypeptide
products described herein results in any of the activities
demonstrated by commercially available polypeptide preparations in
humans. Average quantities of the (modified) non-natural amino acid
polypeptide product may vary and in particular should be based upon
the recommendations and prescription of a qualified physician. The
exact amount of the (modified) non-natural amino acid polypeptide
is a matter of preference subject to such factors as the exact type
of condition being treated, the condition of the patient being
treated, as well as the other ingredients in the composition. The
amount to be given may be readily determined by one skilled in the
art based upon therapy with the (modified) non-natural amino acid
polypeptide.
[0261] A. Administration and Pharmaceutical Compositions
[0262] The non-natural amino acid polypeptides, modified or
unmodified, as described herein (including but not limited to,
synthetases, proteins comprising one or more non-natural amino
acid, etc.) are optionally employed for therapeutic uses, including
but not limited to, in combination with a suitable pharmaceutical
carrier. Such compositions, for example, comprise a therapeutically
effective amount of the non-natural amino acid polypeptides,
modified or unmodified, as described herein, and a pharmaceutically
acceptable carrier or excipient. Such a carrier or excipient
includes, but is not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and/or combinations thereof. The
formulation is made to suit the mode of administration. In general,
methods of administering proteins are well known in the art and can
be applied to administration of the non-natural amino acid
polypeptides, modified or unmodified, as described herein.
[0263] Therapeutic compositions comprising one or more of the
non-natural amino acid polypeptides, modified or unmodified, as
described herein are optionally tested in one or more appropriate
in vitro and/or in vivo animal models of disease, to confirm
efficacy, tissue metabolism, and to estimate dosages, according to
methods known to those of ordinary skill in the art. In particular,
dosages can be initially determined by activity, stability or other
suitable measures of non-natural to natural amino acid homologues
(including but not limited to, comparison of a polypeptide
(modified) to include one or more non-natural amino acids to a
natural amino acid polypeptide), i.e., in a relevant assay.
[0264] Administration is by any of the routes normally used for
introducing a molecule into ultimate contact with blood or tissue
cells. The non-natural amino acid polypeptides, modified or
unmodified, as described herein, are administered in any suitable
manner, optionally with one or more pharmaceutically acceptable
carriers. Suitable methods of administering the non-natural amino
acid polypeptides, modified or unmodified, as described herein, to
a patient are available, and, although more than one route can be
used to administer a particular composition, a particular route can
often provide a more immediate and more effective action or
reaction than another route.
[0265] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions described herein.
[0266] Non-natural amino acid polypeptides may be administered by
any conventional route suitable for proteins or peptides,
including, but not limited to parenterally, e.g. injections
including, but not limited to, subcutaneously or intravenously or
any other form of injections or infusions. Polypeptide compositions
(including the various polypeptides described herein) can be
administered by a number of routes including, but not limited to
oral, intravenous, intraperitoneal, intramuscular, transdermal,
subcutaneous, topical, sublingual, or rectal means. Compositions
comprising non-natural amino acid polypeptides, modified or
unmodified, as described herein, can also be administered via
liposomes. Such administration routes and appropriate formulations
are generally known to those of skill in the art. The non-natural
amino acid polypeptide may be used alone or in combination with
other suitable components such as a pharmaceutical carrier.
[0267] The non-natural amino acid polypeptides, modified or
unmodified, as described herein, alone or in combination with other
suitable components, can also be made into aerosol formulations
(i.e., they can be "nebulized") to be administered via inhalation.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like.
[0268] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. The formulations
of packaged nucleic acid can be presented in unit-dose or
multi-dose sealed containers, such as ampules and vials.
[0269] Parenteral administration and intravenous administration are
preferred methods of administration. In particular, the routes of
administration already in use for natural amino acid homologue
therapeutics (including but not limited to, those typically used
for EPO, IFN, GM-CSF, IFNs, interleukins, antibodies, and/or any
other pharmaceutically delivered protein), along with formulations
in current use, provide preferred routes of administration and
formulation for the non-natural amino acid polypeptides, modified
or unmodified, as described herein.
[0270] The dose administered to a patient, in the context
compositions and methods described herein, is sufficient to have a
beneficial therapeutic response in the patient over time. The dose
is determined by the efficacy of the particular formulation, and
the activity, stability or serum half-life of the non-natural amino
acid polypeptides, modified or unmodified, employed and the
condition of the patient, as well as the body weight or surface
area of the patient to be treated. The size of the dose is also
determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
formulation, or the like in a particular patient.
[0271] In determining the effective amount of the formulation to be
administered in the treatment or prophylaxis of disease (including
but not limited to, cancers, inherited diseases, diabetes, AIDS, or
the like), the physician evaluates circulating plasma levels,
formulation toxicities, progression of the disease, and/or where
relevant, the production of anti-non-natural amino acid polypeptide
antibodies.
[0272] The dose administered, for example, to a 70 kilogram
patient, is typically in the range equivalent to dosages of
currently-used therapeutic proteins, adjusted for the altered
activity or serum half-life of the relevant composition. The
pharmaceutical formulations described herein can supplement
treatment conditions by any known conventional therapy, including
antibody administration, vaccine administration, administration of
cytotoxic agents, natural amino acid polypeptides, nucleic acids,
nucleotide analogues, biologic response modifiers, and the
like.
[0273] For administration, the pharmaceutical formulations
described herein are administered at a rate determined by the LD-50
or ED-50 of the relevant formulation, and/or observation of any
side-effects of the non-natural amino acid polypeptides, modified
or unmodified, at various concentrations, including but not limited
to, as applied to the mass and overall health of the patient.
Administration can be accomplished via single or divided doses.
[0274] If a patient undergoing infusion of a formulation develops
fevers, chills, or muscle aches, he/she receives the appropriate
dose of aspirin, ibuprofen, acetaminophen or other pain/fever
controlling drug. Patients who experience reactions to the infusion
such as fever, muscle aches, and chills are premedicated 30 minutes
prior to the future infusions with either aspirin, acetaminophen,
or, including but not limited to, diphenhydramine. Meperidine is
used for more severe chills and muscle aches that do not quickly
respond to antipyretics and antihistamines. Cell infusion is slowed
or discontinued depending upon the severity of the reaction.
[0275] Non-natural amino acid polypeptides, modified or unmodified,
as described herein, can be administered directly to a mammalian
subject. Administration is by any of the routes normally used for
introducing a polypeptide to a subject. The non-natural amino acid
polypeptides, modified or unmodified, as described herein, include
those suitable for oral, rectal, topical, inhalation (including but
not limited to, via an aerosol), buccal (including but not limited
to, sub-lingual), vaginal, parenteral (including but not limited
to, subcutaneous, intramuscular, intradermal, intraarticular,
intrapleural, intraperitoneal, intracerebral, intraarterial, or
intravenous), topical (i.e., both skin and mucosal surfaces,
including airway surfaces) and transdermal administration, although
the most suitable route in any given case will depend on the nature
and severity of the condition being treated. Administration can be
either local or systemic. The formulations can be presented in
unit-dose or multi-dose sealed containers, such as ampoules and
vials. The non-natural amino acid polypeptides, modified or
unmodified, as described herein, can be prepared in a mixture in a
unit dosage injectable form (including but not limited to,
solution, suspension, or emulsion) with a pharmaceutically
acceptable carrier. The non-natural amino acid polypeptides,
modified or unmodified, as described herein, can also be
administered by continuous infusion (using, including but not
limited to, minipumps such as osmotic pumps), single bolus or
slow-release depot formulations.
[0276] Formulations suitable for administration include aqueous and
non-aqueous solutions, isotonic sterile solutions, which can
contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic, and aqueous and non-aqueous
sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives.
Solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described.
[0277] Freeze-drying is a commonly employed technique for
presenting proteins which serves to remove water from the protein
preparation of interest. Freeze-drying, or lyophilization, is a
process by which the material to be dried is first frozen and then
the ice or frozen solvent is removed by sublimation in a vacuum
environment. An excipient may be included in pre-lyophilized
formulations to enhance stability during the freeze-drying process
and/or to improve stability of the lyophilized product upon
storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawa et al.
Pharm. Res. 8(3):285-291 (1991).
[0278] The spray drying of pharmaceuticals is also known to those
of ordinary skill in the art. For example, see Broadhead, J. et
al., "The Spray Drying of Pharmaceuticals," in Drug Dev. Ind.
Pharm, 18 (11 & 12), 1169-1206 (1992). In addition to small
molecule pharmaceuticals, a variety of biological materials have
been spray dried and these include: enzymes, sera, plasma,
micro-organisms and yeasts. Spray drying is a useful technique
because it can convert a liquid pharmaceutical preparation into a
fine, dustless or agglomerated powder in a one-step process. The
basic technique comprises the following four steps: a) atomization
of the feed solution into a spray; b) spray-air contact; c) drying
of the spray; and d) separation of the dried product from the
drying air. U.S. Pat. Nos. 6,235,710 and 6,001,800, which are
incorporated by reference herein, describe the preparation of
recombinant erythropoietin by spray drying.
[0279] The pharmaceutical compositions described herein may
comprise a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method
used to administer the composition. Accordingly, there is a wide
variety of suitable formulations of pharmaceutical compositions
(including optional pharmaceutically acceptable carriers,
excipients, or stabilizers) for the non-natural amino acid
polypeptides, modified or unmodified, described herein, (see, e.g.,
Remington's Pharmaceutical Sciences, 17.sup.th ed. 1985)). Suitable
carriers include buffers containing succinate, phosphate, borate,
HEPES, citrate, imidazole, acetate, bicarbonate, and other organic
acids; antioxidants including but not limited to, ascorbic acid;
low molecular weight polypeptides including but not limited to
those less than about 10 residues; proteins, including but not
limited to, serum albumin, gelatin, or immunoglobulins; hydrophilic
polymers including but not limited to, polyvinylpyrrolidone; amino
acids including but not limited to, glycine, glutamine, asparagine,
arginine, histidine or histidine derivatives, methionine,
glutamate, or lysine; monosaccharides, disaccharides, and other
carbohydrates, including but not limited to, trehalose, sucrose,
glucose, mannose, or dextrins; chelating agents including but not
limited to, EDTA; divalent metal ions including but not limited to,
zinc, cobalt, or copper; sugar alcohols including but not limited
to, mannitol or sorbitol; salt-forming counter ions including but
not limited to, sodium; and/or nonionic surfactants including but
not limited to Tween.TM. (including but not limited to, Tween 80
(polysorbate 80) and Tween 20 (polysorbate 20), Pluronics.TM. and
other pluronic acids, including but not limited to, and other
pluronic acids, including but not limited to, pluronic acid F68
(poloxamer 188), or PEG. Suitable surfactants include for example
but are not limited to polyethers based upon poly(ethylene
oxide)-poly(propylene oxide)-poly(ethylene oxide), i.e.,
(PEO-PPO-PEO), or poly(propylene oxide)-poly(ethylene
oxide)-poly(propylene oxide), i.e., (PPO-PEO-PPO), or a combination
thereof. PEO-PPO-PEO and PPO-PEO-PPO are commercially available
under the trade names Pluronics.TM., R-Pluronics.TM., Tetronics.TM.
and R-Tetronics.TM. (BASF Wyandotte Corp., Wyandotte, Mich.) and
are further described in U.S. Pat. No. 4,820,352 incorporated
herein in its entirety by reference. Other ethylene/polypropylene
block polymers may be suitable surfactants. A surfactant or a
combination of surfactants may be used to stabilize a (modified)
non-natural amino acid polypeptide against one or more stresses
including but not limited to stress that results from agitation.
Some of the above may be referred to as "bulking agents." Some may
also be referred to as "tonicity modifiers."
[0280] The non-natural amino acid polypeptides, modified or
unmodified, as described herein, including those linked to water
soluble polymers such as PEG can also be administered by or as part
of sustained-release systems. Sustained-release compositions
include, including but not limited to, semi-permeable polymer
matrices in the form of shaped articles, including but not limited
to, films, or microcapsules. Sustained-release matrices include
from biocompatible materials such as poly(2-hydroxyethyl
methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 267-277
(1981); Langer, Chem. Tech., 12: 98-105 (1982), ethylene vinyl
acetate (Langer et al., supra) or poly-D-(-)-3-hydroxybutyric acid
(EP 133,988), polylactides (polylactic acid) (U.S. Pat. No.
3,773,919; EP 58,481), polyglycolide (polymer of glycolic acid),
polylactide co-glycolide (copolymers of lactic acid and glycolic
acid) polyanhydrides, copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556
(1983), poly(ortho)esters, polypeptides, hyaluronic acid, collagen,
chondroitin sulfate, carboxylic acids, fatty acids, phospholipids,
polysaccharides, nucleic acids, polyamino acids, amino acids such
as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl
propylene, polyvinylpyrrolidone and silicone. Sustained-release
compositions also include a liposomally entrapped compound.
Liposomes containing the compound are prepared by methods known per
se: DE 3,218,121; Eppstein et al., Proc. Natl. Acad. Sci. U.S.A.,
82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A.,
77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Pat. No.
4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; Japanese Pat.
Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP
102,324. All references and patents cited are incorporated by
reference herein.
[0281] Liposomally entrapped polypeptides can be prepared by
methods described in, e.g., DE 3,218,121; Eppstein et al., Proc.
Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc.
Natl. Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP
36,676; U.S. Pat. No. 4,619,794; EP 143,949; U.S. Pat. No.
5,021,234; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045
and 4,544,545; and EP 102,324. Composition and size of liposomes
are well known or able to be readily determined empirically by one
of ordinary skill in the art. Some examples of liposomes as
described in, e.g., Park J W, et al., Proc. Natl. Acad. Sci. USA
92:1327-1331 (1995); Lasic D and Papahadjopoulos D (eds): MEDICAL
APPLICATIONS OF LIPOSOMES (1998); Drummond D C, et al., Liposomal
drug delivery systems for cancer therapy, in Teicher B (ed): CANCER
DRUG DISCOVERY AND DEVELOPMENT (2002); Park J W, et al., Clin.
Cancer Res. 8:1172-1181 (2002); Nielsen U B, et al., Biochim.
Biophys. Acta 1591(1-3):109-118 (2002); Mamot C, et al., Cancer
Res. 63: 3154-3161 (2003). All references and patents cited are
incorporated by reference herein.
[0282] The dose administered to a patient in the context of the
compositions, formulations and methods described herein, should be
sufficient to cause a beneficial response in the subject over time.
Generally, the total pharmaceutically effective amount of the
non-natural amino acid polypeptides, modified or unmodified, as
described herein, administered parenterally per dose is in the
range of about 0.01 .mu.g/kg/day to about 100 .mu.g/kg, or about
0.05 mg/kg to about 1 mg/kg, of patient body weight, although this
is subject to therapeutic discretion. The frequency of dosing is
also subject to therapeutic discretion, and may be more frequent or
less frequent than the commercially available products approved for
use in humans. Generally, a polymer:polypeptide conjugate,
including by way of example only, a PEGylated polypeptide, as
described herein, can be administered by any of the routes of
administration described above.
[0283] XI. Isolation and Purification
A. Chromatography
[0284] In any of the embodiments herein, the isolation of peptides,
(modified) non-natural amino acid polypeptides, binding partners or
receptors to polypeptides can occur by chromatography.
Chromatography is based on the differential absorption and elution
of polypeptides. The sample is dissolved in a mobile phase, which
may be a gas, a liquid or a supercritical fluid. This mobile phase
is then forced through an immiscible stationary phase, which is
fixed in a column or on a solid surface. Examples of stationary
phases include liquids adsorbed on a solid, organic species bonded
to a solid surface, solid, ion exchange resin and liquid in
interstices of a polymeric solid. The ability of a polypeptide to
be purified by different chromatographic or other
isolation/purification methods may be modulated by the addition or
substitution of one or more non-natural amino acids with a
non-natural amino acid optionally in combination with one or more
natural amino acid substitutions. Thus, the properties of a
polypeptide may be modified by altering the amino acid composition
enabling an increase or decrease in its interaction with known
matrices. Changes to the amino acid composition include, but are
not limited to, hydrophobic amino acid content, hydrophilic amino
acid content, and change in charge, pI, or other characteristics of
the polypeptide. Such modifications may be useful in isolating
membrane proteins which are difficult to isolate since they are
hydrophobic in nature and keep in their native conformation.
Gas Chromatography
[0285] In one embodiment the isolation of polypeptides can occur by
gas chromatography (GC). The sample is vaporized and injected onto
the head of a chromatographic column. Examples of mobile gas phases
include but are not limited to helium, argon, nitrogen, carbon
dioxide, and hydrogen. In one embodiment, the sample is isolated by
gas-solid chromatography, where the stationary phase is a solid.
Examples of solid stationary phase are molecular sieves and porous
polymers. In another embodiment the polypeptide is isolated by
gas-liquid chromatography, where the stationary phase is a liquid
immobilized on the surface of an inert solid. Examples of liquid
stationary phases include Polydimethyl siloxane, Poly
(phenylmethyldimethyl) siloxane (10% phenyl), Poly(phenylmethyl)
siloxane (50% phenyl), Poly(trifluoropropyldimethyl) siloxane,
Polyethylene glycol and Poly(dicyanoallyldimethyl) siloxane.
[0286] Conventional GC columns are either packed and open tubular
or capillary. GC-chromatographic columns vary in length from less
than 2 m to 50 m or more. Examples of material for their
construction include stainless steel, metal, glass, fused silica
and Teflon. Typically GC columns have an in inner diameter of
roughly of 2 to 4 mm. Micro-GC has an inner diameter of roughly 1
mm. Capillary GC utilizes a capillary with an inner diameter of
roughly 100 to 750 um. Nano-GC is available with an inner diameter
of 50 um-1 mm
Liquid Chromatography
[0287] In one embodiment the isolation of polypeptides can occur by
liquid chromatography (LC). LC involves the use of fluid carrier
over a stationary phase. The majority of LC-columns range in length
from 10 to 30 cm. LC columns are ordinarily constructed from
smooth-bore stainless steel tubing, although heavy glass tubing in
occasionally encountered. Conventional LC columns have an inner
diameter of roughly 4.6 mm and a flow rate of roughly 1 ml/min.
Micro-LC has an inner diameter of roughly 1.0 mm and a flow rate of
roughly 40 .mu.l/min. Capillary LC utilizes a capillary with an
inner diameter of roughly 300 .mu.m and a flow rate of
approximately 5 .mu.l/min. Nano-LC is available with an inner
diameter of 50 .mu.m-1 mm and flow rates of 200 nl/min. Nano-LC can
vary in length, e.g., 5, 15, or 25 cm. Nano-LC stationary phase may
also be a monolithic material, such as a polymeric monolith or a
sol-gel monolith. Two basic types of packing material have been
used in liquid chromatography, non-porous and porous particles. The
beads or particles are generally characterized by particle and pore
size. Particle sizes generally range between 3 and 50 microns.
Larger particles will generate less system pressure and smaller
particles will generate more pressure. The smaller particles
generally give higher separation efficiencies. The particle pore
size is measured in angstroms and generally range between 100-1000
.ANG.. These can be covered with a porous layer of silica, alumina,
ion exchange resin, organic surface layer, polymers, ligands,
carbohydrates or a specific cofactor.
[0288] In one embodiment of the invention, the polypeptides can be
isolated using HPLC technology. In another embodiment of the
invention, the polypeptide can be isolated using column
chromatography. In column chromatography, the solid medium is
packed onto a chromatography column, and the initial mixture
containing the polypeptide is run through the column to allow
binding. A wash buffer is then run through the column, and the
elution buffer is subsequently applied to the column for sample
collection. These steps may be performed at ambient pressure. In
another embodiment, binding of the polypeptides to a solid phase
may be achieved using a Batch treatment, by adding the initial
mixture to the solid phase in a vessel, mixing the two together,
separating the solid phase (i.e. by centrifugation), removing the
liquid phase, washing, re-centrifuging, adding the elution buffer,
re-centrifuging and removing the eluate. In another embodiment of
the invention, a hybrid method is employed in which the binding is
done by the Batch method, the solid phase with the target molecule
bound is then packed onto a column, and washing and elution are
performed on the column. In yet another embodiment of the
invention, the isolation of peptides occur in a microfluidic
device. In another embodiment of the invention, the isolation of
peptides occur in a nanofluidic device.
Partition Chromatography
[0289] In one embodiment the isolation of polypeptides occurs by
partition chromatography. In one embodiment the isolation of the
polypeptides occurs by liquid-liquid partition chromatography. With
liquid-liquid partition chromatography, a liquid stationary phase
is retained on the surface of the packing by physical adsorption.
In another embodiment, the isolation of the polypeptides can occur
by bonded-phase partition chromatography. With bonded-phase
partition chromatography, the stationary phase is bonded chemically
to the support surfaces.
[0290] In another embodiment, normal-phase chromatography is used
to isolate the polypeptides. In normal-phase chromatography, a
polar stationary phase is used together with a non-polar solvent.
Examples of the stationary phase for normal phase chromatography
include but are not limited to, water, alcohols and triethylene
glycol. Examples of non-polar solvents for normal phase
chromatography include but are not limited to, ethyl, ether,
chloroform, tetrahydrofuran, fluoroalkanes, cyclohexane,
1-chlorobutane, carbon tetrachloride, toluene, diethyl ether,
hexane and i-propylether. In one embodiment the partition
chromatography uses reversed-phase packings; this is referred as
reversed-phase chromatography. In reversed-phase chromatography, a
non-polar stationary phase is used together with a polar mobile
phase. Examples of stationary phases for reversed-phased
chromatography include but are not limited to, hydrocarbons, ether,
esters, ketones, aldehydes, amides, and amines. Examples of mobile
stationary phases for reversed-phased chromatography include water,
methanol, ethanol, ethyl acetate, dioxane, nitromethane, ethylene
glycol, tetrahydrofuran and acetonitrile.
[0291] In one embodiment, the type of reversed chromatography that
can be use to isolate polypeptides is ion-pair chromatography. The
mobile phase in ion-pair chromatography consists of an aqueous
buffer containing an organic solvent such as methanol or
acetonitrile and an ionic compound containing a counter ion of
opposite charge to the polypeptides. The counter ion binds to the
polypeptide to form an ion pair, which is a neutral species that is
retained by a reversed-phase packing. Elution of the ion pairs is
then accomplished with an aqueous solution of methanol or another
water soluble organic solvent like the one described above.
Examples of counter-ions are
ClO.sub.4.sup.-C.sub.12H.sub.25SO.sub.3.sup.-,
(C.sub.4H.sub.9).sub.4N.sup.+,
(C.sub.16H.sub.33)(CH.sub.3).sub.3N.sup.+,
(C.sub.4H.sub.9).sub.4N.sup.+, Bis-(2-ethylhexyl)phosphate, and
(C.sub.4H.sub.9).sub.4N.sup.+.
[0292] In one embodiment, the polypeptides can be isolated using
partition chromatography with a chiral stationary phase. Examples
of types of chiral stationary phases include but are not limited
to, protein based stationary phases, small molecular weight chiral,
polymers of cellulose and amylose, macrocyclic glycopeptides and
cyclodextrin based materials.
[0293] Adsorption Chromatography
[0294] In one embodiment the isolation of polypeptides can occur by
adsorption chromatography. Adsorption is a process whereby material
(contained in the mobile phase) interacts by physical forces
(dispersive, polar or ionic) with a stationary phase, thereby,
causing a layer (or layers) of the material to adhere to that
stationary phase. The stationary phase in most cases will be a
solid (e.g. silica gel, alumina, charcoal, etc.) or sometimes a
liquid (e.g. surfactants on water surfaces). The surface layer(s)
may be single, double or multiple. Examples of solvents that can be
use in adsorption chromatography include water, methanol, ethanol,
ethyl acetate, dioxane, nitromethane, ethylene glycol,
tetrahydrofuran, acetonitrile, ethyl, ether, chloroform,
tetrahydrofuran, fluoroalkanes, cyclohexane, 1-chlorobutane, carbon
tetrachloride, toluene, diethyl ether, hexane and
i-propylether.
Ion Exchange Chromatography
[0295] In one embodiment the isolation of polypeptides can occur by
ion-exchange chromatography. In ion-exchange chromatography the
isolation of polypeptides is based upon ion-exchange resin. The ion
exchange resin can be an anion exchange resin or a cation exchange
resin. The ion-exchange resin can be made by natural ion
exchangers, such as clays and zeolites, or from synthetic ion
exchangers. Examples of common active sites for cation exchange
resins are the sulfonic acid group --SO.sub.3.sup.-H.sup.+, the
carboxylic acid group --COO.sup.-H.sup.+ and phosphoric acid
--PO.sub.32.sup.+H.sub.2. Examples of common active sites for anion
exchange resins are quaternary amine groups
--N(CH.sub.3).sup.+OH.sup.- or primary amine groups
--NH.sub.3.sup.+OH.sup.-. The mobile phase in ion-exchange
chromatography is generally an aqueous solution that may contain
moderate amounts of methanol or other water miscible organic
solvents; these mobile phases also contain ionic species in the
form of a buffer.
[0296] In one embodiment the ion exchange column is eluted with a
gradient of salt concentrations. In one example, pumps add
increasing amounts of salt to the buffer as it goes onto the column
so that there is a continuous steady increase in the ionic
concentration going through the column. The proteins then "elute"
or come off the column stationary phase when the ionic strength of
the buffer neutralizes their charge. The least charged molecules
come off first, and the most highly charged come off last. In
another example, the column is thoroughly rinsed with buffers of
increasing ionic strength until the desired protein elutes; this
exact same sequence is repeated each time with the same amounts of
buffer to give reproducible yields and purification of the
protein.
[0297] In one embodiment, the sample will be subject removal of
high salt concentrations after isolation the polypeptide of
interest by ion exchange chromatography. In one embodiment the
removal of high salt concentration will be performed by dialysis.
Dialysis makes use of semi-permeable membranes. The main feature of
the dialysis membrane is that it is porous. However, the pore size
is such that while small salt ions can freely pass through the
membrane, larger protein molecules cannot (i.e. they are retained).
Thus, dialysis membranes are characterized by the molecular mass of
the smallest typical globular protein which it will retain. Removal
of high salt concentration can be achieved in a single or multiple
dialysis steps. In another embodiment, the removal of high salt
concentration is performed by electrodialysis. Electrodialysis is
an electromembrane process in which ions are transported through
ion permeable membranes from one solution to another under the
influence of a potential gradient. Since the membranes used in
electrodialysis have the ability to selectively transportions
having positive or negative charge and reject ions of the opposite
charge, electrodialysis is useful for concentration, removal, or
separation of electrolytes.
[0298] In another embodiment, the removal of high concentration of
salt is achieved by using desalting columns in gravity-flow gel
filtration. Gravity-flow gel filtration involves the
chromatographic separation of molecules of different dimensions
based on their relative abilities to penetrate into a suitable
stationary phase. Desalting columns are packed with small, porous
cellulose beads. These columns have a wet bead with specific
diameters. The diameter of the beads used will depend on the
molecular weight of the peptide of interest. Different levels of
separation can be achieved based on the pore size of the medium
packed into the column. The medium can be chosen to totally exclude
proteins or large molecules, while still including small solutes.
Large molecules are excluded from the internal pores of the gel and
emerge first from the column. The smaller molecules are able to
penetrate the pores, and then progress through the column at a
slower rate. These smaller molecules are subsequently flushed
through the column with additional buffer volume.
[0299] Size-Exclusion Chromatography
[0300] In one embodiment the isolation of polypeptides can occur by
size-exclusion chromatography, also known as gel permeation, or gel
filtration chromatography. Molecules that are larger than the
average pore size of the packing are excluded and thus suffer no
retention. Examples of packings for size exclusion chromatography
include silica, cellulose beads and polymer particles.
Conventionally, porous glasses and silica particles have an average
pore size ranging from 40 .ANG. to 2500 .ANG.. In some embodiments,
the molecular weight exclusion limit of a polymer packing with an
average pore size of 102 .ANG. is 700. In another embodiment the
molecular weight exclusion limit of a polymer packing with an
average pore size of 103 .ANG. is (0.1 to 20).times.104. In another
embodiment the molecular weight exclusion limit of a polymer
packing with an average pore size of 104 .ANG. is (1 to
20).times.10.sup.4. In another embodiment the molecular weight
exclusion limit of a polymer packing with an average pore size of
10.sup.5 .ANG. is (1 to 20).times.10.sup.5. In yet another
embodiment the molecular weight exclusion limit of a polymer
packing with an average pore size of 10.sup.6 .ANG. is (5 to
10).times.10.sup.6. In some embodiments, the molecular weight
exclusion limit of silica packing with an average pore size of 125
.ANG. is (0.2 to 5).times.10.sup.4. In another embodiment, the
molecular weight exclusion limit of silica packing with an average
pore size of 300 .ANG. is (0.03 to 1).times.10.sup.5. In another
embodiment, the molecular weight exclusion limit of silica packing
with an average pore size of 500 .ANG. is (0.05 to
5).times.10.sup.5. In yet another embodiment, the molecular weight
exclusion limit of silica packing with an average pore size of 1000
.ANG. is (5 to 20).times.10.sup.5.
[0301] Thin-Layer Chromatography
[0302] In one embodiment the isolation of polypeptides can occur by
thin-layer chromatography. Thin-layer chromatographic methods
include paper chromatography, thin-layer chromatography and
electrochromatography. Each makes use of a flat, thin layer of
material that is either self supporting or that is coated on a
glass, plastic, or metal surface. The mobile phase moves through
the stationary phase by capillary action, sometimes assisted by
gravity or electrical potential. In one embodiment, planar
separation is performed on flat glass or plastic plates that are
coated with a thin and adherent layer of finely divided particles;
this layer constitutes the stationary phase. The stationary phase
and mobile phase are similar to those discussed in adsorption,
normal- and reversed-phase partition, ion-exchange, and size
exclusion chromatography. In one embodiment, the polypeptides are
located in the plate by spraying a solution that will react with
organic compounds to yield dark products. Examples of this type of
solution include ninhydrin, iodine solutions and sulfuric acid
solution. In another embodiment, the polypeptides are located by
incorporating a fluorescent material to the stationary phase. The
plate is examined under ultraviolet light. The sample components
quench the fluorescent material so that all of the plate fluoresces
except where the non-fluorescing sample components are located.
[0303] Affinity Chromatography
[0304] In one embodiment the isolation of polypeptides can occur by
affinity chromatography. Affinity chromatography relies on the
ability to design a stationary phase that reversibly binds to a
known subset of molecules. Affinity purification generally involves
the following steps: 1) incubate crude sample with the immobilized
ligand support material to allow the target molecule in the sample
to bind to the immobilized ligand, 2) wash away nonbound sample
components from solid support and 3) elute (dissociate and recover)
the target molecule from the immobilized ligand by altering the
buffer conditions so that the binding interaction no longer occurs.
Examples of elution buffers used in affinity chromatography include
but are not limited to 100 mM glycine.HCl, 100 mM citric acid,
50-100 mM triethylamine or triethanolamine, 150 mM ammonium
hydroxide, 3.5-4.0 M magnesium chloride in 10 mM Tris, 5 M lithium
chloride in 10 mM phosphate buffer, 2.5 M sodium iodide, 0.2-3.0
sodium thiocyanate, 2-6 M guanidine.HCl, 2-8 M urea, 1%
deoxycholate, 1% SDS, 10% dioxane, 50% ethylene glycol, 0.1 M
Glycine-NaOH, 0.1 M Glycine-NaOH with 50% ethyleneglycol, 3.0 M
Potassium chloride, 0.1 M Tris-acetate with 2.0 M NaCl, 5.0 M
Potassium iodide, 1% SDS, 1% Sodium deoxycholate, 2.0 M Urea, 6.0 M
Urea, 2.0 M Guanadine-HCl, 1.0 M Ammonium thiocyanate and >0.1 M
counter ligand or analog.
[0305] In one embodiment, the stationary phase includes a ligand
including but not limited to, a specific carbohydrate or a
cofactor. In one embodiment, the polypeptides can then be eluted
with a high concentration of the carbohydrate or a specific
cofactor. Mimics for binding sites can sometimes be used as
affinity stationary phases. The specific sugars, inhibitor or
cofactors used in the stationary phase will vary according to the
properties of the polypeptide. The embodiments of the invention
include any ligand, carbohydrate or cofactor known in the art.
[0306] In another embodiment, the immobilized stationary phase
includes a dye. Examples of dyes commonly used for dye-ligand
chromatography include Reactive Blue 2 (Cibacron.COPYRGT. Blue
3GA), Reactive Red 120 (Procion.COPYRGT. Red HE3B), Reactive Blue 4
(Reactive Blue MRB).sup.TC, Reactive Green 5 (Reactive Green
H4G).sup.TC, Reactive Green 19 (Reactive Green HE4BD).sup.TC, Green
19A (Reactive Green HE4BD).sup.TC, Reactive Yellow 86 (Reactive
Yellow M8G).sup.TC and Reactive Brown 10 (Reactive Brown
M4R).sup.TC.
[0307] In another embodiment, the stationary phase includes a metal
chelate resin. In metal chelate chromatography metal ions such as
Zn.sup.2+, Cu.sup.2+ and Ni.sup.2+ are immobilized to a
chromatography stationary phase by chelate bonding take part in a
reversible interaction with electron donor groups situated in the
surface of polypeptides. At a pH value at which the electron group
donor is present at least partially in non-protonized form the
polypeptide is bonded to the stationary phase and can be
subsequently eluted by means of a buffer with lower pH value at
which the electron group is protonized. Examples of chelate resins
include 8-hydroxyquinoline, salicylic acid, diethylenetriamine,
diethylenetriaminetetraacetic acid, ethylenediaminetetraacetic acid
(EDTA), iminodiacetic acid and nitrilo-triacetic acid.
[0308] In another embodiment, the isolation of polypeptides can
occur by immunoaffinity chromatography. The principle of
immunoaffinity or immunoadsorption chromatography is based on the
highly specific interaction of an antigen with its antibody.
Immunoaffinity chromatography utilizes an antibody or antibody
fragment as a ligand immobilized onto the stationary phase in a
manner that retains its binding capacity. Elution of the retained
polypeptide is achieved by alterations to the mobile-phase
conditions that weaken the antibody-antigen interaction. Elution
conditions are intended to break the ionic, hydrophobic and
hydrogen bonds that hold the antigen and antibody together.
Successful eluting conditions will be dependent upon the specific
antigen-antibody interaction that is occurring.
[0309] Antibodies may be generated that recognize the non-natural
amino acid present in the polypeptide. Such antibodies may be used
in affinity chromatography to purify the non-natural amino acid
polypeptides from a complex mixture or enable conjugation of the
polypeptide with other molecules on a support such as a resin, in
immunoassays to detect the presence of non-natural amino acid
polypeptides, and other assays that use antibodies. Antibodies may
be generated that recognize one or more non-natural amino acids
present at the N or C terminus of a polypeptide or other portions
of the polypeptide.
[0310] Non-natural amino acid polypeptides may be antibodies,
antibody fragments, or antigen-binding polypeptides or fragments
thereof, and used to isolate antigens by affinity
chromatography.
[0311] In one embodiment the isolation of polypeptides can occur by
hydrophobic-interaction chromatography. Polypeptides may contain
hydrophilic and hydrophobic natural amino acids and hydrophilic and
hydrophobic non-natural amino acids. Polypeptides are separated
according to their relative hydrophobicity by their ability to
reversibly bind to hydrophobic compounds. The polypeptides are
eluted from the column with decreasing concentrations of salt in
buffer. Examples of hydrophobic compounds include but are not
limited to, hydrophobic fatty acid chains, compounds with n-butyl
functional groups, compounds with n-octyl functional groups and
compounds with phenyl functional groups.
[0312] Supercritical Fluid Chromatography
[0313] In one embodiment the isolation of polypeptides can occur by
supercritical chromatography (SFC). In SFC, the sample is carried
through a separating column by a supercritical fluid where the
mixture is divided into unique bands based on the amount of
interaction between the individual analytes and the stationary
phase in the column. Conventional SFC columns are either packed and
open tubular or capillary. Open-tubular columns vary in length from
10 m to 20 m or more. Typically open-tubular columns have an inner
diameter of roughly of 0.05 to 4 mm. Pack columns vary in diameter
from 0.5 mm or less to 4.6 mm, with particle diameter ranging from
3 to 10 um. Packed columns contain small deactivated particles to
which the stationary phases adhere. The columns are conventionally
stainless steel. Capillary columns are open tubular columns of
narrow internal diameter made of fused silica, with the stationary
phase bonded to the wall of the column. The coatings are similar to
those used in partition chromatography. Examples of supercritical
fluids used in SFC include but are not limited to, carbon dioxide,
ethane, pentane, nitrous oxide, dichlorodifluoromethane, diethyl
ether, ammonia, and tetrahydrofuran. In some applications, polar
organic modifiers such as methanol are introduced in small
concentrations (1-5%).
[0314] B. Precipitation
[0315] In one embodiment the isolation of peptides, (modified)
non-natural amino acid polypeptides, binding partners or receptors
to polypeptides can occur by precipitation. The solubility of
polypeptides is a function of the ionic strength and pH of the
solution. Polypeptides have isoelectric points at which the charges
of their amino acid side groups balance each other. If the ionic
strength of a solution is either very high or very low, proteins
will tend to precipitate at their isoelectric point. In one
embodiment, the ionic strength of the solution will be increased by
adding salt. Examples of salts used in precipitation methods
include but are not limited to ammonium sulfate and sodium sulfate.
Any salt known in the art for protein precipitation can be used in
any of the embodiments of the inventions. In another embodiment,
polypeptides will be forced out of solution with polymers. One
example of a polymer commonly used to precipitate polypeptides is
polyethylene glycol. Any polymer known in the art for protein
precipitation can be used in any of the embodiments of the
inventions. In one embodiment the precipitated polypeptides are
removed by centrifugation or filtration.
[0316] In one embodiment, after precipitation of the peptide of
interest by the addition salts to the solution the sample will be
subject removal of high salt concentrations. Desalting methods are
discussed in the ion-exchange chromatography section.
[0317] Immunoprecipitation
[0318] In one embodiment of the invention the isolation of
polypeptides can occur by immunoprecipitation (IP). IP refers to
the small-scale affinity purification of antigen using a specific
antibody. Classical immunoprecipitation involves the following
steps: 1) incubate specific antibody with a sample containing
antigen, 2) capture antibody-antigen complex with immobilized
Protein A or G agarose gel (Protein A or G binds the antibody,
which is bound to its antigen), 3) Wash the gel with buffer to
remove non-bound sample components, 4) Elute the antigen (and
antibody).
[0319] In one embodiment of the invention, classical IP is
performed in a microcentrifuge tube with the polypeptide-containing
sample using immobilized Protein A or G gel. The gel is pelleted by
centrifugation after each step (washes and elution), and the
supernatant is removed. Usually the eluted sample will always
contain both antigen and antibody, and reducing gel electrophoresis
of the eluted sample will yield both antigen bands and heavy and
light chain antibody fragment bands. Methods to obtain polypeptides
from electrophoresis gel separated are known to those of ordinary
skill in the art.
[0320] In another embodiment of the invention, to avoid antibody
contamination of the eluted antigen, modifications to the classical
IP method can be made so that the antibody is permanently
immobilized and will not elute with the antigen. In one example,
the antibody is first bound to the Protein A or G gel and then the
antibody is covalently cross-linked to the Protein A or G. In
another example the antibody is directly coupled to an activated
affinity support. Non-natural amino acid polypeptides may be
antigen-binding polypeptides and used in immunoprecipitation.
[0321] In one embodiment the support material is a porous gel such
as cross-linked beaded agarose or co-polymer of cross-linked
bis-acrylamide and azlactone. In one embodiment of the invention
polypeptides can be isolated by magnetic affinity separation.
Samples containing the molecule of interest are incubated with
magnetic beads that are derivatized with an antibody or other
binding partner. A magnetic field is used to pull the magnetic
beads out of solution and onto a surface. The buffer can be
carefully removed, containing any nonbound molecules. Protocols
using magnetic beads for isolation of molecules of interest are
well known in the art. Magnetic beads can be derivatized to contain
active groups, including but not limited to, carboxylic acids or
primary amines, or specific affinity molecules such as streptavidin
or goat anti-mouse, anti-rabbit or anti-rat IgG or Protein A or G.
In another embodiment the support is a microplate.
[0322] C. Electrophoresis
[0323] In any of the embodiments herein, isolation of polypeptides
can occur by electrophoresis. Electrophoresis is the separation of
ionic molecules such as polypeptides by differential migration
patterns through a gel based on the size and ionic charge of the
molecules in an electric field. Electrophoresis can be conducted in
a gel, capillary or on a chip. Examples of gels used for
electrophoresis include starch, acrylamide, agarose or combinations
thereof. A gel can be modified by its cross-linking, addition of
detergents, immobilization of enzymes or antibodies (affinity
electrophoresis) or substrates (zymography) and pH gradient.
Methods to obtain polypeptides from electrophoresis gels are known
to those of ordinary skill in the art.
Capillary Electrophoresis
[0324] In one embodiment the isolation of peptides, (modified)
non-natural amino acid polypeptides, binding partners or receptors
to polypeptides can occur by capillary electrophoresis (CE). CE may
be used for separating complex hydrophilic molecules and highly
charged solutes. Advantages of CE include its use of small samples
(sizes ranging from 0.001 to 10 .mu.L), fast separation, easy
reproducibility, very high efficiencies, meaning hundreds of
components can be separated at the same time, is easily automated,
can be used quantitatively and consumes limited amounts of
reagents. CE technology, in general, relates to separation
techniques that use narrow bore fused-silica capillaries to
separate a complex array of large and small molecules. High
voltages are used to separate molecules based on differences in
charge, size and hydrophobicity. Depending on the types of
capillary and buffers used, CE can be further segmented into
separation techniques such as capillary zone electrophoresis (CZE),
capillary isoelectric focusing (CIEF) and capillary
electrochromatography (CEC).
[0325] Capillary zone electrophoresis (CZE), also known as
free-solution CE (FSCE), is the simplest form of CE. The separation
mechanism of CZE is based on differences in the charge-to-mass
ratio of the analytes. Fundamental to CZE are homogeneity of the
buffer solution and constant field strength throughout the length
of the capillary. The separation relies principally on the
pH-controlled dissociation of acidic groups on the solute or the
protonation of basic functions on the solute.
[0326] Capillary isoelectric focusing (CIEF) allows amphoteric
molecules, such as polypeptides, to be separated by electrophoresis
in a pH gradient generated between the cathode and anode. A solute
will migrate to a point where its net charge is zero. At this
isoelectric point (the solute's pI), migration stops and the sample
is focused into a tight zone. In CIEF, once a solute has focused at
its pI, the zone is mobilized past the detector by either pressure
or chemical means.
[0327] CEC is a hybrid technique between traditional liquid
chromatography (HPLC) and CE. In essence, CE capillaries are packed
with HPLC packing and a voltage is applied across the packed
capillary, which generates an electro-osmotic flow (EOF). The EOF
transports solutes along the capillary towards a detector. Both
differential partitioning and electrophoretic migration of the
solutes occurs during their transportation towards the detector,
which leads to CEC separations. It is therefore possible to obtain
unique separation selectivities using CEC compared to both HPLC and
CE. The beneficial flow profile of EOF reduces flow related band
broadening and separation efficiencies of several hundred thousand
plates per meter are often obtained in CEC. CEC also makes it is
possible to use small-diameter packings and achieve very high
efficiencies.
[0328] Micellar electrokinetic capillary chromatography (MECC) is a
capillary electropheretic method that allows the separation of
uncharged solutes. In this technique, surfactants, such as sodium
dodecyl sulfate, are added to the operating buffer in amounts that
exceed the critical micelle concentration at which micelles form.
The surface of anionic micelles of this type has a large negative
charge, which give them a large electrophoretic mobility toward the
positive electrode. Most buffers, however, exhibit such a high
electroosmotic rate toward the negative electrode that the anionic
micelles are carried toward the negative electrode, but at a much
reduced rate. This form a fast moving aqueous phase and a slower
moving micellar phase. When the sample is introduced into the
system, the components distribute themselves between the aqueous
phase and the hydrocarbon phase at the interior of the
micelles.
[0329] Alternatively, isotachophoresis (ITP) is a method of
concentrating samples by electrophoretic separation using a
discontinuous buffer. In isotachophoresis, two different buffer
systems are used to create zones which the analytes separate into.
During an isotachophoresis experiment it is possible to separate
either cations or anions, not both. In ITP, a large volume of
sample is placed between a leading electrolyte and a terminating
electrolyte. Analytes in the sample stack into narrow bands one
after another according to their mobility. The technique can be
used in conjunction with capillary electrophoresis where a
discontinuous electrolyte system is employed at the site of sample
injection into the capillary.
[0330] Moreover, transient isotachophoresis (tITP) is a variation
of this technique commonly used in conjunction with capillary
electrophoresis (CE). Foret, F., et al. in "Trace Analysis of
Proteins by Capillary Zone Electrophoresis with On-Column Transient
Isotachophoretic Preconcentration". Electrophoresis 1993, 14,
417-428 (1993) describe two electrolyte arrangements for performing
tITP.
[0331] One configuration employs two reservoirs connected by a
capillary. The capillary and one reservoir are filled with a
leading electrolyte (LE), while the second reservoir is filled with
terminating electrolyte (TE). The sample for analysis is first
injected into the capillary filled with LE and the injection end of
the capillary is inserted into the reservoir containing TE. Voltage
is applied and those components of the sample which have mobilities
intermediate to those of the LE and TE stack into sharp ITP zones
and achieve a steady state concentration. The concentration of such
zones is related to the concentration of the LE co-ion but not to
the concentration of the TE. Once a steady state is reached, the
reservoir containing TE is replaced with an LE containing
reservoir. This causes a destacking of the sharp ITP zones, which
allows individual species to move in a zone electrophoretic
mode.
[0332] The other configuration discussed by Foret, F., et al.
employs a similar approach but uses a single background electrolyte
(BGE) in each reservoir. The mobility of the BGE co-ion is low such
that it can serve as the terminating ion. The sample for analysis
contains additional co-ions with high electrophoretic mobility such
that it can serve as the leading zone during tITP migration. After
sample is injected into the capillary and voltage is applied, the
leading ions of higher mobility in the sample form an asymmetric
leading and sharp rear boundary. Just behind the rear boundary, a
conductivity discontinuity forms, and this results in a non-uniform
electric field, and thus stacking of the sample ions. As migration
progresses, the leading zone will broaden due to electromigration
dispersion and the concentration of higher mobility salt will
decrease. The result is decreasing differences of the electric
field along the migrating zones. At a certain concentration of the
leading zone, the sample bands will destack and move with
independent velocities in a zone electrophoretic mode. Isolation of
peptides can involve any procedure known in the art, such as
capillary electrophoresis (e.g., in capillary or on-chip), or
chromatography (e.g., in capillary, column or on a chip).
[0333] D. Procedures for Removal of Contaminants
[0334] In some embodiments of the invention following the primary
purification procedure to obtain a polypeptide of interest,
secondary purification steps to remove contaminants may be
required. The contaminants can be inhibitors, interfering
substances or inappropriate buffers. In one embodiment of the
invention removal of contaminants will be achieved by specifically
purifying their protein of interest away from a complex mixture of
biological molecules. In another embodiment of the invention the
removal of contaminants will be achieved by specifically removing
contaminants from a sample containing a protein of interest. For
example, immobilized Protein A can be used to selectively remove
immunoglobulins from a sample where they are considered to be a
contaminant. In yet another embodiment filters can be used to
remove undesired components from a sample. Examples include but are
not limited to size exclusion chromatography and ultrafiltration
membranes that separate molecules on the basis of size and
molecular weight. In yet another embodiment, ultracentrifugation is
used for removing undesired components from a sample.
Ultracentrifugation can involve centrifugation of a sample while
monitoring with an optical system the sedimentation (or lack
thereof) of particles. In another embodiment of the invention,
electrodialysis is used to remove undesired components from the
sample. Electrodialysis is an electromembrane process in which ions
are transported through ion permeable membranes from one solution
to another under the influence of a potential gradient. Since the
membranes used in electrodialysis have the ability to selectively
transportions having positive or negative charge and reject ions of
the opposite charge, electrodialysis is useful for concentration,
removal, or separation of electrolytes.
[0335] Removal of Endotoxin
[0336] In some embodiments of the invention it may be necessary to
remove endotoxins from the sample. Endotoxins are pyrogenic
lipopolysaccharide (LPS) components of Gram-negative bacteria.
Because these bacteria are ubiquitous, it is not surprising that
endotoxins are frequent contaminants of biochemical preparations.
Endotoxin contamination usually is measured as endotoxin units
(EU), where 1 EU corresponds to a concentration of endotoxin
(usually about 0.1 ng/kg body weight) sufficient to generate a
pyrogenic reaction. In one embodiment removal of endotoxin is
performed by ultracentrifugation. In another embodiment removal of
endotoxin is performed by using immobilized polymixin B. Methods
for reducing endotoxin levels are known to one of ordinary skill in
the art and include, but are not limited to, purification
techniques using silica supports, glass powder or hydroxyapatite,
reverse-phase, affinity, size-exclusion, anion-exchange
chromatography, hydrophobic interaction chromatography, a
combination of these methods, and the like. Methods for measuring
endotoxin levels are known to one of ordinary skill in the art and
include, but are not limited to, Limulus Amebocyte Lysate (LAL)
assays.
[0337] Removal of Detergent
[0338] In some embodiments of the invention it may be necessary to
remove some or all of the detergent in the sample. For example,
although many water-soluble polypeptides are functional in
detergent-solubilized form, other polypeptides may be modified and
inactivated by detergent solubilization. In one embodiment
detergent removal can occur by dialysis. Dialysis is effective for
removal of detergents that have high CMCs (critical micelle
concentrations) and/or small aggregation numbers, such as the
N-octyl glucosides. In another embodiment removal of detergent from
the sample can occur by sucrose density gradient separation. In yet
another embodiment, detergents can be removed from the sample by
size exclusion chromatography.
[0339] E. Recombinant polypeptides
[0340] In one embodiment of the invention isolation of polypeptides
may use genetic engineering techniques to synthesize of hybrid
proteins. By fusing the coding sequence of a polypeptide of
interest with the coding sequence of a polypeptide with high
affinity to a ligand, a hybrid protein with an affinity tag can be
produced directly by a microorganism. Examples of expression
systems are Escherichia coli, Bacillus subtilis, Pseudomonas
fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, yeast,
mammalian cells and the baculovirus system in insect cells. The
affinity tag can then be used to recover the product from a culture
medium, cell lysate, estract, inclusion bodies, periplasmic space
of the host cells, cytoplasm of the host cells, or other material
by affinity chromatography.
[0341] In one embodiment of the invention non-natural amino acid
polypeptides which are secreted into the medium can be obtained by
centrifugation or filtration. These solutions may be suitable for
direct application to chromatography columns. In another embodiment
of the invention polypeptides which are accumulated intracellularly
are extracted prior to purification by chromatography. In one
embodiment polypeptides are extracted by cell disruption. Examples
of cell disruption techniques include mechanical desintegrators,
such as glass bead mills and high-pressure homoganizers. In another
embodiment of the invention polypeptides are extracted by cell
permeabilization. Examples of permeabilization agents include but
are not limited to guanidine hydrochloride and Triton X-100. In
addition to chemical permeabilization cells can be permeabilized by
enzymatic lysis. The clarification of the cell homogenate or crude
extract obtained after cell permeabilization can be done by
centrifugation or by different filtration methods, such as
microfiltration or ultrafiltration.
[0342] Purification tags have been developed to be applied in ion
exchange, hydrophobic interaction, affinity, immunoaffinity, and
metal-chelate chromatography. For example, hybrid polypeptides with
a polyarginine tag can be purified by ion exchange chromatography,
hybrid peptides with a polyphenylalanine tag can be isolated by
hydrophobic chromatography, hybrid peptides with a
.beta.-Galactosidase tag can be isolated by affinity
chromatography, hybrid peptides with a protein A tag can be
isolated by IgG-affinity chromatography, hybrid peptides with an
antigenic tag can be isolated by immunoaffinity chromatography and
hybrid peptides with a polyhistidine can be isolated by metal
chelate chromatography. Tags may be removed by chemical or
enzymatic means. In some embodiments, the tag is removed via an
intramolecular reaction. A linker molecule may or may not be
released.
[0343] Similarly, non-natural amino acids may be used to generate
purification tags and hybrid polypeptides with these tags can be
purified using chromatography or other techniques. In one
embodiment, multiple non-natural amino acids are included at a
terminus of the polypeptide. Purification of this polypeptide with
multiple non-natural amino acids may be purified by affinity
chromatography or by other means depending on the properties of the
non-natural amino acids.
[0344] To conjugate polypeptides with multiple non-natural amino
acid tags with another molecule, the following procedure may be
performed. After the binding of the polypeptide to a resin that
binds to the non-natural amino acid tag, a reaction is performed to
conjugate the polypeptide to another molecule such as PEG. The
conjugated product may be released from the resin as a result of
the conjugation or after the conjugation is complete. The
conjugation may be performed under denaturing conditions and
refolding of the polypeptide may be performed on the resin. The
second molecule may be conjugated to the polypeptide at a natural
or non-natural amino acid present in the polypeptide. The second
molecule may be conjugated to the polypeptide at a natural or
non-natural amino acid present in the non-natural amino acid
tag.
[0345] In another embodiment, the multiple non-natural amino acids
included at a terminus of the polypeptide are metal-binding amino
acids. Purification of this polypeptide may be performed using
methods similar to those used for His-tagged proteins. In another
embodiment, the polypeptide comprises two or more non-natural amino
acids in that one or more non-natural amino acid is used to bind
the polypeptide to a resin and the second non-natural amino acid is
used to conjugate the polypeptide to another molecule, including
but not limited to, PEG. Other materials useful in purification
techniques may be used instead of resins. Tags may be removed by
chemical or enzymatic means. In some embodiments, the tag is
removed via an intramolecular reaction. A linker may or may not be
released.
[0346] In another embodiment, a hybrid polypeptide may have a
non-natural amino acid at the junction of the polypeptide and the
tag. This non-natural amino acid may be used to separate the
polypeptide from the tag by chemical cleavage, for example during
or after the binding of the tag to a column. This non-natural amino
acid may be used to separate the polypeptide from the tag by
enzymatic cleavage or by an intramolecular chemical reaction.
[0347] In another embodiment, a "prodrug" type approach is used. A
non-natural amino acid polypeptide is bound to a purification
matrix, and a portion or all of the polypeptide is released after
an event, including but not to, an intramolecular reaction,
exposure to UV light (light activated molecule for release),
chemical cleavage, or enzymatic cleavage.
[0348] In another embodiment a specific cleavage site at the
junction between parts of a polypeptide could be introduced. This
enables, for example, cleavage of the hybrid molecule to yield the
protein of interest free of an affinity tag. Removal of a fusion
sequence may be accomplished by enzymatic or chemical cleavage. To
split off the affinity tag from the polypeptide of interest, a
specific chemical or enzymatic cleavage site may be engineered into
the fusion proteins. Enzymatic removal of fusion sequences may be
accomplished using methods known to those of ordinary skill in the
art. The choice of enzyme for removal of the fusion sequence will
be determined by the identity of the fusion, and the reaction
conditions will be specified by the choice of enzyme as will be
apparent to one of ordinary skill in the art. Chemical cleavage may
be accomplished using reagents known to those of ordinary skill in
the art, including but not limited to, cyanogen bromide, TEV
protease, and other reagents. Examples of cleavage reagents include
but are not limited to, formic acid, hydroxylamine, collagenase,
factor Xa, enterokinase, renin, carboxypeptidase A and
carboxypeptidase B. The cleaved hGH polypeptide may be purified
from the cleaved fusion sequence and cleavage reagents by methods
known to those of ordinary skill in the art. Such methods will be
determined by the identity and properties of the fusion sequence
and the polypeptide, as will be apparent to one of ordinary skill
in the art. Methods for purification may include, but are not
limited to, size-exclusion chromatography, hydrophobic interaction
chromatography, ion-exchange chromatography or dialysis or any
combination thereof.
[0349] With an increasing number of protein and peptide
therapeutics in development, there is a demand for an efficient,
economic, and large-scale protein purification method that is not
costly and difficult to scale up. Resins or other materials known
to those skilled in the art may be used to isolate polypeptides.
FIG. 10 shows an example of a purification method for a non-natural
amino acid polypeptide utilizing a resin that reacts with the
non-natural amino acid. A covalent linkage is formed between a
chemically specific affinity tag on the resin and a non-natural
amino acid present in the protein. Such linkages are stable under a
broad range of pH and purification conditions. The separation step
may be performed in alternate modes, including but not limited to a
bath mode, enabling the large-scale purifications. The resin and
the affinity tags are physically and chemically stable, and thus,
can be reused to reduce the cost of protein purification upon
scale-up. The separation can be performed in conjunction with
conjugation of the polypeptide to molecules including but not
limited to, PEG. This "one-pot" method further simplifies the
conjugation process and reduces the cost of production of proteins,
including but not limited to target therapeutic proteins (FIG. 11).
Resins can be selected and functionalized according to the
non-natural amino acid present in the polypeptide. FIG. 12 shows an
example of resin selection and functionalization. Resins or other
matrixes for purification can be functionalized with different
functional groups depending on the non-natural amino acid in the
polypeptide. For example, FIG. 13 shows an example of affinity
purification of a non-natural amino acid polypeptide using
hydroxylamine resin. FIG. 14 shows an example of purification of a
non-natural amino acid polypeptide using an aldehyde resin. The
ability to regenerate the matrix used in purification methods also
provides advantages for large-scale production.
[0350] In some embodiments, the purification process changes one or
more non-natural amino acids present in the polypeptide to one or
more natural amino acids. FIG. 15 shows an example of purification
of native proteins from a non-natural amino acid precursor. The
non-natural amino acid is converted to tyrosine after release from
the resin used in the purification process. FIG. 16 shows
non-limiting examples of non-natural amino acids.
[0351] Non-natural amino acids present in a set of two or more
proteins may be used to purify complexes of polypeptides. The
non-natural amino acids may be bonded to each other or joined via a
linker, a polymer, or another molecule to enable purification of a
complex of polypeptides. Polypeptides that may be isolated in this
fashion include but are not limited to multiple subunit receptors
or enzymes. Techniques used to isolate complexes may utilize one or
more additional non-natural amino acids present in one or more of
the polypeptides. Techniques for isolating large proteins are known
to one of ordinary skill in the art. Dissociation of the
polypeptide complex may be performed using one or more non-natural
amino acids present in one or more of the polypeptides. One or more
of the non-natural amino acids may be reacted with another molecule
with a functional group that causes separation of the polypeptides
in the complex.
[0352] In some embodiments, the polypeptides may form a complex due
to non-covalent interactions that involve one or more non-natural
amino acids present in the polypeptide.
[0353] In some embodiments, electro/chemical interaction such as
electrical or magnetic fields may be used to purify polypeptides
due to one or more non-natural amino acids present in the
polypeptide. In other embodiments, single cell purification or
isolation may be achieved using non-natural amino acid
polypeptide.
[0354] XII. Library Screening
[0355] 1. High Throughput Screening
[0356] The technological approaches for the screening process of
the non-natural amino acids, non-natural amino acid polypeptides,
modified non-natural amino-acid polypeptides and fragments thereof
disclosed herein, include, but not limited to, multiwell-plate
based screening systems, cell-based screening systems,
microfluidics-based screening systems, and screening of soluble
targets against solid-phase synthesized drug components.
[0357] Automated multiwell formats are developed high-throughput
screening systems. Automated 96-well plate-based screening systems
are widely used. The plate based screening systems can be made to
reduce the volume of the reaction wells further, thereby increasing
the density of the wells per plate. Other types of high-throughput
assays, such as miniaturized cell-based assays can also be used in
the present invention. Miniaturized cell-based assays have the
potential to generate screening data of quality and accuracy, due
to their in vivo nature. Microfluidics-based screening systems that
measure in vitro reactions in solution make use of ten to
several-hundred micrometer wide channels. Micropumps,
electroosmotic flow, integrated valves and mixing devices control
liquid movement through the channel network.
[0358] Libraries for screening can be grouped as, by way of example
only, General Screening or Template-Based such as Groups with
common heterocyclic lattices; Targeted such as Mechanism based
selections, for example, Kinase Modulators, GPCR Ligands,
Anti-infectives, Potassium Channel Modulators, and Protease
Inhibitors; Privileged Structure such as Compounds containing
chemical motifs that are more frequently associated with higher
biological activity than other structures; Diversity such as
Compounds pre-selected from available stock with maximum chemical
diversity; Plant Extracts; Natural Products/Natural
Product-Derived, etc.
[0359] A. Chemical Libraries
[0360] Combinatorial chemical libraries are a means to assist in
the generation of new chemical compound leads. A combinatorial
chemical library is a collection of diverse chemical compounds
generated by either chemical synthesis or biological synthesis by
combining a number of chemical "building blocks" such as reagents.
Millions of chemical compounds can be synthesized through such
combinatorial mixing of chemical building blocks. LogP, molecular
weight, number of H-bond donors and acceptors, as set forth in the
Lipinski "rule of five" requirements, help to determine strong
candidates for drug-like characteristics. Lipinski "rule of five"
requires the compound to have these properties: five or fewer
hydrogen bond donors, molecular weight less than or equal to 500
Da, calculated LogP less than or equal to 5), and ten or fewer
hydrogen bonding acceptors. High throughput screening technologies
coupled with compound libraries obtained through combinatorial
chemistry and/or high throughput synthesis methods can be utilized
to rapidly identify and optimize ligands for non-natural amino
acids, non-natural amino acid polypeptides, modified non-natural
amino acid polypeptides and fragments thereof, as disclosed
herein.
[0361] Chemical diversity libraries of organic compounds include,
but are not limited to: benzodiazepines, diversomers such as
hydantoins, benzodiazepines and, analogous organic syntheses of
small compound libraries, oligomeric libraries such as peptide,
N-alkyl glycine, polycarbamate and polyureas, oligocarbamates,
and/or peptidyl phosphonates, carbohydrate libraries, chiral
compound libraries, and small organic molecule libraries. A wide
variety of heterocyclic compound libraries have been synthesized by
solid phase methods. These include, by way of example only,
benzodiazepins, pyrrolidines, hydantoins, 1,4-dihydropyridines,
isoquinolinones, diketopiperazines, benzylpiperazines, quinolones,
dihydro- and tetrahydroisoquinolines, 4-thiazolidinones, b-lactams,
benzisothiazolones, pyrroles and imidazoles.
[0362] Combinatorial libraries of inorganic compounds include, but
not limited to, (a) Oxides of metals and main group elements,
including transition metal oxides such as zirconia, titania,
manganese oxide, rare earth oxides such as ceria and lanthanum
oxide; binary, ternary, and more complex solid state oxides and
ceramic phases; various forms of alumina, silica, aluminosilicates
and aluminophosphates; (b) Natural and synthetic forms of
aluminosilicate and silicate zeolites such as ZSM-5, Beta, zeolite
Y, and ferrierite, various forms of molecular sieves such as
aluminophosphates and titanosilicates; natural or synthetic clays
and related minerals such as kaolin, attapulgite, talc,
montmorillonite, and Laponite.RTM.; (c) Non-oxide ceramics such as
metal carbides and nitrides; (d) Various forms of carbons such as
activated carbon, carbon molecular sieves, graphite, fullerenes,
carbon nanotubes, and carbon black; (e) Various organic polymers,
oligomers, or resins, such as polyethylene, polypropylene,
polystyrene, polyamides, halo hydrocarbon polymers, polyesters,
etc.; (f) Metals such as precious metals and/or transition metals
deposited, mixed with, or exchanged into any support such as any of
the materials described in (a)-(e) above. Examples of such phases
include Pt/alumina, Pd/alumina, and Cu-ZSM-5.
[0363] B. Biological Libraries
[0364] Peptide library by using microorganisms--Antibodies and
immune cell receptors of the immune system are representative
biological libraries. In the immune system, all the processes of
library design, synthesis, and optimization are controlled by the
organism itself. Only structures of antigens and genetic
information to form embryonic factors are external conditions, but
the rest is controlled spontaneously by internal factors. Because
the immune system uses protein structure libraries, they are
libraries using amino acids as basic factors. Because peptides or
proteins made of amino acids are the first products of synthesis by
translating genetic information, through genetic engineering
technologies, proteins of desired sequences can be easily obtained
by inserting modified genetic information into microorganisms like
bacteria or virus. Microorganism library synthesis brings several
advantages. It is possible to clone microorganisms to make only one
kind of proteins per microorganism, and even though only one cell
is acquired, the number of clones can be easily increased by cell
multiplication. The other advantage of using microorganisms is that
they can self-propagate whenever there is enough supply. After
synthesizing a DNA strand that makes the desired protein sequence,
its complementary strand is synthesized, by enzymes if needed. For
synthesized DNA to replicate and translate properly in
microorganisms, it needs to be packed with vector and inserted into
microorganisms. Proteins expressed on the surface of the
microorganism, and to find desired proteins is the next step.
[0365] To make library various genetic information is needed.
Random DNA synthesis or cutting cDNA or the whole genomic DNA of a
particular organism can be used. A portion of DNA sequence that
makes particular protein can be modified to make mutated protein
library. Considering volume limitations and expression rates of
microorganism incubation, 109 (one billion) kinds of libraries can
be made. Compared to 106 to 107 kinds of synthesis libraries, it is
a huge number. The number of 5-unit peptides is 205 (3.2 millions),
that of 6-unit ones is 64 millions, and for 7-unit peptides the
number passes one billion. Therefore, if more than 7 amino acids
are changed incomplete library that does not contain all the
possible combinations is made. For long proteins, 7 different amino
acids can be selected separately and replaced. When DNA is randomly
synthesized, DNA codes can be repeated and designate the same amino
acid, and generation frequency changes. Therefore, to make all the
possible combinations, much more quantities of clones are
required.
[0366] A linear combinatorial biological library such as a
polypeptide library is formed by combining a set of chemical
building blocks called amino acids in every possible way for a
given compound length (i.e., the number of amino acids in a
polypeptide compound). The proteins may be members of a protein
family such as a receptor family (examples: growth factor
receptors, catecholamine receptors, amino acid derivative
receptors, cytokine receptors, lectins), ligand family (examples:
cytokines, serpins), enzyme family (examples: proteases, kinases,
phosphatases, ras-like GTPases, hydrolases), transcription factors
(examples: steroid hormone receptors, heat-shock transcription
factors, zinc-finger, leucine-zipper, homeodomain), HIV proteases
or hepatitis C virus (HCV) proteases, and antibody or antibody
fragment (Fab, for example). Other examples are, such as, peptoids,
encoded peptides, random biooligomers, dipeptides, vinylogous
polypeptides, nonpeptidal peptidomimetics with Beta D Glucose
scaffolding, antibody libraries, and peptide nucleic acid
libraries.
[0367] Bacteriophage library--It is one of a number of protein
library methods. Bacteriophage is living in a host bacterium and a
kind of virus with genetic materials and capsids. M13 and Lambda
viruses are the most famous.
[0368] A M13 is a thin, long virus and due to its small genome
size, numerous libraries can be made easily. Different from other
viruses, it can come out to outside of host cells without damaging
them or inhibiting their growth. It is known that M13 amplifies its
genetic information in the host cell and wears the capsid when
emerging. It makes 10 kinds of proteins and pVIII and pIII capsids
are commonly used in library synthesis among them. A pVIII protein
surrounds the whole body and has about 50 amino acids. Usually 2700
per a virus are expressed. Because its amino end protrudes toward
outside of the capsid, it can be modified to express a different
peptide on it. Usually a long peptide cannot be expressed, but it
is possible for 6-unit peptides. Because large amount of the same
library molecules are expressed at the same time, in spite of its
relatively short size, it is appropriate for a reaction with
various ligands. A pIII protein is expressed at the end of a virus,
and usually 3 to 5 proteins of 406 amino acids are expressed. It
can express quite large proteins so that it is used for the whole
protein or antibody molecule libraries. A normal antibody uses Fab,
an antigen recognition region, or a Fvs chain. Bacteriophage
Library and hybridoma are the most famous methods to make
antibodies. M13 is ideal to make random peptide libraries and the
virus is stable enough to be precipitated and concentrated so that
screening 10.sup.9 libraries in a volume of 1-10 .mu.L is
possible.
[0369] Different from the M13, a Lambda virus coats itself with a
capsid in the cytoplasm and comes out of its host cell when there
is an enough number, instead of wearing a capsid when emerging. In
other words, if a different protein is expressed, it will probably
emerge in a folded shape with proper functions. A pV and D proteins
are commonly used for the library synthesis. As proteins that can
be expressed on a bacteriophage surface, there are random peptide,
natural protein fragments, mutated particular protein libraries,
and partial antibody fragments and they are used for chromatography
materials, protein-protein mutual reactions, receptor binding site
searching, and drug discoveries.
[0370] Phage display is a widely utilized technique to make peptide
libraries. These peptide libraries are useful for screening to
identify peptides that have a particular desired activity, such as
binding to another polypeptide or other molecule. In phage display
the peptide library is fused to a bacteriophage protein, typically
a coat protein, that is displayed on the surface of the phage. The
library of peptide bearing phage is contacted with an immobilized
binding partner, such as a cell surface or a purified protein, and
specific binders are then isolated. Phage display techniques and
libraries are described in U.S. Pat. Nos. 5,580,717, 5,702,892,
5,750,344, 5,821,047, 5,962,255, 6,140,471, 6,475,806, 5,427,908,
5,667,988, 5,733,743, 5,750,373, 5,824,520, 6,096,551, 6,225,447,
6,492,160, which are incorporated in their entirety by reference
herein. U.S. Pat. No. 5,750,373, which is incorporated by reference
herein, describes a method for selecting novel proteins such as
growth hormone and antibody fragment variants having altered
binding properties for their respective receptor molecules. The
method comprises fusing a gene encoding a protein of interest to
the carboxy terminal domain of the gene III coat protein of the
filamentous phage M13.
[0371] Bacteria and yeast libraries--Not only viruses with capsids,
but also bacteria with cell walls and membranes can be used for
library expression as well. Both the gram-positive bacteria and
gram-negative bacteria can be used to express proteins on cell
surfaces, and E. coli, a gram-negative bacterium, is commonly used.
Bacteria library can find an antigen that strongly binds to a
certain antibody and use it as a vaccine, or it can express
diagnostic antibodies or receptor libraries for analysis of
particular materials.
[0372] It is called translational modification that the higher
animal's protein is modified by phosphorylation or sugar addition
after the protein synthesis. But a bacterium, a prokaryote, does
not have such a function, and when even a protein is synthesized,
it either precipitates due to its bad solubility or is inactivated
in most cases. Therefore, S. cerevisiae, a eukaryote, is used. Even
though S. cerevisiae is unicellular like bacteria, it has
translational modification function and very similar proteins to
the original can be made.
[0373] Different from viruses, it has a micron size cell so that
FACS (fluorescence-activated cell sorting) can be used.
Fluorescence labeled target molecules are added to the library of
proteins expressed on a cell surface and flow through thin tubes of
FACS machine. FACS sorts each cells by fluorescent colors and
intensities as alive. It is possible to screen different target
molecules with different colors and also possible to sort cells of
different intensities and selectivity. Another advantage is a
liquid-phase screening. It is not necessary to separate strongly
clung molecules. Sorted cells multiply again and they are
re-screened.
[0374] Yeast surface display techniques are also widely utilized to
product and display peptide libraries. Yeast surface display may be
utilized in combination with fluorescence activated cell sorting to
select cells displaying the desired peptides. Yeast surface display
techniques and libraries are described in U.S. Pat. Nos. 6,083,693,
6,406,863, 6,410,271, 6,232,074, 6,410,246, 6,610,472, which are
incorporated in their entirety by reference herein.
[0375] Bacterial surface display has been used in a variety of
forms to display peptides on the cell surface or in the periplasm.
A variety of bacterial hosts are available for use in this system,
as are a variety of polypeptide anchoring domains to anchor the
displayed peptide to the cell surface. Bacterial surface display
techniques and libraries are described in U.S. Pat. Nos. 5,348,867,
5,866,344, 6,277,588, 5,635,182, 6,180,341, which are incorporated
in their entirety by reference herein.
[0376] Other in vivo systems are utilized to make libraries of
polypeptides and identify changes in activities, such as target
protein binding modulation, resulting from changes in amino acid
sequences. Examples of in vivo systems include, but are not limited
to, the yeast two hybrid system (Schneider, S et al., Nat.
Biotechnol., 17, 170-175 (1990)), and the dihydrofolate reductase
protein-fragment complementation assay (Pellitier, N. J. et al.,
Nat. Biotechnol., 17, 683-690, (1990)), which are hereby
incorporated by reference herein.
[0377] Bio-panning--A synthesized microorganism library may be used
to find a peptide that binds to a particular molecule with high
affinity.
[0378] Target molecules, such as non-natural amino acids,
non-natural amino acid polypeptides, modified non-natural amino
acid polypeptides and fragments thereof as disclosed herein, may be
evenly placed on a test plate. The prepared microorganism library
may be added to the plate. Only the microorganisms that strongly
bind to the target molecules will remain and the rest will be in
the solution. After a while, unbound microorganisms may be
discarded, and then weakly or accidentally bound microorganisms may
be washed with appropriate solutions. The target molecule's binding
affinity determines the washing process. Still remaining
microorganisms can be taken apart by addition of low pH or high
concentrated target molecules, and the quantity is amplified by
re-incubation. Sometimes it may be difficult to separate them
without killing bacteria when the affinity is too strong. If it is
a bacteriophage, instead of separation, one can infect its host
cell directly. Because there still can be some undesired
microorganisms bound accidentally, the first amplified
microorganisms may go through repeated screening and amplification
processes to increase the number of clones containing active
proteins. Finally after they are incubated in low concentration,
each clone may be separated and usually tens of clones may be
selected and used for DNA sequence analysis. It is successful if
peptide structures from DNA information are recognizable and most
of clones show accord peptide sequences. However, because proteins
can have toxicity up to kinds of clones and DNA expression rate can
vary, there may be a possibility that faster multiplying and
well-expressed clones are selected than desired screening results.
Therefore, a confirmation step is necessary by measurement of
peptide synthesis and binding affinity.
[0379] The microorganism protein library technology fundamentally
uses a living organism's self-reproduction ability. That is, by
amplifying (feeding) a small quantity of obtained candidate
molecules, one can increase purity and quantity.
[0380] Ribosome display--Ribosome display and mRNA display
techniques are also widely utilized to make peptide libraries.
Ribosome display and mRNA display are in vitro techniques that
couple the mRNA encoding a peptide to the encoded peptide either on
the ribosome or by using puromycin. Ribosome display and mRNA
display techniques and libraries are described in U.S. Pat. Nos.
6,416,950, 6,436,665, 6,602,685, 6,660,473, 6,429,300, 6,489,116,
6,623,926, 6,589,741, 6,348,315, 6,207,446, 6,258,558, 6,416,950,
6,440,695, 6,228,994, 6,281,344, 6,429,300, 6,660,473, 5,580,717,
5,688,670, 6,238,865, 6,261,804, 6,518,018, 6,281,344, 6,258,558,
6,214,553, which are incorporated in their entirety by reference
herein.
[0381] DNA, RNA library--Development of PCR, DNA amplification
technology, has enabled using nucleic acids as libraries. Because
DNA and RNA are made of 4 units, 10 oligomers have 410 (about
10.sup.6=a million) kinds and 20 oligomer library can have about
1012. By using automated solid-phase DNA synthesizer, 5' end and 3'
end are fixed in a sequence and A, T, C, and G are randomly placed
as each take about 25% of the sequence. When one strand is made, it
may be replicated by using enzymes or amplified by PCR. Commonly
about 1014-15 molecules are made and used, but occasionally there
are about 40 places (1024 kinds) for random introduction, sometimes
they start with incomplete set of library. For DNA library, DNA
themselves are simply used, but for RNA library, T7 RNA Polymerase
is needed to transcript.
[0382] Prepared libraries are sorted by target molecule binding
screening; amplified by PCR for DNA and by RT-PCR for RNA.
Non-natural aminoacids, non-natural amino acid polypeptides,
modified non-natural amino acid polypeptides and fragments thereof
as disclosed herein, can be used as target molecules. Screening and
amplification of the amplified library is repeated until the
beginning number of 10.sup.14-15 is narrowed to several hundreds,
and then sequences of acquired candidate molecules are analyzed and
each binding affinity is measured. Such acquired DNA and RNA are
called aptamers, and they show strong affinity toward protein
target molecules. The aptamer inhibits the target molecule's
function in vivo, but it is quickly destroyed by in vivo nucleases.
To solve the problem, some parts of library are substituted with
artificial nucleic aids to increase resistance against
nucleases.
[0383] Few examples of biological libraries include, but not
limited to, Bioactive Lipid Library; Endocannabinoid
Library-compounds having activity at cannabinoid (CB) and vanniloid
(VR) receptors which includes various classes of ligands, for
example, Amides, Ethanolamides, Lipo-amino acids, Acyl-GABAs, and
Acyl-dopamines etc.; Known Bioactives Library, such as, GPCR
ligands, second messenger modulators, nuclear receptor ligands,
actin & tubulin modulators, kinase inhibitors, protease
inhibitors, ion channel blockers, gene regulation agents, lipid
biosynthesis inhibitors, etc.; Ion Channel Ligand Library;
Kinase/Phosphatase Inhibitor Library; Natural Products
Library-Natural products are an unsurpassed source of chemical
diversity and are an ideal starting point for any screening program
for pharmacologically active small molecules; Neurotransmitter
Library-CNS Receptor Ligands, such as, Adrenergics, Dopaminergics,
Serotonergics, Opioids (& Sigma ligands), Cholinergics,
Histaminergics (& Melatonin Ligands), Ionotropic
Glutamatergics, Metabotropic Glutamatergics, GABAergics, and
Purinergics (& Adenosines) etc.; Nuclear Receptor Ligand
Library-Nuclear Receptor Ligand Library contains compounds with at
nuclear receptors. Receptor agonists and antagonists may be
included; Orphan Ligand Library-Orphan ligand library contains
compounds with biological activity but whose protein binding
partners have not been identified. For example, trace Amines,
neurotransmitter metabolites, endogenouse .beta.-carbolines,
urinary metabolites, nicotine congeners, and D-Amino Acids etc.
[0384] 2. Methods of Screening
[0385] The present invention provides methods to identify candidate
agents that bind to a protein or act as a modulator of the binding
characteristics or biological activity of a protein. Assays may be
conducting in a variety of ways including screening a library of
non-natural amino acid polypeptides with a known molecule or vice
versa. In one embodiment, the method is performed in single test
tubes or on a modest scale. In another embodiment, the method is
performed in plurality simultaneously. For example, the method can
be performed at the same time on multiple assay mixtures in a
multi-well screening plate. Thus, in one aspect, the invention
provides a high throughput screening system. With regards to
assaying for interactions in one embodiment, fluorescence or
absorbance readouts are utilized to determine activity. Other
biological activities to assays by way of example only are
acetylation, carboxylation, acylation, phosphorylation,
dephosphorylation, ubiquitination, glycosylation, lipid
modification, ADP-ribosylation, bioavailability and half-life.
[0386] There are many methods known to those skilled in the art
which can also be used to detecting interaction between a
non-natural amino acid polypeptide and another molecule within a
screening assay. These methods may include by way of example only,
fluorescent bind-binding assays, thermal shift assays,
electrophoretic mobility shift assays, protein-protein binding
assays, biochemical screening assays, immunoassays (i.e.
immunoprecipitation) and cell based assays (i.e. two- or
three-hybrid screens, GST pull down, TAP-TAG system), expression
assays, protein-DNA binding assays, functional assays
(phosphorylation assays, etc.) and the like. See, e.g., U.S. Pat.
No. 6,495,337, incorporated herein by reference. Other methods may
also include protein chip systems which can screen enzymes,
receptor proteins or antibodies which aid conducting
protein-protein interaction studies, ligand binding studies, or
immunoassays (MacBeath and Schreiber, Science 2000 289: 1760-1763).
Another embodiment may involve, profiling drug which can effect in
intact cells, that are introduced with functional non-natural amino
acid polypeptides, by probing the cell physiology using fluorescent
stains for DNA and other proteins known to interact with the
non-natural amino acid polypeptide and using fluorescent
microscopes generated pictures so as to measure changes in the
cells' behavior (Mayer, T. U., Kapoor, T. M., Haggarty, S. J.,
King, R. W., Schreiber, S. L., Mitchison, T. J. (1999). Science.
286, 971-4.)
[0387] In particular, there are numerous methods by which detection
of binding of a test ligand to a non-natural amino acid polypeptide
(and, thus, by which identification of a ligand of the non-natural
amino acid polypeptide) can be carried out. Useful methods are
those by which the folded non-natural amino acid polypeptide can be
distinguished from unfolded non-natural amino acid polypeptide. The
methods described below are by way of example only some of the
means by which this can be done. In each case, the detection method
is carried out on a test combination (test ligand-non-natural amino
acid polypeptide combination) after sufficient time has passed for
binding of a non-natural amino acid polypeptide to its ligand and
on a control combination (which is the same as the test combination
except that no test ligand is present).
[0388] A. Methods for Determining the Presence of Folded
Non-Natural Amino Acid Polypeptide
[0389] In the present method, a test ligand may be combined with a
non-natural amino acid polypeptide for which a ligand (i.e., an
agent which binds the non-natural amino acid polypeptide) is to be
identified. The resulting combination is a test ligand-non-natural
amino acid polypeptide combination or test combination. In general,
the test ligand is present in excess molar amounts, relative to the
non-natural amino acid polypeptide. The present method can be
carried out in solution or, in some embodiments of the method, the
non-natural amino acid polypeptide can be present on a solid phase
(e.g., linked covalently through a linker or otherwise to a bead).
The test ligand and non-natural amino acid polypeptide are combined
under conditions (e.g., temperature, pH, salt concentration, time)
appropriate for binding of the non-natural amino acid polypeptide
to a ligand. In addition, conditions under which test ligand and
non-natural amino acid polypeptide are combined are generally such
that, for non-natural amino acid polypeptide that unfolds
reversibly, a substantial fraction of non-natural amino acid
polypeptide is present in the absence of the test ligand in the
unfolded form, although the fraction can vary, depending on the
detection method used. In the case of non-natural amino acid
polypeptide which unfold irreversibly, conditions are generally
such that the non-natural amino acid polypeptide unfolds at a
substantial rate in the absence of ligand. These conditions are
chosen to ensure that the non-natural amino acid polypeptide
unfolds to an appropriate extent; thus, the observed signal (e.g.,
digestion by a protease; binding to antibody, chaperonin or
surface) can be measured conveniently. If too little non-natural
amino acid polypeptide is unfolded, the observed signal will occur
at too low a level or rate to be conveniently measured. For each
test ligand-non-natural amino acid polypeptide combination
assessed, the conditions under which the present method is carried
out will be determined empirically, using known methods. Such
conditions include reaction temperature and the chaotropic agent(s)
or denaturant(s) used. The temperature at which the method is
carried out is determined by the non-natural amino acid polypeptide
being used and can be determined empirically using known methods.
To adjust or optimize the fraction of unfolded non-natural amino
acid polypeptide, denaturing conditions may be required for some
non-natural amino acid polypeptide. Such denaturing conditions
might include the use of elevated temperatures, the addition of
protein denaturants (e.g., urea, guanidine) to the incubation
mixture or use of both. In addition, the stability of some
non-natural amino acid polypeptide might be adjusted through
engineering destabilizing or stabilizing amino acid substitutions
in the non-natural amino acid polypeptide. The test ligand and
non-natural amino acid polypeptide are combined, maintained under
appropriate conditions and for sufficient time for binding of the
non-natural amino acid polypeptide to a ligand. The time necessary
for binding of non-natural amino acid polypeptide to ligand will
vary depending on the test ligand, non-natural amino acid
polypeptide and other conditions used. In some cases, binding will
occur instantaneously (e.g., essentially simultaneous with
combination of test ligand and non-natural amino acid polypeptide),
while in others, the resulting test ligand-non-natural amino acid
polypeptide combination is maintained for a longer time before
binding is detected. In the case of non-natural amino acid
polypeptide which unfolds irreversibly, the rate of unfolding must
also be taken into consideration in determining an appropriate time
for binding of test ligand. Binding of a test ligand to the
non-natural amino acid polypeptide is assessed in one of several
ways: by determining the extent to which folded non-natural amino
acid polypeptide is present in the test ligand-non-natural amino
acid polypeptide combination; by determining the extent to which
unfolded non-natural amino acid polypeptide is present in the test
ligand-non-natural amino acid polypeptide combination or by
determining the ratio of folded non-natural amino acid polypeptide
to unfolded non-natural amino acid polypeptide in the combination.
That is, the difference between the amount of folded non-natural
amino acid polypeptide, the amount of unfolded non-natural amino
acid polypeptide or the ratio of folded non-natural amino acid
polypeptide to unfolded non-natural amino acid polypeptide in the
presence of the test ligand and in its absence is determined. If a
test ligand binds the non-natural amino acid polypeptide (i.e., if
the test ligand is a ligand for the non-natural amino acid
polypeptide), there will be more folded non-natural amino acid
polypeptide and less unfolded non-natural amino acid polypeptide
(and, thus, a higher ratio of folded to unfolded non-natural amino
acid polypeptide and a lower ratio of unfolded to folded
non-natural amino acid polypeptide) than is present in the absence
of a test ligand which binds the non-natural amino acid
polypeptide. It is not necessary to determine the quantity or
fraction of a folded and unfolded non-natural amino acid
polypeptide. It is only necessary to know that there is a
difference in the amount of folded or unfolded protein (a change in
equilibrium of the two forms) in the presence and absence of a
ligand or a change in the rate of unfolding. This difference can be
determined by comparing the extent to which folded and/or unfolded
non-natural amino acid polypeptide is present in a test combination
(test ligand-non-natural amino acid polypeptide combination) With
the extent to which they are present in a control combination
(non-natural amino acid polypeptide in the absence of test ligand).
Alternatively, for reversible unfolding, the difference between the
extent to which the two forms occur in the absence of a test ligand
can be assessed by determining their occurrence initially (e.g.,
prior to addition of a test ligand to a solution of non-natural
amino acid polypeptide or to solid support-bound test protein) and
then after the test ligand has been combined with the non-natural
amino acid polypeptide under conditions appropriate for non-natural
amino acid polypeptide-ligand binding to occur. In either case,
determination of the two forms of non-natural amino acid
polypeptide can be carried out using a variety of known methods,
which are described below. A test ligand which is shown by the
present method to bind a non-natural amino acid polypeptide is
referred to as a ligand of the non-natural amino acid
polypeptide.
[0390] 1. Determining Ligand Binding Using Proteolysis
[0391] In one embodiment of the present method, binding of test
ligand to non-natural amino acid polypeptide is detected through
the use of proteolysis. In this embodiment, a protease which acts
preferentially upon unfolded non-natural amino acid polypeptide is
combined with the test ligand-non-natural amino acid polypeptide
combination (test combination) and the resulting test
combination-protease mixture is assayed after an appropriate period
of incubation, using one of the methods described in detail below,
to determine the difference between intact or degraded non-natural
amino acid polypeptide in the presence and in the absence of the
test ligand. An identical assay is performed on a test
ligand-non-natural amino acid polypeptide combination and on a
control combination and results of the two assays are compared.
More intact protein or less degraded protein in the test
combination than in the control combination indicates that the test
ligand has bound the non-natural amino acid polypeptide and, thus,
indicates that the test ligand is a ligand of the non-natural amino
acid polypeptide. Similarly, a higher ratio of intact non-natural
amino acid polypeptide to degraded protein in the test combination
than in the control indicates the test ligand is a ligand of the
non-natural amino acid polypeptide.
[0392] A wide variety of proteases, such as trypsin, chymotrypsin,
V8 protease, elastase, carboxypeptidase, proteinase K, thermolysin
and subtilisin, can be used in this embodiment. It is only
necessary that the protease used be able to act upon (hydrolyze the
peptide bonds of) the non-natural amino acid polypeptide used under
the chosen incubation conditions and that this action be
preferentially directed toward the unfolded form of the protein. To
avoid interference by target ligands which directly inhibit the
protease, more than one protease can be used simultaneously or in
parallel assays.
[0393] In order to be efficiently digested the peptide bonds, the
peptide substrate--the non-natural amino acid polypeptide--must
have access to the enzyme active site of the chosen protease.
Because the atoms in a folded protein molecule are tightly packed,
the majority of the susceptible peptide bonds are sterically
blocked from entering a protease active site when the protein is in
the folded state. In the unfolded state, the peptide bonds are more
exposed and are therefore relatively more susceptible to protease
action.
[0394] Consequently, the addition of a test ligand which binds the
folded non-natural amino acid polypeptide, stabilizing it in the
protease-resistant form, changes the rate of proteolysis. Thus, by
incubating the test ligand with the non-natural amino acid
polypeptide, adding a protease to preferentially degrade the
unfolded proteins, and then employing an assay to quantify the
intact or the degraded non-natural amino acid polypeptide, it is
possible to ascertain whether the test ligand bound the non-natural
amino acid polypeptide and, thus, is a ligand of the non-natural
amino acid polypeptide, indicating that it is potentially
therapeutically useful.
[0395] Alternatively, the protease may be intrinsic to the
unpurified or partially purified non-natural amino acid polypeptide
sample.
[0396] 2. Determining Ligand Binding Through Detection of Surface
Binding
[0397] In another embodiment of the present method, the propensity
of unfolded proteins to adhere to surfaces is utilized. This
embodiment relies on the fact that folded proteins are held in
specific three dimensional arrangements and, thus, are not as
likely as their unfolded counterparts to bind a surface. If a test
ligand binds a non-natural amino acid polypeptide (i.e., is a
ligand of the non-natural amino acid polypeptide), it will
stabilize the folded form of the non-natural amino acid
polypeptide. Thus, the ability of a test ligand to bind a
non-natural amino acid polypeptide can be determined by assessing
the extent to which non-natural amino acid polypeptide is bound to
an appropriate solid surface in the presence and in the absence of
the test ligand. The methods described in detail below can be used
for this purpose.
[0398] In this embodiment, the non-natural amino acid polypeptide,
a test ligand and a surface that preferentially binds unfolded
protein are combined and maintained under conditions appropriate
for binding of the non-natural amino acid polypeptide to a ligand
and binding of unfolded non-natural amino acid polypeptide to the
surface. There are numerous suitable surfaces for this purpose,
including microtiter plates constructed from a variety of treated
or untreated plastics, plates treated for tissue culture or for
high protein binding, nitrocellulose filters and PVDF filters.
[0399] If a test ligand binds the non-natural amino acid
polypeptide, more folded non-natural amino acid polypeptide and
less unfolded non-natural amino acid polypeptide is present in the
test ligand-non-natural amino acid polypeptide combination than is
present in a comparable control combination. That is, in the
presence of a test ligand that is a ligand for a non-natural amino
acid polypeptide, less unfolded protein is available to bind a
surface that preferentially binds unfolded protein than in the
absence of a ligand for the non-natural amino acid polypeptide.
Determination of the amount of surface-bound non-natural amino acid
polypeptide or the amount of non-natural amino acid polypeptide
remaining in solution can be carried out using one of the methods
described below. If more non-natural amino acid polypeptide is not
surface bound (i.e., if more non-natural amino acid polypeptide is
in solution) in the presence of a test ligand than in the absence
of the test ligand, the test ligand is a ligand of the non-natural
amino acid polypeptide. The ratio of non-natural amino acid
polypeptide in solution to surface-bound non-natural amino acid
polypeptide is greater if a test ligand is a ligand for the
non-natural amino acid polypeptide than if it is not. Conversely,
the ratio of surface-bound non-natural amino acid polypeptide to
non-natural amino acid polypeptide in solution is less if a test
ligand is a ligand for the non-natural amino acid polypeptide than
if it is not.
[0400] 3. Determining Ligand Binding Using Antibody Binding
[0401] In a third embodiment, the extent to which folded and
unfolded non-natural amino acid polypeptide are present and, thus,
binding of test ligand to non-natural amino acid polypeptide, are
assessed through the use of specific antibodies directed against
only the unfolded state ("denatured-specific antibodies" or "DS
antibodies") or only the folded state ("nature specific antibodies"
or "Nantibodies"). When a non-natural amino acid polypeptide is in
the folded state, and stabilized in that state by test ligand which
is a ligand for the non-natural amino acid polypeptide, the DS
antibody's apparent binding affinity will be reduced (Breyer,
(1989) "Production and Characterization of Mono-clonal Antibodies
to the N-terminal Domain of the Lambda Repressor", J. Biol. Chem.,
264(5):13348-13354) and that of the NS antibody will be enhanced.
If DS antibody binding to non-natural amino acid polypeptide is
less or if NS antibody binding is greater in the presence of a test
ligand than in its absence the test ligand is a ligand for the
non-natural amino acid polypeptide.
[0402] There are numerous methods known in the art for producing
antibody that binds to a particular protein (Harlow, E. & D.
Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor
Laboratory, 1988, incorporated herein by reference). To prepare
antibody specific for the denatured state, animals can be immunized
with a peptide from a region of the protein that is buried in the
native state. If the structure of the protein is unknown,
antibodies can be prepared against several peptides and then the
antibodies can be screened for preferential binding to the
denatured state. Antibody production is by standard techniques,
such as the technique for production of mono-clonal antibodies
described in detail in Zola, Monoclonal Antibodies: A Manual of
Techniques, CRC Press, Inc., Boca Raton, Fla. (1987), incorporated
herein by reference.
[0403] There are at least three basic methods by which DS or NS
antibodies can be utilized to detect a ligand-induced change in the
occurrence of folded non-natural amino acid polypeptide, the
occurrence of unfolded proteins or the ratio of one to the
other.
[0404] In one approach, a test solution containing the DS antibody
directed against the unfolded non-natural amino acid polypeptide,
the non-natural amino acid polypeptide, and the test ligand is
incubated, such as in a microtiter plate coated with the denatured
non-natural amino acid polypeptide or a peptide fragment thereof,
under conditions appropriate for binding of the non-natural amino
acid polypeptide with its ligand and binding of the DS antibody to
unfolded non-natural amino acid polypeptide. A control solution,
which is the same as the test solution except that it does not
contain test ligand, is processed in the same manner as the test
solution. By comparing the amount of antibody bound to the plate or
the amount remaining in solution in the test and control solutions,
the difference in non-natural amino acid polypeptide folding is
detected. The amount of antibody bound to the plate or remaining in
solution can be measured as described below.
[0405] In a second approach, a test solution containing the DS
antibody, the test ligand, and the non-natural amino acid
polypeptide is incubated in a plate coated with a second antibody,
referred to as a solid phase antibody, which cannot bind to the
non-natural amino acid polypeptide simultaneously with the DS
antibody, and is specific for the non-natural amino acid
polypeptide, but is either specific for the folded state ("native
specific" or "NA antibody") or unable to differentiate between the
native and denatured states ("non-differentiating" or "ND
antibody"). The resulting test combination or solution is
maintained under conditions appropriate for binding of the
non-natural amino acid polypeptide with a ligand of the non-natural
amino acid polypeptide and for binding of the antibodies to the
proteins they recognize (are specific for). A control solution,
which is the same as the test solution except that it does not
contain test ligand, is processed in the same manner as the test
solution. In both solutions, denatured (unfolded) non-natural amino
acid polypeptide binds the DS antibody and is inhibited from
binding the solid phase antibody. The ability of the test ligand to
bind the non-natural amino acid polypeptide can be gauged by
determining the amount of non-natural amino acid polypeptide that
binds to the solid phase antibody in the test solution and
comparing it with the extent to which non-natural amino acid
polypeptide binds to the solid phase antibody in the absence of
test ligand, which in turn reflects the amount of non-natural amino
acid polypeptide in the folded state. The amount of non-natural
amino acid polypeptide bound to the plate via the second antibody
or remaining in solution can be detected by the methods described
below. This approach may be used in a comparable manner with NS
antibody as the in solution antibody and DS or ND antibody on the
solid phase.
[0406] In a third approach, a test solution containing the
non-natural amino acid polypeptide and the test ligand is incubated
in a container, such as a microtiter well which has been coated
with a DS or NS antibody and maintained under conditions
appropriate for binding of non-natural amino acid polypeptide to
its ligand and for binding of the antibody to non-natural amino
acid polypeptide. Alternatively, the antibody can be present on the
surfaces of beads. The ability of the test ligand to bind the
non-natural amino acid polypeptide is gauged by determining the
extent to which non-natural amino acid polypeptide remains in
solution (unbound to the antibody) or on the solid surface (bound
to the antibody), or the ratio of the two, in the presence and in
the absence of test ligand. If the test ligand binds the
non-natural amino acid polypeptide (is a ligand of the non-natural
amino acid polypeptide), there will be less non-natural amino acid
polypeptide bound to a DS antibody or more bound to an NS antibody
(i.e., more non-natural amino acid polypeptide will be in solution
in the case of DS antibody or less in solution for NS antibody)
than is bound to the antibody in the control solution. In a further
embodiment, the antibody can be present in solution and the
non-natural amino acid polypeptide can be attached to a solid
phase, such as a plate surface or bead surface.
[0407] 4. Determining Ligand Binding Using Molecular Chaperones
[0408] In a fourth embodiment, molecular chaperones are used to
determine binding of a test ligand to a non-natural amino acid
polypeptide. Chaperones are a variety of protein that bind unfolded
proteins as part of their normal physiological function. They are
generally involved in assembling oligomeric proteins, in ensuring
that certain proteins fold correctly, in facilitating protein
localization, and in preventing the formation of proteinaceous
aggregates during physiological stress. Hardy, (1991) "A Kinetic
Partitioning Model of Selective Binding of Nonnative Proteins by
the Bacterial Chaperone SecB", Science 251:439-443 These proteins
have the ability to interact with many unfolded or partially
denatured proteins without specific recognition of defined sequence
motifs.
[0409] One molecular chaperone, found in E. coli, is SecB. SecB has
a demonstrated involvement in export of a subset of otherwise
unrelated proteins. Competition experiments have shown that SecB
binds tightly to all the unfolded proteins tested, including
proteins outside of its particular export subset, but does not
appear to interact with the folded protein.
[0410] In this embodiment, a test solution containing the test
ligand and the target is incubated on a microtiter plate or other
suitable surface coated with molecular chaperones, under conditions
appropriate for binding of non-natural amino acid polypeptide with
its ligand and binding of the molecular chaperones used to unfolded
non-natural amino acid polypeptide. The unfolded non-natural amino
acid polypeptide in the solution will have a greater tendency to
bind to the molecular chaperone-covered surface relative to the
ligand-stabilized folded non-natural amino acid polypeptide. Thus,
the ability of the test ligand to bind non-natural amino acid
polypeptide can be determined by determining the amount of
non-natural amino acid polypeptide remaining unbound, or the amount
bound to the chaperone-coated surface, using the methods detailed
below.
[0411] Alternatively, a competition assay for binding to molecular
chaperones can be utilized. A test solution containing purified
non-natural amino acid polypeptide, the test ligand, and a
molecular chaperone can be incubated in a container, such as a
microtiter well coated with denatured (unfolded) non-natural amino
acid polypeptide, under conditions appropriate for binding
non-natural amino acid polypeptide with its ligand and binding of
the molecular chaperones to unfolded non-natural amino acid
polypeptide. A control solution which is the same as the test
solution except that it does not contain test ligand is processed
in the same manner. Denatured non-natural amino acid polypeptide in
solution will bind to the chaperonin and, thus, inhibit its binding
to the denatured non-natural amino acid polypeptide bound to the
container surface (microtiter well surface). Binding of a test
ligand to non-natural amino acid polypeptide will result in a
smaller amount of unfolded non-natural amino acid polypeptide, and,
thus, more chaperones will be available to bind to the solid-phase
denatured non-natural amino acid polypeptide than is the case in
the absence of binding of test ligand. Thus, binding of test ligand
can be determined by assessing chaperones bound to the surface or
in solution in the test solution and i.sub.D the control solution
and comparing the results. Binding of chaperone to solid-phase
denatured non-natural amino acid polypeptide to a greater extent in
the test solution than in the control solution is indicative of
test ligand-non-natural amino acid polypeptide binding (i.e., is
indicative of identification of a ligand of the non-natural amino
acid polypeptide). In this assay, the molecular shaperones are
generally not provided in excess, so that competition for their
binding can be measured.
[0412] Alternatively, test solution containing the non-natural
amino acid polypeptide, the test ligand and a molecular chaperone
can be incubated in a container, such as a microtiter well, whose
surface is coated with antisera or a monoclonal antibody specific
for the folded non-natural amino acid polypeptide (NS antibody) and
unable to bind the non-natural amino acid polypeptide bound to the
chaperone. Unfolded non-natural amino acid polypeptide will bind
chaperone in solution and thus be inhibited from binding the solid
phase antibody. By detecting non-natural amino acid polypeptide in
the solution or bound to the well walls and comparing the extent of
either or both in an appropriate control (the same combination
without the test ligand), the ability of the test ligand to bind
non-natural amino acid polypeptide can be determined. If the test
ligand is a ligand for the non-natural amino acid polypeptide, more
non-natural amino acid polypeptide will be bound to the antisera or
monoclonal antibody bound to the container surface in the test
solution than in the control solution. Conversely, less non-natural
amino acid polypeptide will be present unbound (in solution) in the
test solution than in the control solution. Detection and
comparison of bound non-natural amino acid polypeptide, unbound
non-natural amino acid polypeptide or a ratio of the two in the
test solution and control solution indicate whether the test ligand
is a ligand of the non-natural amino acid polypeptide or not.
[0413] 5. Determining Ligand Binding Through Measurements of
Protein Aggregation
[0414] The higher the fraction of protein in the folded form, the
greater the amount of protein that is available to bind to a ligand
that binds exclusively to the folded state. Consequently, if a
protein has a known ligand, it is possible to increase the binding
of the protein to the known ligand by adding a ligand that binds
another site on the protein. In this approach, a ligand known to
bind to the non-natural amino acid polypeptide is immobilized on a
solid substrate. A solution containing the non-natural amino acid
polypeptide is then added, along with test ligand or ligands. An
increase in the amount of non-natural amino acid polypeptide that
binds to the immobilized ligand relative to an identical assay in
the absence of test ligand indicates that the test ligand binds the
non-natural amino acid polypeptide. The amount of non-natural amino
acid polypeptide bound to the solid substrate can be assessed by
sampling the solid substrate or by sampling the solution, using the
detection methods outlined below.
[0415] 6. Determining Ligand Binding Through Measurements of
Protein Aggregation
[0416] For proteins that unfold irreversibly, unfolded protein
often forms insoluble aggregates. The extent of protein aggregation
can be measured by techniques outlined below such as light
scattering, centrifugation, and filtration. In this approach,
non-natural amino acid polypeptide and test ligand are incubated
and the amount of protein aggregation is measured over time or
after a fixed incubation time. The extent of protein aggregation in
the test mixture is compared to the same measurement for a control
assay in the absence of test ligand. If a test ligand binds a
non-natural amino acid polypeptide, the rate of unfolding of
non-natural amino acid polypeptide will be lower than in the
absence of test ligand. For measurements over time, the rate of
increase of unfolded protein and hence of aggregated protein will
be lower if the test ligand is a ligand for the non-natural amino
acid polypeptide than if it is not. For measurements at a fixed
time, there will be less unfolded protein add therefore less
aggregated protein if the test ligand is a ligand for the
non-natural amino acid polypeptide than if it is not. Thus, the
ability of a test ligand to bind a non-natural amino acid
polypeptide can be determined by assessing the extent of protein
aggregation in the presence and absence of test ligand.
[0417] XIV. Protein Detection Techniques
[0418] Methods known in the art to detect the presence or absence
of protein, small peptides or free amino acids can be used in the
present method for detecting non-natural amino acids, non-natural
amino acid polypeptides, modified non-natural amino acid
polypeptides and fragments thereof. The method used can be
determined by the product (proteins, peptides, free amino acids) to
be detected. For example, techniques for detecting protein size can
be used to determine the extent of proteolytic degradation of the
non-natural amino acid polypeptide. Radio-labeling, fluorescence
labeling, and enzyme-linked labeling can detect the presence or
absence either in solution or on a substrate by measurement of
radioactivity, fluorescence or enzymatic activity. Immunologic
methods can detect the presence or absence of a known non-natural
amino acid polypeptide in solution or on a substrate such as by
binding of an antibody specific for that protein. FIG. 1a presents
various protein detection techniques that can be used to detect
non-natural amino acids, non-natural amino acid polypeptides,
modified non-natural amino acid polypeptides and fragments
thereof.
[0419] A. Fluoroscence Microscopy
[0420] Methods for protein detection disclosed herein, include
fluorescence microscopy to detect non-natural amino acids,
non-natural amino acid polypeptides, modified non-natural amino
acid polypeptides and fragments thereof. Fluorescence Microscopy is
a widely used microscopy technique that enables the molecular
composition of the structures being observed to be identified
through the use of fluorescently-labelled probes of high chemical
specificity. Such probes may be antibodies, antibody fragments, or
antigen-binding polypeptides that comprise a non-natural amino
acid. Fluorescence microscopy may be used in studies of fixed
specimens. For proteins that can be extracted and purified in
reasonable abundance, a fluorophore may be conjugated to a protein
and the conjugate introduced into a cell. A fluorophore may be
conjugated to a non-natural amino acid in the polypeptide. It is
assumed that the fluorescent analogue behaves like the native
protein and can therefore serve to reveal the distribution and
behavior of this protein in the cell. Along with NMR, infrared
spectroscopy, circular dichroism and other techniques, protein
intrinsic fluorescence decay and its associated observation of
fluorescence anisotropy, collisional quenching and resonance energy
transfer are key techniques for protein detection.
[0421] Measuring the fluorescence decay allows the dynamics of
structural changes in a protein to be observed directly. Moreover,
excitation of the native fluorescence of proteins emanating from
the amino acids tyrosine and tryptophan eliminates the possibility
of perturbation of the local environment when using extrinsic
fluorescent probes.
[0422] A development in the use of fluorescent probes for
biological studies has been the use of naturally fluorescent
proteins as fluorescent probes. Naturally occurring dyes, so-called
fluorescent proteins (GFP, YFP, CFP, TOPAS, GFT, RFP), were
discovered in the late 1990s (Clonetech, USA). These dyes are
distinguished by their reduced influence on specimens. They are
therefore particularly suitable for labeling cell regions in living
preparations.
[0423] The jellyfish Aequorea victoria produces a naturally
fluorescent protein known as green fluorescent protein (GFP). The
fusion of these fluorescent probes to a target protein enables
visualization by fluorescence microscopy and quantification by flow
cytometry. Because they are genetically encoded and require no
auxiliary cofactors, GFP tags can be used to analyze protein
expression and localization in living cells and whole organisms.
The gene for this protein has been cloned and can be transfected
into other organisms. GFP tags may be used for localizing regions
in which a particular gene is expressed in an organism, or in
identifying the location of a particular protein. In many cases
these chimeric proteins preserve their original function. It is
therefore often possible, for example, to use this technique to
visualize the intracellular distribution of a protein, including
but not limited to a cytoskeletal protein. With GFP, unstained or
unfixed samples can be observed. There are presently several
variants of GFP which provide spectrally separable emission colors.
Mutations to GFP have resulted in blue-, cyan- and
yellow-fluorescent light emitting versions. Fluorescent proteins
which can be used to label the present non-natural amino acid
peptides, polypeptides, antibodies, and antibody fragments include
but are not limited to, green fluorescent protein (GFP), cyan
fluorescent protein (CFP), red fluorescent protein (RFP), yellow
fluorescent protein (YFF), enhanced GFP (EGFP), enhanced YFP
(EYFP), and the like. New versions of GFP have been developed via
mutation, including a "humanized" GFP DNA, the protein product of
which has increased synthesis in mammalian cells (see Cormack, et
al., (1996) Gene 173, 33-38; Haas, et al., (1996) Current Biology
6, 315-324; and Yang, et al., (1996) Nucleic Acids Research 24,
4592-4593). One such humanized protein is "enhanced green
fluorescent protein" (EGFP). GFP, variants of GFP, or other
naturally occurring dyes may be coupled to non-natural amino acid
polypeptides.
[0424] GFP can be used as a biosensor, reporting the results of
levels of ions or pH by fluorescing in characteristic ways. One
molecule that can be used to sense the level of zinc ions is a blue
fluorescent protein shown as PDB (Protein Data Bank) entry 1kys.
The protein fluoresces twice as brightly creating an easily
detectable visible signal once zinc binds to the modified
chromophore. Construction of other peptide and protein biosensors
comprising a non-natural amino acid may exhibit altered
fluorescence properties in response to changes in their
environment, oligomeric state, conformation upon ligand binding,
structure, or direct ligand binding. Appropriately labeled
fluorescent biomolecules allow spatial and temporal detection of
biochemical reactions inside living cells. See for example
Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol Struct. 1995,
24:405-434; Day, R. N. Mol. Endocrinol. 1998, 12:1410-9; Adams, S.
R., et al., Nature 1991, 349:694; Miyawaski, A., et al., Nature
1997, 388:882-7; Hahn, K., et al., Nature 1992, 359:736; Hahn, K.
M., et al., J. Biol. Chem. 1990, 265:20335; and Richieri, G. V., et
al., Mol. Cell. Biochem. 1999, 192:87-94. U.S. Pat. No. 6,951,947,
which is incorporated by reference herein, discusses biosensors and
fluorophores that detect environmental changes.
[0425] At present the technology is driven by new applications of
existing probes and the design and synthesis of new and innovative
probes. Without limiting the scope of the present invention, some
of the probes are as following:
[0426] Labels: Sensitivity and safety (compared to radioactive
methods) of fluorescence has been increasingly used for specific
labelling of nucleic acids, proteins and other biomolecules.
Besides Fluorescein, there are other fluorescent labels that cover
the whole range from 400 to 820 nm. By way of example only, some of
the labels include, but are not limited to, Fluorescein and its
derivatives, Carboxyfluoresceins, Rhodamines and their derivatives,
Atto labels, Fluorescent red and Fluorescent orange: Cy3/Cy5.TM.
alternatives, Lanthanide complexes with long lifetimes, Long
wavelength labels--up to 800 nm, DY cyanine labels, Phycobili
proteins. Fluorescent molecules that are capable of absorbing
radiation at one wavelength and emitting radiation at a longer
wavelength include but are not limited to Alexa-532,
Hydroxycoumarin, Aminocoumarin, Methoxycoumarin, Coumarin, Cascade
Blue, Lucifer Yellow, P-Phycoerythrin, R-Phycoerythrin, (PE),
PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, Fluorescein,
BODIPY-FL, BODIPY TR, BODIPY TMR, Cy3, TRITC, X-Rhodamine,
Lissamine Rhodamine B. PerCP, Texas Red, Cy5, Cy7, Allophycocyanin
(APC), TruRed, APC-Cy7 conjugates, Oregon Green,
Tetramethylrhodamine, Dansyl, Dansyl aziridine, Indo-1, Fura-2, FM
1-43, DilC18(3), Carboxy-SNARF-1, NBD, Indo-1, Fluo-3, DCFH, DHR,
SNARF, Monochlorobimane, Calcein,
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amine (NBD),
ananilinonapthanele, deproxyl, phthalamide, amino pH phthalamide,
dimethylamino-naphthalenesulfonamide, probes comparable to Prodan,
Lordan or Acrylodan and derivatives thereof. Coumarin fluorescent
dyes include, for example, amino methylcoumarin,
7-diethylamine-3-(4'-(1-maleimidyl)phenyl)-4-methylcoumarin (CPM)
and N-(2-(1-maleimidyl)ethyl)-7-diethylaminocoumarin-3-Carboxamide
(MDCC). Other useful molecules include those that display
fluorescence resonance energy transfer (FRET). Many such
donor-acceptor pairs are known, and include fluorescein to
rhodamine, coumarin to fluorescein or rhodamine, etc. Still another
class of useful label pairs includes fluorophore-quencher pairs in
which the second group is a quencher, which decreases the
fluorescence intensity of the fluorescent group. Some known
quenchers include acrylamide groups, heavy atoms such as iodide and
bromate, nitroxide spin labels such as TEMPO, etc. Labels such as
these may be conjugated to non-natural amino acid polypeptides.
[0427] Fluorophores that are be conjugated to a non-natural amino
acid polypeptide may fluoresce all of the time or only when the
polypeptide is bound to a target. Other types of fluorophores
include
[0428] Conjugates: By way of example only, some of the conjugates
include but are not limited to, Isothiocyanate conjugates,
streptavidin conjugates, and Biotin conjugates. Antibody conjugates
have been widely used to track biomolecules in living cells and
whole organisms They can be generated with specificity for
virtually any epitope and are therefore, in principle, applicable
to imaging a wide range of biomolecules. Conjugates including but
not limited to antibody conjugates may comprise a non-natural amino
acid.
[0429] Enzyme Substrates: Enzyme substrates include but are not
limited to fluorogenic and chromogenic substrates.
[0430] Micro- and Nanoparticles: Various techniques allow the
preparation of a wide variety of fluorescent microspheres ranging
in size, matrix chemistry, type of fluorochrome, fluorescence
intensity, and surface functional groups. By way of example only,
some of the fluorochromes used are: FITC (green fluorescence,
Excitation/Emission=506/529 nm), Rhodamine B (orange fluorescence,
Excitation/Emission=560/584 nm), Nile Blue A (red fluorescence,
Excitation/Emission=636/686 nm)
[0431] Fluorescent nanoparticles are promising tools for both
optical data storage and other technical applications, for example,
in biochemical, bioanalytical and medical areas. Current medical
and biological fluorescent imaging methods are mainly based on dye
markers, which are limited in light emission per molecule, as well
as photostability. Nanoparticles overcome those problems offering
strong and stable fluorescence. Fluorescent nanoparticles have been
successfully used for various types of immunoassays. Fluorescent
nanoparticles are based on different materials, such as,
polyacrylonitrile, and polystyrene, etc.
[0432] Molecular Rotors: Fluorescent Molecular Rotors are sensors
of microenvironmental restriction that become fluorescent only if
their rotation is constrained. The change of fluorescence intensity
is caused by the restriction of intramolecular rotational
relaxation about the donor-acceptor bond of the fluorophores.
Examples of molecular constraint include but are not limited to
increased dye (aggregation), binding to antibodies, or being
trapped in the polymerization of actin.
[0433] IEF-Markers: IEF (Isoelectric Focusing) is a powerful
analytical tool for the separation of ampholytes, mainly proteins.
In order to ensure the high performance of analysis, standards of
pI (pI markers) are needed. An advantage for IEF-Gel
electrophoresis with Fluorescent IEF-Marker is the possibility to
directly observe the formation of gradient. Fluorescent IEF-Marker
can also be detected by UV-absorption at 280 nm (20.degree.
C.).
[0434] Any or all of these fluorescent probes can be used for the
detection of non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof. FIG. 9 presents non-limiting examples of
molecules that are site specifically attached to proteins through
oxime formation between carbonyl of non-natural amino acid
incorporated into a polypeptide and the hydroxylamine of the
molecule. The molecules shown are fluorophores, biotin, and
chelators.
[0435] Bio-Orthogonal Chemical reporters: Small molecules have
better access to intracellular and extravascular compartments.
Their use as imaging agents requires a means to selectively target
the small probe to a desired biomolecule. Nucleophilic
functionality occurs in most types of biopolymers, permitting
facile derivatization with biotin, fluorophores and numerous other
small-molecule reporters. Established bioconjugation protocols have
made these operations trivial for purified biopolymers in vitro. It
is an alternative strategy for tagging biomolecules that blends the
simplicity of genetically encoded tags with the specificity of
antibody labeling and the versatility of small-molecule probes.
This approach involves the incorporation of unique chemical
functionality--a bioorthogonal chemical reporter--into a target
biomolecule using the cell's own biosynthetic machinery.
Bioorthogonal chemical reporters are non-native, non-perturbing
chemical handles that can be modified in living systems through
highly selective reactions with exogenously delivered probes. This
two-step labeling process can be used to outfit a target
biomolecule for detection or isolation, depending on the nature of
the probe.
[0436] Examples of bio-orthogonal coupling reactions include but
are not limited to, the Staudinger ligation of azides with triaryl
phosphines, the ketone/aldehyde-hydrazine reaction, and Huisgen's
1,3-dipolar azide-alkyne cycloaddition. Replacement of the bulky
fluorescent tag with a sterically inconspicuous azide group may
furnish probes that are more able to distribute in an unbiased
manner within a living cell, tissue, or organism. Likewise, the
variable and often antagonistic effect of the fluorescent tag on
probe binding affinity for specific proteins is also eliminated.
Finally, the use of azide-alkyne cycloaddition chemistry can
streamline probe synthesis by removing the need to generate and
purify large quantities of structurally diverse fluorophore-tagged
reagents. Coupling reactions utilizing non-natural amino acid
polypeptides may provide probes that are alternatives to
fluorescently tagged polypeptides. Huisgen's 1,3-dipolar
azide-alkyne cycloaddition may be used to attach other molecules or
provide other methods for polypeptide purification or
detection.
[0437] Peptide libraries can be synthesized on solid supports and,
by using coloring receptors, dyed solid supports can be selected
one by one. If receptors cannot indicate any colors, their binding
antibodies can be dyed. Because it is possible to separate solid
supports by tweezers under microscopes or even magnifiers, the
method can be not only be used on protein receptors, but also on
screening binding ligands of synthesized artificial receptors and
screening new metal binding ligands as well. This method is useful
to search new lead compounds, because it enables the screening of a
large amount of compounds.
[0438] However, determination of activity depending on dye
intensity may not be accurate, and large amount of solid supports
may not be always treated one by one. Therefore, automated methods
for high throughput screening (HTS) are required and a FACS
(Fluorescence Activated Cell Sorter) method can be used. This
machine originally runs cells through a capillary tube and
separates cells by detecting their fluorescent intensities. The
same method may be used on solid supports instead of cells. Because
it is designed for cells, small resins of cell size may be run, but
normal sizes of solid supports (50.about.200 pmol) need specially
modified machines. Partial or entire isolation of compounds may
also be done. For partial isolation of compounds, time controlled
photodecomposition or several functional groups to cleave in
different conditions are used. In the meanwhile, one can scatter
solid supports on soft agar and isolate some of compounds by
photodecomposition. The isolated compounds then spread out around
solid supports so that screening and solid support separation can
be done at a time.
[0439] B. Immunoassays
[0440] Methods for protein detection disclosed herein, include
immunoassays to detect non-natural amino acids, non-natural amino
acid polypeptides, modified non-natural amino acid polypeptides and
fragments thereof. Immunoassays combine the principles of chemistry
and immunology enabling scientific tests, e.g. enzyme immunoassays
and immunoblotting for a specific and sensitive detection of the
analytes (non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof) of interest. The basic principle of these assays
is the specificity of the antibody-antigen reaction. Similar to the
Western blot, a single protein can be identified by its antibody
with immunoblotting. Competitive binding immunoassays may be done
in which analyte competes with a labelled antigen for a limited
pool of antibody molecules (eg. radioimmunoassay, EMIT).
Immunoassays can be non-competitive such that antibody is present
in excess and is labelled. As analyte antigen is increased, the
amount of labeled antibody-antigen complex also increases (e.g.
ELISA). Antibodies can be polyclonal if produced by antigen
injection into experimental animal, or monoclonal if produced by
cell fusion and cell culture techniques. In immunoassays the
antibody serves as a specific reagent for the analyte antigen. The
antigen may be non-natural amino acid polypeptides, modified
non-natural amino acid polypeptides and fragments thereof). On the
other hand, the antibodies or fragments thereof used in
immunoassays may be non-natural amino acid polypeptides, and may be
used in the detection of antigens that may or may not comprise a
non-natural amino acid.
[0441] Without limiting the scope and content of the present
invention, some of the types of immunoassays are, by way of example
only, RIAs (Radioimmunoassay) and enzyme immunoassays like ELISA
(Enzyme-linked immunosorbent assay), EMIT (Enzyme Multiplied
Immunoassay Technique), Microparticle Enzyme Immunoassay (MEIA),
LIA (luminescent immunoassay), and FIA (fluorescent immunoassay).
These techniques can be used to detect non-natural amino acids,
non-natural amino acid polypeptides, modified non-natural amino
acid polypeptides and fragments thereof. The antibodies--either
used as primary or secondary antibodies--may be labeled with
radioisotopes (e.g. .sup.125I), fluorescent dyes (e.g. FITC) or
enzymes (e.g. HRP or AP) which catalyze fluorogenic or luminogenic
reactions.
[0442] 1. EMIT (Enzyme Multiplied Immunoassay Technique)
[0443] EMIT is a competitive binding immunoassay that avoids a
separation step. A type of immunoassay in which the protein is
labeled with an enzyme, and the enzyme-protein-antibody complex is
enzymatically inactive, allowing quantitation of unlabeled
protein.
[0444] 2. ELISA (Enzyme Linked Immunosorbent Assay)
[0445] Methods for protein detection disclosed herein, include
ELISA to detect non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof. Enzyme linked immunosorbent assays are based on
selective antibodies attached to solid supports combined with
enzyme reactions to produce systems capable of detecting low levels
of proteins. It is also known as enzyme immunoassay or EIA. The
antigen, including but not limited to a protein, is detected by
antibodies that have been made against it; that is, for which it is
the antigen. Monoclonal antibodies are often used.
[0446] The test may require the antibodies to be fixed to a solid
surface, such as the inner surface of a test tube; and a
preparation of the same antibodies coupled to an enzyme. The enzyme
is one (e.g., .beta.-galactosidase) that produces a colored product
from a colorless substrate. The test, for example, is performed by
filling the tube with the antigen solution (e.g., protein) to be
assayed. Any antigen molecules present may bind to the immobilized
antibody molecules. The antibody-enzyme conjugate is added to the
reaction mixture. The antibody part of the conjugate binds to any
antigen molecules that were bound previously, creating an
antibody-antigen-antibody "sandwich". After washing away any
unbound conjugate, the substrate solution is added. After a set
interval, the reaction is stopped (e.g., by adding 1 N NaOH) and
the concentration of colored product formed by reaction of the
substrate with molecules conjugated to the secondary antibody is
measured in a spectrophotometer. The intensity of color is
proportional to the concentration of bound antigen.
[0447] ELISA can also be adapted to measure the concentration of
antibodies, in which case, the wells are coated with the
appropriate antigen. The solution (e.g., serum) containing antibody
is added. After it has had time to bind to the immobilized antigen,
an enzyme-conjugated anti-immunoglobulin is added, consisting of an
antibody against the antibodies being tested for. After washing
away unreacted reagent, the substrate is added. The intensity of
the color produced is proportional to the amount of enzyme-labeled
antibodies bound (and thus to the concentration of the antibodies
being assayed).
[0448] 3. Radioimmunoassays
[0449] Methods for protein detection disclosed herein, include
radioimmunoassays to detect non-natural amino acids, non-natural
amino acid polypeptides, modified non-natural amino acid
polypeptides and fragments thereof. Radioimmunoassays are highly
sensitive. Using antibodies of high affinity (eg.,
K.sub.0=10.sup.8-10.sup.11 M.sup.-1), it is possible to detect a
few picograms (10-12 g) of antigen in the tube.
[0450] Radioactive isotopes can be used to study in vivo
metabolism, distribution, and binding of small amount of compounds.
Radioactive isotopes of .sup.1H, .sup.12C, .sup.31P, .sup.32S,
.sup.127I are used such as .sup.3H, .sup.14C, .sup.32P, .sup.35S,
.sup.125I. Radioactive isotopes have almost same chemical
properties as unradioactive ones, so that they can be converted
easily. Also because their radiation energy is relatively large,
only a little amount is needed.
[0451] Receptor Fixation Method--For a 96 well plate format,
receptors are fixed in each well by using antibody or chemical
methods, and radioactive labeled ligands are added to each well to
induce binding. Unbound ligands are washed out and then the
standard is determined by the quantitative analysis of the
radioactivity of bound ligands or that of washed-out ligands. The
addition of target compounds for screening induces competitive
binding reactions with receptors. If target compounds show higher
affinity to receptors than standard radioactive ligands, most of
the radioactive ligands do not bind to receptors and are left in
solution. Therefore, by analyzing the quantity of bound radioactive
ligands (or washed-out ligands), the affinity of target compounds
to receptors can be easily indicated.
[0452] A filter membrane method may be used when receptors cannot
be fixed to 96 well plates or ligand binding must be performed in
solution phase. With this method, after the ligand-receptor binding
reaction is done in solution, the reaction solution is filtered
through nitrocellulose filter paper. Small molecules including
ligands will go through the filter paper, and only protein
receptors will be left on the paper. Only ligands that are strongly
bound to receptors will stay on the filter paper, and the relative
affinity of added compounds can be identified by quantitative
analysis of the standard radioactive ligands. This method can also
be used to screen protein kinase inhibitors as well. In this case,
.gamma.-.sup.32P-ATP can be used as a phosphoric acid group
supplier, and by checking radioactive labeled protein substrate,
enzymatic activity can be analyzed. Radioactive ATP that does not
react will be filtered and removed.
[0453] By way of example only, radioimmunoassays can be performed
by preparing a mixture of radioactive antigen and antibodies
against that antigen. Iodine atoms can be introduced into tyrosine
residues in a protein, the radioactive isotopes .sup.125I or
.sup.131I are often used. Known amounts of unlabeled ("cold")
antigen can be added to samples of the mixture. These compete for
the binding sites of the antibodies. At increasing concentrations
of unlabeled antigen, an increasing amount of radioactive antigen
is displaced from the antibody molecules. The antibody-bound
antigen is separated from the free antigen in the supernatant
fluid, and the radioactivity of each is measured. From these data,
a standard binding curve can be drawn. The samples to be assayed
("the unknowns") are run in parallel. After determining the ratio
of bound to free antigen in each unknown, the antigen
concentrations can be read directly from the standard curve.
[0454] Other methods of radioimmunoassays that can be used for
detecting non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof are, by way of example only, precipitating the
antigen-antibody complexes by adding a "second" antibody directed
against the first. For example, if a rabbit IgG is used to bind the
antigen, the complex can be precipitated by adding an
anti-rabbit-IgG antiserum (e.g., raised by immunizing a goat with
rabbit IgG). Alternatively, the antigen-specific antibodies can be
coupled to the inner walls of a test tube. After incubation, the
unbound contents are removed; the tube is washed, and the
radioactive of the unbound and bound material are both measured.
The antigen-specific antibodies can be coupled to particles, like
Sephadex. Centrifugation of the reaction mixture separates the
bound counts (in the pellet) from the free counts in the
supernatant fluid.
[0455] 4. Fluorescence Immunoassays
[0456] Methods for protein detection disclosed herein, include
fluorescence immunoassays to detect non-natural amino acids,
non-natural amino acid polypeptides, modified non-natural amino
acid polypeptides and fragments thereof. Fluorescence based
immunological methods are based upon the competitive binding of
labeled ligands versus unlabeled ones on highly specific receptor
sites. It is a very important tool for clinical and analytical
biochemistry in the analysis of proteins.
[0457] This technique can be used for immunoassays based on changes
in fluorescence lifetime with changing analyte concentration. This
technique works with dyes with a short lifetime like fluorescein
isothiocyanate (FITC) (the donor) whose fluorescence is quenched by
energy transfer to Eosin (the acceptor). A number of molecular
species have been used for causing energy transfer from a donor
molecule to an acceptor molecule. In particular, sandwich type
immuno-complex formation can be used with this technique.
[0458] A number of photoluminescent compounds may be used in the
method of the invention and include the compounds listed above in
fluorescence microscopy, as well as groups such as cyanines,
oxazines, thiazines, porphyrins, phthalocyanines, fluorescent
infrared-emitting polynuclear aromatic hydrocarbons,
phycobiliproteins, squaraines and organo-metallic complexes,
hydrocarbons and azo dyes.
[0459] Fluorescence based immunological methods can be, for
example, heterogenous or homogenous. Heterogenous Immunoassays
comprise a physical separation of bound from free labeled analyte.
The analyte or antibody may be attached to a solid surface. The
technique can be competitive (for a higher selectivity) or
noncompetitive (for a higher sensitivity). Detection can be direct
(only one type of antibody used) or indirect (a second type of
antibody is used). Homogenous Immunoassays comprise no physical
separation. Double-Antibody Fluorophore-labeled antigen
participates in an equilibrium reaction with antibodies directed
against both the antigen and the fluorophore. Labeled and unlabeled
antigens compete for a limited number of anti-antigen
antibodies.
[0460] Simple Fluorescence Labelling method--It can be used for
receptor-ligand binding, enzymatic activity by using pertinent
fluorescence, and as a fluorescent indicator of various in vivo
physiological changes such as pH, ion concentration, and electric
pressure. Self-fluorescence of amino acids such as tyrosine and
tryptophan result in background radiation, and to overcome such
weak points fluorescent compounds of absorption UV length longer
than 520 nm such as cyanine are often used.
[0461] FRET: Fluorescence Resonance Energy Transfer--FRET may be
used to measure the interaction of two proteins in vivo and can
measure nanometer scale distances and distance (conformation)
changes. Therefore, it has been used to measure simple
protein-protein interactions and changes in protein folding,
conformation, and stability (see Philipps, B.; Hennecke, J.;
Glockshuber R. Mol Biol. 2003, 327, 239-249; Riven, I.; Kalmanzon,
E.; Segev, L.; Reuveny E. Neuron. 2003, 38, 225-235). Two different
fluorescent molecules (fluorophores) are conjugated to the two
proteins of interest. Non-natural amino acid polypeptides
conjugated to fluorophores may be used in FRET. When two
fluorescent compounds are used instead of a single fluorescent
compound, non-fluorescent energy transfer occurs. When the emission
wavelength of a fluorescent donor is similar to absorption
wavelength of an acceptor, the donor in its excited state will
transfer its energy to the acceptor instead to emitting fluorescent
light, and consequently emission occurs at emission wavelength of
the acceptor. A number of different fluorophore pairs have been
used for FRET analysis including GFP (green fluorescent protein)
variants CFP (cyan) and YFP (yellow) fused to the proteins of
interest.
[0462] Distance R0 of 50% FRET effect depends on the overlap of the
emission range of donors, absorption range of the acceptors, and
the acceptor's quantum yields and solvent. If two fluorescent
molecules are at a shorter distance from each other than R0, when
the donor's absorption light is emitted, theoretically the
acceptor's fluorescence will be stronger. If the distance becomes
longer than R0, when the same light is emitted, the donor's
fluorescence will be detected as stronger. Therefore, enzymatic
activity can be measured easily if fluorescent molecules are linked
to the ends of small peptides, which can be used as kinases such as
protease. BRET (Bioluminescene resonance energy transfer) was
developed by Xu et al (Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
151-156). It acts on a principle similar to FRET and is based on
the finding that the emission spectrum of Renilla luciferase is
similar to that of CFP. These techniques allow the study of
interactions within specific subcellular compartments, including
membrane protein-protein interactions, when utilizing organelle
targeted fluorescent protein variants. Also post-translational
modification events can be studied in mammalian cells.
[0463] TRF: Time Resolved Fluorescence--To reduce fluorescent
background, Time Resolved Fluorescence was developed. The lifetime
of excited states of common fluorescent molecules is usually only a
few microseconds, but Lanthanide series elements have milliseconds
of life time. TRF is a method that selectively measures the
fluorescence of the Lanthanide series after the emission of other
fluorescent molecules has finished. TRF can be also with FRET, and
Lanthanide series become donors or acceptors.
[0464] 5. Various Assay Formats
[0465] Various assay formats may be used for the detection of the
non-natural amino acids, non-natural amino acid polypeptides,
modified non-natural amino acid polypeptides, and fragment thereof,
disclosed herein, including "sandwich" immunoassays and probe
assays. For example, in a first assay format, a polyclonal or
monoclonal antibody or fragment thereof, or a combination of these
antibodies, which has been coated on a solid phase, is contacted
with a test sample, to form a first mixture. This first mixture is
incubated for a time and under conditions sufficient to form
antigen/antibody complexes. Then, an indicator reagent comprising a
monoclonal or a polyclonal antibody or a fragment thereof, or a
combination of these antibodies, to which a signal generating
compound has been attached, is contacted with the antigen/antibody
complexes to form a second mixture. This second mixture then is
incubated for a time and under conditions sufficient to form
antibody/antigen/antibody complexes. The presence of antigen in the
test sample and captured on the solid phase, if any, is determined
by detecting the measurable signal generated by the signal
generating compound. The amount of antigen present in the test
sample is proportional to the signal generated.
[0466] In an alternative assay format, a mixture is formed by
contacting: (1) a polyclonal antibody, monoclonal antibody, or
fragment thereof, which specifically binds to antigen, or a
combination of such antibodies bound to a solid support; (2) the
test sample; and (3) an indicator reagent comprising a monoclonal
antibody, polyclonal antibody, or fragment thereof, which
specifically binds to a different epitope (or a combination of
these antibodies) to which a signal generating compound is
attached. This mixture is incubated for a time and under conditions
sufficient to form antibody/antigen/antibody complexes. The
presence, if any, of antigen present in the test sample and
captured on the solid phase is determined by detecting the
measurable signal generated by the signal generating compound. The
amount of antigen present in the test sample is proportional to the
signal generated.
[0467] In another assay format, one or a combination of at least
two monoclonal antibodies of the invention can be employed as a
competitive probe for the detection of antibodies to antigen. For
example, unnatural amino acid polypeptides disclosed herein, either
alone or in combination, are coated on a solid phase. A test sample
suspected of containing antibody to antigen then is incubated with
an indicator reagent comprising a signal generating compound and at
least one monoclonal antibody for a time and under conditions
sufficient to form antigen/antibody complexes of either the test
sample and indicator reagent bound to the solid phase or the
indicator reagent bound to the solid phase. The reduction in
binding of the monoclonal antibody to the solid phase can be
quantitatively measured.
[0468] In yet another detection method, the monoclonal or
polyclonal antibodies can be employed in the detection of antigens
in tissue sections, as well as in cells, by immunohistochemical
analysis. The tissue sections can be cut from either frozen or
chemically fixed samples of tissue. If the antigens are to be
detected in cells, the cells can be isolated from blood, urine,
breast aspirates, or other bodily fluids. The cells may be obtained
by biopsy, either surgical or by needle. The cells can be isolated
by centrifugation or magnetic attraction after labeling with
magnetic particles or ferrofluids so as to enrich a particular
fraction of cells for staining with the antibodies. Cytochemical
analysis wherein these antibodies are labeled directly (with, for
example, fluorescein, colloidal gold, horseradish peroxidase,
alkaline phosphatase, etc.) or are labeled by using secondary
labeled anti-species antibodies (with various labels as exemplified
herein) to track the histopathology of disease also are within the
scope of the present invention.
[0469] Combinations of the monoclonal antibodies (and fragments
thereof) also may be used together as components in a mixture or
"cocktail" along with antibodies which specifically bind to other
regions of unnatural amino acid polypeptides disclosed herein, each
antibody having different binding specificities. The polyclonal
antibodies used in the assays can be used either alone or as a
cocktail of polyclonal antibodies. Since the cocktails used in the
assay formats are comprised of either monoclonal antibodies or
polyclonal antibodies having different binding specificity to
unnatural amino acid polypeptides disclosed herein, they are useful
for the detecting, diagnosing, staging, monitoring,
prognosticating, in vivo imaging, preventing or treating, or
determining the predisposition to, various diseases and
conditions.
[0470] It is contemplated and within the scope of the present
invention that unnatural amino acid amino acids disclosed herein,
may be detected in assays by use of a recombinant antigen as well
as by use of a synthetic polypeptide or purified polypeptide, which
polypeptide comprises an amino acid sequence of unnatural amino
acid polypeptides disclosed herein. It also is within the scope of
the present invention that different synthetic, recombinant or
purified polypeptides, identifying different epitopes of unnatural
amino acid polypeptides disclosed herein, can be used in
combination in an assay for the detecting, diagnosing, staging,
monitoring, prognosticating, in vivo imaging, etc. In this case,
all of these polypeptides can be coated onto one solid phase; or
each separate polypeptide may be coated onto separate solid phases,
such as microparticles, and then combined to form a mixture of
polypeptides which can be later used in assays. Polypeptides coated
on solid phases or labeled with detectable labels are then allowed
to compete with those present in a sample for a limited amount of
antibody. A reduction in binding of the synthetic, recombinant, or
purified peptides to the antibody (or antibodies) is an indication
of the presence of unnatural amino acid polypeptides disclosed
herein. Variations of assay formats are known to those of ordinary
skill in the art.
[0471] 6. Scanning Probe Microscopy (SPM) for Immunoassays
[0472] Methods for protein detection disclosed herein, include SPM
to detect non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof. In scanning probe microscopy, in the capture
phase, for example, at least one of the monoclonal antibodies is
adhered to a solid phase and a scanning probe microscope is
utilized to detect antigen/antibody complexes which may be present
on the surface of the solid phase. The use of scanning tunneling
microscopy eliminates the need for labels which normally must be
utilized in many immunoassay systems to detect antigen/antibody
complexes.
[0473] The use of SPM to monitor specific binding reactions can
occur in many ways. In one embodiment, one member of a specific
binding partner (analyte specific substance which is the monoclonal
antibody) is attached to a surface suitable for scanning. The
attachment of the analyte specific substance may be by adsorption
to a test piece which comprises a solid phase of a plastic or metal
surface. Covalent attachment of a specific binding partner (analyte
specific substance) to a test piece which test piece comprises a
solid phase of derivatized plastic, metal, silicon, or glass may be
utilized. Covalent attachment methods are known to those skilled in
the art and include a variety of means to irreversibly link
specific binding partners to the test piece. If the test piece is
silicon or glass, the surface must be activated prior to attaching
the specific binding partner. Also, polyelectrolyte interactions
may be used to immobilize a specific binding partner on a surface
of a test piece by using techniques and chemistries. The preferred
method of attachment is by covalent means. Following attachment of
a specific binding member, the surface may be further treated with
materials such as serum, proteins, or other blocking agents to
minimize non-specific binding. The surface also may be scanned
either at the site of manufacture or point of use to verify its
suitability for assay purposes. The scanning process is not
anticipated to alter the specific binding properties of the test
piece.
[0474] C Spectroscopy
[0475] I. Nuclear Magnetic Resonance (NMR)
[0476] Methods for protein detection disclosed herein, include NMR
to detect non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof.
[0477] NMR spectroscopy is capable of determining the structures of
biological macromolecules like proteins and nucleic acids at atomic
resolution. In addition, it is possible to study time dependent
phenomena with NMR, such as intramolecular dynamics in
macromolecules, reaction kinetics, molecular recognition or protein
folding. Methods for protein detection disclosed herein, include
NMR to detect non-natural amino acid polypeptides and modified
non-natural amino acid polypeptides and fragments thereof.
[0478] Progress in the theoretical and practical capabilities of
NMR, led to increasingly efficient utilization of the information
content of NMR spectra. Parallel developments in the biochemical
methods (recombinant protein expression) allow the simple and fast
preparation of protein samples. Heteronuclei like .sup.15N,
.sup.13C and .sup.2H, can be incorporated in proteins by uniformly
or selective isotopic labeling. Spectra from these samples can be
drastically simplified. Additionally, some new information about
structure and dynamics of macromolecules can determined with these
methods. All these developments currently allow the structure
determination of proteins with a mass of up to 30 kDa or more.
[0479] 2. X-Ray Crystallography
[0480] Methods for protein detection disclosed herein, include
X-ray crystallography to detect non-natural amino acids,
non-natural amino acid polypeptides, modified non-natural amino
acid polypeptides and fragments thereof.
[0481] X-ray crystallography is a technique in crystallography in
which the pattern produced by the diffraction of X-rays through the
closely spaced lattice of atoms in a crystal is recorded and then
analyzed to reveal the nature of that lattice. This generally leads
to an understanding of the material and molecular structure of a
substance. The spacings in the crystal lattice can be determined
using Bragg's law. The electrons that surround the atoms, rather
than the atomic nuclei themselves, are the entities which
physically interact with the incoming X-ray photons. This technique
is widely used in chemistry and biochemistry to determine the
structures of an immense variety of molecules, including inorganic
compounds, DNA and proteins. X-ray diffraction is commonly carried
out using single crystals of a material, but if these are not
available, microcrystalline powdered samples may also be used,
although this requires different equipment and is much less
straightforward.
[0482] For X-ray crystallography, the molecule must be
crystallized. One photon diffracted by one electron cannot be
reliably detected, however, because of the regular crystalline
structure; the photons are diffracted by corresponding electrons in
many symmetrically arranged molecules. Because waves of the same
frequency whose peaks match reinforce each other, the signal
becomes detectable. To determine a structure, crystals of the
molecule of interest are grown using some method of
crystallization. The crystals are harvested and often frozen with
liquid nitrogen. Freezing crystals both reduces radiation damage
incurred during data collection and decreases thermal motion within
the crystal. Crystals are placed on a diffractometer, a machine
that emits a beam of X-rays. The X-rays diffract off the electrons
in the crystal, and the pattern of diffraction is recorded on film
and scanned into a computer. These diffraction images are combined
and eventually used to construct a map of the electron density of
the molecule that was crystallized, atoms are then fitted to the
electron density map and various parameters such as position are
refined to best fit the observed diffraction data.
[0483] 3. Fluorescence Spectroscopy
[0484] Methods for protein detection disclosed herein, include
fluorescence spectroscopy to detect non-natural amino acids,
non-natural amino acid polypeptides, modified non-natural amino
acid polypeptides and fragments thereof.
[0485] Besides the standard fluorescence measurements a variety of
other methods have been developed. Conventional Fluorometry
involves measurements of emission light intensities at defined
wavelengths for a certain emission maxima of a fluorophore. Total
Fluorometry involves a collection of data for a continuum of
absorption as well as emission wavelengths. In Fluorescence
Polarization, polarized light is used for excitation and binding of
fluorochrome-labeled antigens to specific antibodies affects
polarization extent. Line Narrowing Spectroscopy involves
low-temperature solid-state spectroscopy that derives its
selectivity from the narrow-line emission spectra it provides.
[0486] Time-dependent Fluorescence Spectroscopy comprises
time-resolved measurements containing more information than
steady-state measurements, since the steady-state values represent
the time average of time-resolved determinations. It is a single
photon timing technique in which the time between an excitation
light pulse and the first photon emitted by the sample is
measured.
[0487] Frequency-Domain Fluorescence Spectroscopy is an alternative
to the time-resolved methods. The time decay of fluorescence is
typically measured using a light source with an intensity modulated
sinusoidally at a given frequency, by determining the phase delay
and the relative modulation of the fluorescence signal with respect
to the exciting light.
[0488] 4. Matrix Assisted Laser Desorption Ionization
Time-of-Flight Mass Spectrometry (MALDI TOF-MS)
[0489] Methods for protein detection disclosed herein, include
MALDI TOF-MS to detect non-natural amino acids, non-natural amino
acid polypeptides, modified non-natural amino acid polypeptides and
fragments thereof.
[0490] Linear TOF-MS--Mass spectrometry has emerged as an important
tool for analyzing and characterizing large biomolecules of varying
complexity. The matrix assisted laser desorption/ionization (MALDI)
technique, developed in 1987, has increased the upper mass limit
for mass spectrometric analyses of biomolecules to over 300,000 Da
and has enabled the analyses of large biomolecules by mass
spectrometry to become easier and more sensitive. TOF mass
spectrometers operate on the principle that when a temporally and
spacially well defined group of ions of differing mass/charge (m/z)
ratios are subjected to the same applied electric field
(K.E.=[mv2]/2=zeEs where K.E.=kinetic energy; m=the mass of the
ion; v velocity of the ion; z=number of charges; e=the charge on an
electron in coulombs; E=electric field gradient; and s=the distance
of the ion source region) and allowed to drift in a region of
constant electric field, they will traverse this region in a time
which depends upon their m/z ratios.
[0491] Reflectron TOF-MS--Improved mass resolution in MALDI TOF-MS
has been obtained by the utilization of a single-stage or a
dual-stage reflectron (RETOF-MS). The reflectron, located at the
end of the flight tube, is used to compensate for the difference in
flight times of the same m/z ions of slightly different kinetic
energies by means of an ion reflector. This results in focusing the
ion packets in space and time at the detector. In the reflectron
mass spectrum, the isotopic multiplet is well resolved producing a
full width half maximum (FWHM) mass resolution of about 3400. Mass
resolutions up to 6000 (FWHM) have been obtained for peptides up to
about 3000 Da with RETOF-MS. Enhancing the mass resolution can also
increase the mass accuracy when determining the ion's mass.
[0492] Historically, both linear and reflectron MALDI-TOF-MS have
been utilized primarily for molecular weight determinations of
molecular ions and enzymatic digests leading to structural
information of proteins. These digests are typically mass analyzed
with or without purification prior to molecular weight
determinations. Varieties of methodologies have been developed to
obtain primary sequence information for proteins and peptides
utilizing MALDI TOF-MS. Two different approaches can be taken. The
first method is known as protein ladder sequencing and is employed
to produce structurally informative fragments of the analyte prior
to insertion into the TOF mass spectrometer and subsequent
analysis. The second approach utilizes the phenomenon of metastable
ion decay that occurs inside the TOF mass spectrometer to produce
sequence information.
[0493] Ladder Sequencing with TOF-MS-Proteins or peptides can be
sequenced using MALDI-TOF-MS with a ladder sequencing technique
which consists of either a time-dependent or
concentration-dependent chemical degradation from either the N- or
C-terminus of the protein or peptide into fragments, each of which
differs by one amino acid residue. The mixture is mass analyzed in
a single MALDI-TOF-MS experiment with mass differences between
adjacent mass spectral peaks corresponding to a specific amino acid
residue. This type of analysis can be thought of as simply
determining the masses of a series of peptides/proteins that are
present in a single MALDI sample. The order of occurrence in the
mass spectrum defines the sequence of amino acids in the original
protein or peptide.
[0494] Post-Source Decay with RETOF-MS MALDI--It has historically
been considered a "soft" ionization technique that produces almost
exclusively intact protonated pseudomolecular ion species. A
significant degree of metastable ion decay occurs after ion
acceleration and prior to detection. The ion fragments produced
from the metastable ion decav of peptides and proteins typically
include both neutral molecule losses (such as water, ammonia and
portions of the amino acid side chains) and random cleavage at
peptide bonds. The observance of these metastable ion decay
products in MALDI mass spectra is dependent on the TOF instrumental
configuration.
[0495] MALDI TOF-MS has developed into a valuable tool in the
biosciences for obtaining both accurate mass determinations and
primary sequence information. Methods for protein detection
disclosed herein, include MALDI TOF-MS to detect non-natural amino
acid polypeptides and modified non-natural amino acid polypeptides
and fragments thereof. The sequence information obtained from the
mass spectra whose sequence was known a priori by no means implies
a straightforward scheme to deduce an unknown peptide or protein
sequence from its metastable ion decay mass spectrum. These MALDI
techniques are envisioned to be most useful in conjunction with
conventional biochemical techniques such as protein digests. They
may be applicable to identifying blocked amino termini,
post-translational modifications and mutation sites in known
proteins in this way. Also, with a total unknown, a significant
amount of preliminary structure determination should be possible on
very small (less than 10 pmol) amounts of analyte. For ladder
sequencing and in-source fragmentation studies, it is important to
minimize potential peptide impurities.
[0496] In-Source Decay with Linear TOF-MS--An alternative approach
to RETOF-MS for studying metastable ion decay of MALDI generated
ions is to utilize DE with linear TOF-MS. By employing the DE
technique, primary structural information for peptides and proteins
can also be obtained. Prompt ion fragmentation produced at the time
of the desorption event (i.e., ion formation) is generally absent
for MALDI generated peptide or protein ions. By incorporating a
time delay between ion formation and ion extraction, ions in the
source are allowed to fragment in a relatively short period of time
(<100 ns) into smaller ions and neutrals prior to extraction. A
drawout potential is then applied extracting the fragmented ions.
Coherent mass spectral peaks are produced from these metastable
decayed ions giving rise to significant structural information for
peptides and proteins.
[0497] 5. Surface-Enhanced Laser Desorption Ionization--Time of
Flight (SELDI-TOF)
[0498] Another proteomic technology involved in quantitative
analysis of protein mixtures is known as surface-enhanced laser
desorption ionization--time of flight (SELDI-TOF). Methods for
protein detection disclosed herein, include SELDI-TOF to detect
non-natural amino acids, non-natural amino acid polypeptides,
modified non-natural amino acid polypeptides and fragments
thereof.
[0499] This technique utilizes stainless steel or aluminum-based
supports, or chips, engineered with chemical (hydrophilic,
hydrophobic, pre-activated, normal-phase, immobilized metal
affinity, and cationic or anionic) or biological (antibody, antigen
binding fragments (including but not limited to, scFv), DNA,
enzyme, or receptor) bait surfaces of 1-2 mm in diameter. These
varied chemical and biochemical surfaces allow differential capture
of proteins based on the intrinsic properties of the proteins
themselves. Solubilized tissue or body fluids in volumes as small
as 0.1 .mu.l are directly applied to these surfaces, where proteins
with affinities to the bait surface will bind. Following a series
of washes to remove non-specifically or weakly bound proteins, the
bound proteins are laser desorbed and ionized for MS analysis.
Masses of proteins ranging from small peptides of less than 1000 Da
up to proteins of greater than 300 kDa are calculated based on
time-of-flight. As mixtures of proteins will be analyzed within
different samples, a unique sample fingerprint or signature will
result for each sample tested. Consequently, patterns of masses
rather than actual protein identifications are produced by SELDI
analysis. These mass spectral patterns are used to differentiate
patient samples from one another, such as diseased from normal.
While protein fingerprints can be analyzed for differential
biomarker expression, this technology is currently unable to
specifically identify proteins within a sample using MS. However,
this situation is rapidly evolving as prototypes are being tested
which couple the SELDI-TOF technology with tandem mass
spectrometers. Coupling of these types of instruments will enable
amino acid sequencing and subsequent protein identification.
[0500] 6. UV-Vis
[0501] Methods for protein detection disclosed herein, include
UV-Vis to detect non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof.
[0502] Optical absorption spectroscopy (UV/VIS) plays an important
role for the determination of concentrations (proteins, DNA,
nucleotides etc.). Organic dyes can be used to enhance the
absorption and to shift it into the visible range (e.g. coomassie
blue reagents). Understanding the forces that govern the
interaction of proteins with one another assists in the
understanding of such processes as macromolecular assembly,
chaperone-assisted protein folding and protein translocation.
[0503] Resonance Raman Spectroscopy (RRS) is a tool which can be
used to study molecular structure and dynamics. Resonance Raman
scattering requires excitation within an electronic absorption band
and results in a large increase of scattering. Few molecules have
visible absorption bands; however everything absorbs in the deep
UV. By using UV light it is possible to study a wide variety of
colorless chromophores, and have the additional benefit of avoiding
interference from fluorescence. Furthermore, electrons of different
functional groups with different excitation wavelengths can be
selectively excited. This approach helps to investigate specific
parts of macromolecules by using different excitation
wavelengths.
[0504] 7. Liquid Chromatography (LC)
[0505] Liquid chromatography has been a powerful tool for isolating
proteins, peptides, and other molecules from complex mixtures.
Methods for protein detection disclosed herein, include LC to
detect non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof. Liquid chromatography can be affinity
chromatography, gel filtration chromatography, anion exchange
chromatography, cation exchange chromatography, diaode array-LC and
high performance liquid chromatography (HPLC).
[0506] Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size. Molecules move through a
bed of porous beads, diffusing into the beads to greater or lesser
degrees. Smaller molecules diffuse further into the pores of the
beads and therefore move through the bed more slowly, while larger
molecules enter less or not at all and thus move through the bed
more quickly. Both molecular weight and three dimensional shape
contribute to the degree of retention. Gel Filtration
Chromatography may be used for analysis of molecular size, for
separations of components in a mixture, or for salt removal or
buffer exchange from a preparation of marcromolecules.
[0507] Affinity chromatography is the process of bioselective
adsorption and subsequent recovery of a compound from an
immobilized ligand. This process allows for the highly specific and
efficient purification of many diverse proteins and other
compounds. The process requires the utilization of an appropriately
selective ligand which will bind the desired compound generally
with a dissociation constant in the range of 10.sup.-4 to
10.sup.-8, while permitting recovery under mild conditions. The
ligand is generally immobilized on a beaded and porous matrix which
may be in the form of a column packing or batchwise adsorption
medium.
[0508] Ion exchange chromatography separates molecules based on
differences between the overall charge of the proteins. It is
usually used for protein purification but may be used for
purification of oligonucleotides, peptides, or other charged
molecules, The protein of interest must have a charge opposite that
of the functional group attached to the resin in order to bind. For
example, immunoglobulins, which generally have an overall positive
charge, will bind well to cation exchangers, which contain
negatively charged functional groups. Because this interaction is
ionic, binding must take place under low ionic conditions. Elution
is achieved by increasing the ionic strength to break up the ionic
interaction, or by changing the pH of the protein.
[0509] HPLC can be used in the separation, purification and
detection of non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof disclosed herein. Peptides: Use of
reversed-phased chromatography (RPC) has become a common and
important step in synthetic peptide production. RPC has also been
used to purify natural sequences. Although analytical columns are
used to carry out the process, the procedure can be preparative in
nature due to the limited amount of "active" proteins in tissue.
Some other advantages are that recovery of post-purification
biological activity and reformation of secondary or tertiary
structure after exposure to RPC are favored due to the abbreviated
size of the peptides. Crude tissue extracts may be loaded directly
onto the RPC system and mobilized by gradient elution.
Rechromatography under the identical conditions is an option if
further purification is warranted or necessary. RPC can also be
utilized in the process of protein structure determination. The
normal procedure of this process is 1) fragmentation by proteolysis
or chemical cleavage; 2) purification; and 3) sequencing. A common
mobile phase for RPC of peptides is a gradient of 0.1%
trifluoroacetic acid (TFA) in water to 0.1% TFA in an organic
solvent, such as acetonitrile, since the organic solvent 1)
solubilizes the peptide, 2) allows detection at approximately
230-240 nm, and 3) can evaporate away from the sample. Biologically
Active Proteins: The use of size-exclusion chromatography (SEC) and
ion-exchange chromatography (IEC) is well-suited for use with
biologically active proteins, such as enzymes, hormones, and
antibodies, since each protein has its own unique structure and the
techniques may be performed in physiological conditions. Full
recovery of activity after exposure to the chromatography may be
achieved, and currently, availability of SEC columns is diverse
enough to allow fractionation from 10 to 1000 kilodaltons.
Extremely basic or hydrophobic proteins may not exhibit true SEC
character since the columns tend to have slight hydrophobicity and
anionic character. The use of gradient elution with the IEC column
is favorable because of equivalent resolution as polyacrylamide gel
electrophoresis (PAGE) and increased loading capability when
compared to SEC. In liquid affinity chromatography (LAC)
interaction is based on binding of the protein due to mimicry of
substrate, receptor, etc. The protein is eluted by introducing a
competitive binding agent or altering the protein configuration
which facilitates dissociation. Membrane Proteins: Membrane
proteins are either peripheral (situated on the outer surface) or
integral (partially span, entirely span, or lie completely within
the membrane). The lipophilicity of the bilayer conveys the
lipophilic character (i.e., hydrophobic amino acids) of the
proteins within the membrane. RPC would be a logical choice in
analysis and purification of these proteins, but IEC is also
employed. Another procedure used in the separation of membrane
proteins is the use of nonionic detergents, such as Triton X-100,
or protein solubilization by organic solvents with IEC. HPLC may be
coupled with MS.
[0510] Diode array detector-liquid chromatography (DAD-LC) provides
complete, multiple spectra for each HPLC peak, which, by
comparison, can provide indication of peak purity. These data can
also assign presence of Tyr, Trp, Phe, and possibly others (His,
Met, Cys) and can quantitate these amino acids by 2nd derivative or
multi-component analysis. By a post-column derivatization, DAD-LC
can also identify and quantitate Cys, His and Arg in individual
peptides. Thus, it is possible to analyze for 6 of the 20 amino
acids of each separated peptide in a single LC run, and information
can be obtained about presence or absence of these amino acids in a
given peptide in a single step. This is assisted by knowing the
number of residues in each peptide. Also, by correction at 205 nm
absorbance for side-chain chromophores, this technique can give
much better estimation of relative amounts of each peptide.
[0511] D. Electrophoresis
[0512] Methods for protein detection disclosed herein, include
electrophoresis to detect non-natural amino acids, non-natural
amino acid polypeptides, modified non-natural amino acid
polypeptides and fragments thereof. Electrophoresis can be gel
electrophoresis or capillary electrophoresis.
[0513] Gel Electrophoresis: Gel electrophoresis is a technique that
can be used for the separation of proteins. Separation of large
(macro) molecules depends upon two forces: charge and mass. When a
biological sample, such as proteins, is mixed in a buffer solution
and applied to a gel, these two forces act together. The electrical
current from one electrode repels the molecules while the other
electrode simultaneously attracts the molecules. The frictional
force of the gel material acts as a "molecular sieve," separating
the molecules by size. During electrophoresis, macromolecules are
forced to move through the pores when the electrical current is
applied. Their rate of migration through the electric field depends
on the strength of the field, size and shape of the molecules,
relative hydrophobicity of the samples, and on the ionic strength
and temperature of the buffer in which the molecules are moving.
After staining, the separated macromolecules in each lane can be
seen in a series of bands spread from one end of the gel to the
other. Using this technology it is possible to separate and
identify protein molecules that differ by as little as a single
amino acid. Its advantage is that proteins can be visualized as
well as separated, permitting a researcher to estimate quickly the
number of proteins in a mixture or the degree of purity of a
particular protein preparation. Also, gel electrophoresis allows
determination of crucial properties of a protein such as its
isoelectric point and approximate molecular weight.
[0514] Electrofocusing, or isoelectric focusing, is a technique for
separating different molecules by their electric charge differences
(if they have any charge). It is most commonly used on proteins. It
is a type of zone electrophoresis that takes advantage of the fact
that a molecule's charge changes as the pH of its surroundings
changes. Molecules are distributed over a medium that has a pH
gradient (usually created by aliphatic ampholytes). An electric
current is passed through the medium, creating a "positive" and
"negative" end. Negatively charged particles migrate through the pH
gradient toward the "positive" end while positively charged
particles move toward the "negative" end. As a particle moves into
a pH that neutralizes its charge, it will stop following the
current. Particles of the same initial charge will deposit (or
focus) around the same place on the pH gradient.
[0515] Capillary Electrophoresis: Capillary electrophoresis is a
collection of a range of separation techniques which involve the
application of high voltages across buffer filled capillaries to
achieve separations. The variations include separation based on
size and charge differences between analytes (termed Capillary Zone
Electrophoresis, CZE, or Free Solution CE, FSCE), separation of
neutral compounds using surfactant micelles (Micellar
electrokinetic capillary chromatography, MECC or sometimes referred
to as MEKC) sieving of solutes through a gel network (Capillary Gel
Electrophoresis, GCE), separation of cations (or anions) based on
electrophoretic mobility (Capillary Isotachophoresis, CITP), and
separation of zwitterionic solutes within a pH gradient (Capillary
Isoelectric Focusing, CIEF). Capillary electrochromatography (CEC)
is an associated electrokinetic separation technique which involves
applying voltages across capillaries filled with silica gel
stationary phases. Separation selectivity in CEC is a combination
of both electrophoretic and chromatographic processes. Many of the
CE separation techniques rely on the presence of an electrically
induced flow of solution (electroosmotic flow, EOF) within the
capillary to pump solutes towards the detector. GCE and CIEF are of
importance for the separation of biomolecules such as proteins.
Generally CE is performed using aqueous based electrolytes however
there is a growing use of non-aqueous solvents in CE.
[0516] Operation of a CE system involves application of a high
voltage (typically 10-30 kV) across a narrow bore (25-100 mm)
capillary. The capillary is filled with electrolyte solution which
conducts current through the inside of the capillary. The ends of
the capillary are dipped into reservoirs filled with the
electrolyte. Electrodes made of an inert material such as platinum
are also inserted into the electrolyte reservoirs to complete the
electrical circuit. A small volume of sample is injected into one
end of the capillary. The capillary passes through a detector,
usually a UV absorbance detector, at the opposite end of the
capillary. Application of a voltage causes movement of sample ions
towards their appropriate electrode usually passing through the
detector. The plot of detector response with time is generated
which is termed an electropherogram. A flow of electrolyte, known
as electroendosmotic flow, EOF, results in a flow of the solution
along the capillary usually towards the detector. This flow can
significantly reduce analysis times or force an ion to overcome its
migration tendency towards the electrode it is being attracted to
by the sign of its charge.
[0517] E. Arrays
[0518] Methods for protein detection disclosed herein, include
arrays to detect non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof.
[0519] Arrays involve performing parallel analysis of multiple
samples against known protein targets. The development of various
microarray platforms has remarkably enabled and accelerated the
determination of protein abundance, localization, and interactions
in a cell or tissue. Microarrays provide a platform that allows
identification of protein interaction or function against a
characterized set of proteins, antibodies, or peptides.
[0520] Protein-based chips array proteins on a small surface and
can directly measure the levels of proteins in tissues using
fluorescence-based imaging. Proteins can be arrayed on either flat
solid phases or in capillary systems (microfluidic arrays), and
several different proteins can be applied to these arrays. The most
popular ones currently rely on antibody-antigen interactions, which
can also detect antigen-protein interactions. The potential of
antibody arrays is currently limited by the availability of
antibodies that have both high specificity (to eliminate cross
reactions with non-specific proteins within the sample) and high
affinity for the target of interest (to allow detection of small
quantities within a sample). Another challenge of protein array
technology is the ability to preserve proteins in their
biologically active shape and form. In addition to the use of
antibodies as array probes, single-stranded oligonucleotides, whose
specificity is optimized by in vitro elution (aptamers), offer a
viable alternative. Aptamers allow their covalent attachment to
cognate proteins by photo-crosslinking, thus reducing background.
Nonspecific protein stains are then used to detect bound proteins.
International Publication No. WO 04/58946 entitled "Protein
Arrays," which is incorporated by reference herein, describes the
attachment of non-natural amino acid polypeptides to solid
supports.
[0521] Arrays include, but not limited to, bead arrays, bead based
arrays, bioarrays, bioelectronic arrays, cDNA arrays, cell arrays,
DNA arrays, gene arrays, gene expression arrays, frozen cell
arrays, genome arrays, high density oligonucleotide arrays
hybridization arrays, microcantilever arrays, microelectronic
arrays, multiplex DNA hybridization arrays, nanoarrays,
oligonucleotide arrays, oligosaccharide arrays, planar arrays,
protein arrays, solution arrays, spotted arrays, tissue arrays,
exon arrays, filter arrays, macroarrays, small molecule
microarrays, suspension arrays, theme arrays, tiling arrays, and
transcript arrays.
[0522] F. Sensors
[0523] Methods for protein detection disclosed herein, include
sensors to detect non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof. Sensors can be used for both in vivo and in
vitro detection. Sensors may be used to detect events such as
binding of a non-natural amino acid polypeptide to its target,
conformational changes in a non-natural amino acid polypeptide, and
or measure other interactions, modifications, or changes to a
non-natural amino acid polypeptide or its environment.
[0524] Sensors can be chemical sensors, optical sensors, and
biosensors. Chemical sensors are miniaturized analytical devices
which deliver real-time and online information on the presence of
specific compounds or ions in complex samples. Optical sensors are
based on measurement of either intrinsic optical properties of
analytes, or of optical properties of indicator dyes or labeled
biomolecules attached to solid supports. Biosensors can be affinity
biosensor based on capabilities of enzymes to convert "substrates"
into products; or catalytic biosensors.
[0525] The binding of a non-natural amino acid polypeptide to its
target, including but not limited to, an antibody, antibody
fragment, or antigen-binding polypeptide or fragment thereof, may
be measured. The non-natural amino acid polypeptide is conjugated
to a molecule such as a nanotransmitter. While bound to its target
in-vivo, the nanotransmitter emits a signal that is read ex vivo by
a medical imaging instrument.
[0526] G. Methods for Identifying Proteins from a Library
Screen
[0527] In order to identify the protein(s) that interact with the
non-natural amino acid polypeptide, many methods may be used.
Protein separation aids to separate a complex mixture so that
individual proteins are more easily processed with other
techniques. Protein identification methods include but is not
limited to low-throughput sequencing through Edman degradation,
mass spectrometry techniques, peptide mass fingerprinting, de novo
sequencing, antibody-based assays and protein quantification assays
such as fluorescent dye gel staining, tagging or chemical
modification methods (i.e. isotope-coded affinity tags--ICATS,
combined fractional diagonal chromatography--COFRADIC). The
purified protein may also be used for determination of
three-dimensional crystal structure, which can be used for modeling
intermolecular interactions. Common methods for determining
three-dimensional crystal structure include x-ray crystallography
and NMR spectroscopy. Detailed below are a few of the methods for
identifying proteins.
[0528] Protein sequencing: N-terminal sequencing and C-terminal
sequencing. N-terminal sequencing aids in the identification of
unknown proteins; confirm recombinant protein identity and fidelity
(reading frame, translation start point, etc.); aid the
interpretation of NMR and crystallographic data; demonstrate
degrees of identity between proteins; or provide data for the
design of synthetic peptides for antibody generation, etc.
N-terminal sequencing utilizes the well-established Edman
degradative chemistry, sequentially removing amino acid residues
from the N-terminus of the protein and identifying them by
reverse-phase HPLC. Sensitivity is at the level of 100 s femtomoles
and long sequence reads (20-40 residues) can often be obtained from
a few 10 s picomoles of starting material. Pure proteins (>90%)
generate easily interpreted data, but insufficiently purified
protein mixtures may also provide useful data, subject to rigorous
data interpretation. N-terminally modified (especially acetylated)
proteins cannot be sequenced directly, as the absence of a free
primary amino-group prevents the Edman chemistry. However, limited
proteolysis of the blocked protein (e.g. using cyanogen bromide)
may allow a mixture of amino acids to be generated in each cycle of
the instrument, which can be subjected to database analysis in
order to interpret meaningful sequence information.
[0529] C-terminal sequencing is recognized as an important
post-translational modification, sometimes critically affecting the
structure and activity of a protein. Various disease situations
have been associated with impaired protein processing and
C-terminal sequencing provides an additional tool for the
investigation of protein structure and processing mechanisms.
[0530] Proteome analyses: With proteomics proteins can be
identified primarily by computer search algorithms that assign
sequences to a set of empirically acquired mass/intensity data
which are generated from conducting electrospray ionization (ESI),
matrix-assisted laser desorption/ionization (MALDI), time-of-flight
(TOF) instruments, or a three-dimensional quadrupole ion traps on
the protein of interest.
Other Methods of Detection
[0531] Additional detection methods involve bipyridines, metal
coordination, nanotechnology (gold), biotin-streptavidin/avidin,
UV/Vis, 2 step systems that involve a binding event and a coupling
event due to proximity of a non-natural amino acid to a target
resulting in exmission from a fluorophore, small molecule based
fluorescent/fluorogenic molecules bound to a non-natural amino acid
present in a polypeptide, lipocalins (beta barrel), fatty acid
binding proteins, and dark to light or light to dark
fluorophores.
[0532] XV. Imaging and Diagnostics
[0533] Methods for imaging and diagnostics utilizing non-natural
amino acids, non-natural amino acid polypeptides, modified
non-natural amino acid polypeptides and fragments thereof, are
disclosed herein.
[0534] Molecular Imaging is a multidisciplinary field involving the
efforts from molecular and cell biology to identify the molecular
imaging target, radiochemistry and bioconjugation chemistry to
develop suitable imaging probes, pharmacology to optimize the
probes for optimal targeting efficacy and favorable in vivo
kinetics, and image-capture techniques to non-invasively monitor
the fate of molecular imaging probes in vivo. Aside from its basic
diagnostic applications, molecular imaging also plays roles in
treatment efficacy assessment, drug discovery, and understanding of
molecular mechanisms in living systems. Molecular imaging probes
(monoclonal antibodies, minibodies, proteins, peptides and
peptidomimetics) can be used for visualization and quantification
of molecular targets. The combination of anatomical (microMRI and
microCT) and molecular imaging techniques (microPET, microSPECT,
and NIR fluorescence imaging) can allow obtaining molecular and
functional information, and monitor specific molecular therapeutic
efficacy. Bio-imaging methods can be used to detect spatial
organization (i.e., distribution) and to quantify cellular and
tissue natural constituents, structures, organelles and
administered components such as tagging probes (e.g., fluorescent
probes) and drugs using light transmission, reflection, scattering
and fluorescence emission strategies, with high spatial and
spectral resolutions.
[0535] In-vivo competition assays of unlabeled compounds with
labeled probes for agents with known pharmacological
characteristics and efficacy can be used in the drug evaluation
process. Noninvasive characterization of drug targeting, receptor
occupancy, concentrations required for effective receptor or enzyme
inhibition, etc., can speed up the evaluation of lead compounds. As
new drug candidates proceed through pharmacodynamic and
pharmacokinetic studies, imaging analyses can quantitatively and
repetitively monitor target accessibility, duration of retention at
the target site and its correlation with drug efficacy, and
clearance from irrelevant tissues.
[0536] In clinical trials, imaging assays can facilitate evaluation
of non-natural amino acids, non-natural amino acid polypeptides,
modified non-natural amino acid polypeptides and fragments thereof,
for both their pharmacological properties and their therapeutic
effectiveness in patients. By combining imaging probes with
multimodality-imaging instruments that merge structural and
functional data, physicians can perform multiple functional-imaging
assays simultaneously with anatomic analyses. Information derived
from structural studies and from noninvasive, repetitive monitoring
of drug distribution and concentration can then be correlated with
biological effects on signal transduction pathways, target enzyme
activities, antigen levels, receptor activation, cell
proliferation, proteasome activity, etc. These noninvasive assays
can permit real-time monitoring and modification of targeted
interventions and therapeutic strategies. Molecular-imaging
technologies can be used to study mouse models in pre-clinical
studies. For example, many drugs for cancer and other disorders
exert their therapeutic effects by inducing apoptosis. The ability
to repetitively image apoptotic responses in living animals can
facilitate preclinical evaluation of these drugs. For studying
transgenic mice, identification of founder mice that can express
the transgene in the proper spatial and temporal pattern by
noninvasive imaging can permit the identification of founders
without breeding.
[0537] Molecular imaging can provide the location, magnitude, and
duration of expression of the therapeutic gene for the optimization
of gene-therapy protocols. Optical imaging can be coupled with
targeted gene transfer. Molecular imaging of reporter genes can
also be used to monitor the biodistribution and efficacy of
cell-based therapies.
[0538] Imaging Probes
[0539] Imaging probes can be molecules labeled with radioisotopes
or light- or nearinfrared (NIR)--emitting molecules. The
concentration and/or spectral properties of molecular imaging
probes are altered by the specific biological process under
investigation. Two types of probes that can be used in functional
imaging studies are, by way of example only, direct binding probes
and indirect probes. Direct binding probes and indirect probes may
be non-natural amino acid polypeptides. Examples of direct binding
probes include but are not limited to antibodies, antibody
fragments, antigen-binding polypeptides and fragments thereof and
receptor ligands. Direct probes can be used to detect
concentrations of their targets, since their binding is
stoichiometric. Therefore, direct probes are useful in
investigating targets that are overexpressed in pathological
conditions, for example, before and after therapy. Indirect probes
are used to monitor activities of their macromolecular targets,
including catalytic activities. Examples of such probes are
described by Herschman in Science 2003 302:605-608.
[0540] Probes can be developed to monitor endogenous targeted
molecules and biological processes. Such probes may be (modified)
non-natural amino acid polypeptides. Key mediators and/or
indicators of endogenous processes may be investigated using
imaging probes. Substrates for enzymes such as kinases or proteases
may be labeled via radionuclides or fluorescent molecules such that
events such as phosphorylation or protease cleavage are detected by
molecular-imaging assays. Such fluorescent probes that emit NIR
fluorescent light after protease cleavage may be referred to as
"activatable" optical imaging probes.
[0541] Direct and indirect probes may be discovered by
high-throughput screening of chemical libraries. Direct probes may
also be discovered by screening large recombinant antibody and
phage libraries. Such libraries may be composed of (modified)
non-natural amino acid polypeptides.
[0542] Quantum dots: Methods for imaging and diagnostics utilizing
non-natural amino acids, non-natural amino acid polypeptides,
modified non-natural amino acid polypeptides and fragments thereof
disclosed herein include fluorescent semiconductor nanocrystals
(also known as quantum dots or qdots). Qdots can be used for the
study of intracellular processes at the single-molecule level,
high-resolution cellular imaging, long-term in vivo observation of
cell trafficking, tumor targeting, and diagnostics.
[0543] Colloidal semiconductor quantum dots are single crystals a
few nanometers in diameter whose size and shape can be precisely
controlled by the duration, temperature, and ligand molecules used
in the synthesis. This process may yield qdots that have
composition- and size-dependent absorption and emission. Absorption
of a photon with energy above the semiconductor band gap energy may
result in the creation of an electron-hole pair (or exciton). The
absorption may have an increased probability at higher energies
(i.e., shorter wavelengths) and result in a broadband absorption
spectrum, in marked contrast to standard fluorophores. For
nanocrystals smaller than the so-called Bohr exciton radius (a few
nanometers), energy levels may be quantized, with values directly
related to the qdot size (an effect called quantum confinement,
hence the name "quantum dots"). The radiative recombination of an
exciton (characterized by a long lifetime, >10 ns) may lead to
the emission of a photon in a narrow, symmetric energy band. The
long fluorescence lifetime of qdots may enable the use of
time-gated detection to separate their signal from that of shorter
lived species (such as background autofluorescence encountered in
cells).
[0544] Single qdots can be observed and tracked over an extended
period of time with, for example, confocal microscopy, total
internal reflection microscopy, or basic wide-field epifluorescence
microscopy. Fluorescence correlation spectroscopy may allow
determination of the brightness per particle and also provide a
measurement of the average qdot size. Qdots can also be used as
probes for two-photon confocal microscopy because they are
characterized by a very large absorption cross section. They can be
used simultaneously with standard dyes. Qdots have a potential as
customizable donors of a fluorescence resonance energy transfer
(FRET) pair.
[0545] For applications such as qdot tagging of a target molecule
such as a non-natural amino acid polypeptide, a single recognition
moiety can be grafted to the qdot (e.g., DNA oligonucleotide or
aptamer, antibody, antibody fragment, antigen-binding polypeptide,
etc.) or, used as the qdot solubilization ligand. Qdot ligands
containing either an amine or a carboxyl group, for example, may
offer a possibility of cross-linking molecules containing a thiol
group or an N-hydroxysuccinimyl ester moiety by means of standard
bioconjugation reactions. Another approach can be to use
electrostatic interactions between qdots and charged adapter
molecules, or between qdots and proteins modified to incorporate
charged domains. These functionalization steps can be repeated to
add or change functionality. For instance, streptavidin-coated
qdots can be used in combination with biotinylated proteins or
antibodies. A three-layer approach such as, using (i) an antibody
against a specific target, (ii) a biotinylated secondary antibody
against the first, and (iii) a streptavidin-coated qdot can allow
qdot labeling of non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof, as disclosed herein.
[0546] A number of potential surface attachment groups can be used
to "graft" different functionalities to individual qdots, resulting
in multipotent probes. For instance, in addition to a recognition
moiety, qdots can be equipped with a membrane-crossing or
cell-internalization capability, and/or an enzymatic function.
Peptides can be customized, and with a choice of sequence, a
single-step surfactant exchange can yield necessary functions: (i)
protect the core/shell structure and maintain the original qdot
photophysics, (ii) solubilize qdots, (iii) provide a biological
interface, and (iv) allow the incorporation of multiple functions.
The resulting particles can have colloidal properties,
photophysics, and biocompatibility, and this "peptide toolkit" can
be tailored to provide additional functionalities. Such
functionalities can be improved by molecular evolution.
[0547] Live-cell experiments such as, whole-cell labeling, labeling
of membrane-bound proteins, and cytoplasmic or nuclear target
labeling can be used for cell or pathogen detection, cell tracking,
and cell lineage studies. This can be achieved without any
functionalization through microinjection, electroporation, or
phagocytosis of qdots. Different types of functionalization can be
explored as a way to target qdots to cell surface proteins. Some
examples include streptavidin, secondary, or primary antibodies,
receptor ligands such as epidermal growth factor (EGF) or
serotonin, recognition peptides, and affinity pairs such as
biotin-avidin after engineering of the target protein. Another
strategy may consist of cross-linking primary antibodies to qdots.
Some proteins can be recognized by peptides, so peptides can be
used for qdot functionalization. Microinjection can allow the
delivery of qdots functionalized with the appropriate targeting
peptide sequence to mitochondria or the cell nucleus. The long-term
stability and brightness of qdots make them a candidate for live
animal targeting and imaging.
[0548] In synthesis, new compositions could entail qdots with
properties such as (i) sensitivity to electric or magnetic fields;
(ii) narrower fluorescence emission and longer lifetimes (using
lanthanide-doped qdots); (iii) smaller sizes and extension to the
NIR spectrum, as demonstrated by ternary alloys; (iv) end-specific
functionalizations of nanorod qdots; (v) suppression of blinking
and quantum yield enhancement; and (vi) built-in on-off switches or
photoelectric biotransducers.
[0549] Biotransducer, light-excited qdots could transfer their
charge to bound enzymes functioning as electron or hole acceptors,
enabling their control by light activation. Reciprocally, qdots
could be lit up by electron or hole donor enzymes through
chemiluminescence. Peptide coating of nano-materials can be a tool
for imparting novel functions to the organic-inorganic interface.
The simultaneous engineering of the semiconductor's band gap (by
rational design) with the peptide's redox potential (by molecular
evolution) could be used to optimize qdot compositions and peptide
sequences for binding and desired optical, electronic, magnetic,
and chemical properties. In summary, different shapes, end
specificities, and compositions can lead to more complex
bioinorganic architectures that could be exploited as an
optoelectronic interface to the cellular machinery.
[0550] Qdots can be used as contrast reagents for functional
imaging with a combination of MRI, PET, computed tomography, and IR
fluorescence imaging (the latter by direct imaging through the
epidermis or by a catheter-based confocal fiber microscope). In
vivo optical biopsy could confirm the pathology, and therapy could
then be performed selectively, locally, and temporally by
depositing energy (monochromatic x-rays for k-shell absorption or
laser IR radiation) into the targeted qdots. Alternatively, it may
be possible to graft therapeutic enzymes to the qdot surface and
activate them by light, or produce free radicals (such as singlet
oxygen) by optically cycling the qdots.
[0551] Imaging Instrumentation
[0552] Various instrumentation can be used for imaging and
diagnostics of non-natural amino acids, non-natural amino acid
polypeptides, modified non-natural amino acid polypeptides and
fragments thereof, as disclosed herein.
[0553] Monitoring the probes may consist of (1) a measurement
system, and (2) an analysis software. The measurement system may
include all of the optics, electronics and the manner in which the
sample is illuminated (e.g., light source selection), the mode of
measurement (e.g., fluorescence or transmission), as well as the
calibration best suited for extracting the desired results from the
measurement. The analysis software may include all of the software
and mathematical algorithms necessary to analyze and display
important results in a meaningful way. The measurement can be
carried out using virtually any optical system attached to the
system, for example, an upright or inverted microscope, a
fluorescence microscope, a macro lens, an endoscope and a fundus
camera. Furthermore, any standard experimental method can be used,
including light transmission (bright field and dark field),
auto-fluorescence and fluorescence of administered probes, etc.
Fluorescence measurements can be made with any standard filter cube
(consisting of a barrier filter, excitation filter and a dichroic
mirror), or any customized filter cube for special applications,
provided the emission spectra fall within the spectral range of the
system sensitivity.
[0554] Spectral bio-imaging can also be used in conjunction with
any standard spatial filtering method such as dark field and phase
contrast, and even with polarized light microscopy.
Radionuclide-labeled probes can be detected by PET or SPECT
(single-photon emission tomography), probes emitting light
(fluorescence, bioluminescence, or NIR emissions) can be detected
by optical imaging, and radiowave emissions can be detected by MRI.
Small-animal devices can be used for radionuclide-based imaging
(e.g., microSPECT and microPET), optical imaging of visible light
(using sensitive, cooled charged-coupled device (CCD) cameras), and
NIR emissions. The combination of anatomical (microMRI and microCT)
and molecular imaging techniques (microPET, microSPECT, and NIR
fluorescence imaging) can help obtain molecular and functional
information, and monitor specific molecular therapeutic
efficacy.
[0555] Noninvasive reporter gene assays can be used for
molecular-imaging studies of living animals. Radionuclide-labeled
probes can be used to monitor, in living mice, the expression of
reporter genes using the direct-binding FESP probe, or the herpes
simplex virus type 1-thymidine kinase (HSV1-TK). HSV1-TK can be
monitored with positron-labeled thymidine analogs. Like FDG, the
indirect substrate probe for hexokinase, positron-labeled
substrates for HSV1-TK can be retained in cells as a result of
enzyme dependent phosphorylation. For optical-imaging assays, the
light produced by the enzymes from their substrates can be
monitored with sensitive CCD cameras. New reporter genes encoding
fusion proteins that can be imaged with fluorescent,
bioluminescent, or radionuclide probes can allow study of a single
animal with a number of different imaging probes and
instrumentation appropriate for distinct applications.
[0556] MicroPET instrumentation can provide better anatomic
discrimination of functional assays: for example, pinpointing the
locations of tumors within organs, determining the location of cell
migration more accurately, etc. Fluorescence-mediated tomography
can improve the resolution and quantitation of optical imaging
procedures. Spectral-imaging technologies can discriminate
emissions from multiple fluorescent probes, permitting simultaneous
analysis of distinct optical probes and dramatically reducing
background autofluorescence.
Non-Natural Amino Acid-Scanning of Polypeptides and Libraries.
[0557] The identification of amino acids to be substituted in order
to modulate activities or properties of the polypeptide may be done
by site-directed mutagenesis. Amino acids in the polypeptides and
polypeptide libraries of the present invention that modulate
function can be identified or modulated by substituting a
non-naturally encoded amino acid in place of a natural amino acid
at any or all positions of the polypeptide. Naturally encoded amino
acids may be substituted into a selected position of a polypeptide
by methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (See, e.g., Cunningham et al. 1989),
which disclosure is hereby incorporated by reference in its
entirety. The alanine-scanning mutagenesis procedure introduces
single alanine mutations at selected or every residue in the
molecule. Instead of substituting the naturally encoded amino acid
alanine, a non-naturally encoded amino acid is substituted for a
naturally encoded amino acid in the polypeptide chain. The
resulting mutant polypeptide molecules comprising a non-naturally
encoded amino acid are then tested for biological activity using
assays appropriate for measuring the function of the particular
polypeptide or protein. Of special interest may be substitutions of
non-naturally encoded charged amino acids or non-naturally encoded
neutral amino acids for the naturally encoded charged and/or
neutral amino acids. These substitutions may produce proteins with
highly desirable improved or modulated characteristics, such as
modulated receptor binding, modulated enzymatic activity, modulated
antigen binding, or modulated aggregation or solubility.
EXAMPLES
[0558] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
[0559] This example describes conjugates that may be formed with
non-natural amino acid polypeptides. Molecules may be directly
bonded to one or more non-natural amino acids in a polypeptide or
may be attached via a linker, polymer, water soluble polymer, or
biologically active molecule.
[0560] FIG. 9 presents non-limiting examples of molecules that are
site specifically attached to polypeptides via a reaction that
forms an oxime bond between the carbonyl of a non-natural amino
acid incorporated into a polypeptide and the hydroxylamine of the
molecule. Molecules including but not limited to, fluorophores,
biotin, and chelators may be attached to non-natural amino acid
polypeptides.
Example 2
[0561] Resins or other materials known to those skilled in the art
may be used to isolate polypeptides. FIG. 10 shows an example of a
purification method for a non-natural amino acid polypeptide
utilizing a resin that reacts with the non-natural amino acid. A
covalent linkage is formed between a chemically specific affinity
tag on the resin and a non-natural amino acid present in the
protein. Such linkages are stable under a broad range of pH and
purification conditions. The separation step may be performed in
alternate modes, including but not limited to a bath mode, enabling
the large-scale purifications. The resin and the affinity tags are
physically and chemically stable, and thus, can be reused to reduce
the cost of protein purification upon scale-up.
[0562] The separation can be performed in conjunction with
conjugation of the polypeptide to molecules including but not
limited to, PEG. This "one-pot" method further simplifies the
conjugation process and reduces the cost of production of proteins,
including but not limited to target therapeutic proteins (FIG. 11).
Other molecules that can be conjugated include but are not limited
to fluorophores.
[0563] Resins or other materials for purification can be selected
and functionalized according to the non-natural amino acid present
in the polypeptide. FIG. 12 shows an example of resin selection and
functionalization.
[0564] Resins or other materials for purification can be
functionalized differently depending on the non-natural amino acid
in the polypeptide. For example, FIG. 13 shows an example of
affinity purification of a non-natural amino acid polypeptide using
hydroxylamine resin. FIG. 14 shows an example of purification of a
non-natural amino acid polypeptide using an aldehyde resin.
Non-limiting examples of hydroxylamine and aldehyde resins are
shown.
[0565] In some embodiments, one or more steps of the purification
process modify one or more non-natural amino acids present in the
polypeptide to one or more natural amino acids. FIG. 15 shows an
example of purification of native proteins from a non-natural amino
acid precursor. The non-natural amino acid is converted to tyrosine
after release from the resin used in the purification process. FIG.
16 shows non-limiting examples of non-natural amino acids.
Example 3
Non-Natural Amino Acid-Scanning Mutagenesis
[0566] This example details cloning and expression of a hGH
polypeptide including a non-naturally encoded amino acid in E.
coli. This example also describes one method to assess the
biological activity of modified hGH polypeptides.
[0567] Methods for cloning hGH and fragments thereof are detailed
in U.S. Pat. Nos. 4,601,980; 4,604,359; 4,634,677; 4,658,021;
4,898,830; 5,424,199; and 5,795,745, which are incorporated by
reference herein. cDNA encoding the full length hGH or the mature
form of hGH lacking the N-terminal signal sequence are shown in SEQ
ID NO: 21 and SEQ ID NO: 22 respectively. For the complete
full-length naturally-occurring GH amino acid sequence as well as
the mature naturally-occurring GH amino acid sequence and naturally
occurring mutant, see SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:
3,
[0568] An introduced translation system that comprises an
orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA
synthetase (O-RS) is used to express hGH containing a non-naturally
encoded amino acid. The O-RS preferentially aminoacylates the
O-tRNA with a non-naturally encoded amino acid. In turn the
translation system inserts the non-naturally encoded amino acid
into hGH, in response to an encoded selector codon.
TABLE-US-00001 TABLE 1 O-RS and O-tRNA sequences. SEQ ID NO: 4 M.
jannaschii mtRNA.sub.CUA.sup.Tyr tRNA SEQ ID NO: 5 HLAD03; an
optimized amber supressor tRNA tRNA SEQ ID NO: 6 HL325A; an
optimized AGGA frameshift supressor tRNA tRNA SEQ ID NO: 7
Aminoacyl tRNA synthetase for the incorporation of
p-azido-L-phenylalanine RS p-Az-PheRS(6) SEQ ID NO: 8 Aminoacyl
tRNA synthetase for the incorporation of p-benzoyl-L-phenylalanine
RS p-BpaRS(1) SEQ ID NO: 9 Aminoacyl tRNA synthetase for the
incorporation of propargyl-phenylalanine RS Propargyl-PheRS SEQ ID
NO: 10 Aminoacyl tRNA synthetase for the incorporation of
propargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO: 11 Aminoacyl
tRNA synthetase for the incorporation of propargyl-phenylalanine RS
Propargyl-PheRS SEQ ID NO: 12 Aminoacyl tRNA synthetase for the
incorporation of p-azido-phenylalanine RS p-Az-PheRS(1) SEQ ID NO:
13 Aminoacyl tRNA synthetase for the incorporation of
p-azido-phenylalanine RS p-Az-PheRS(3) SEQ ID NO: 14 Aminoacyl tRNA
synthetase for the incorporation of p-azido-phenylalanine RS
p-Az-PheRS(4) SEQ ID NO: 15 Aminoacyl tRNA synthetase for the
incorporation of p-azido-phenylalanine RS p-Az-PheRS(2) SEQ ID NO:
16 Aminoacyl tRNA synthetase for the incorporation of
p-acetyl-phenylalanine (LW1) RS SEQ ID NO: 17 Aminoacyl tRNA
synthetase for the incorporation of p-acetyl-phenylalanine (LW5) RS
SEQ ID NO: 18 Aminoacyl tRNA synthetase for the incorporation of
p-acetyl-phenylalanine (LW6) RS SEQ ID NO: 19 Aminoacyl tRNA
synthetase for the incorporation of p-azido-phenylalanine RS
(AzPheRS-5) SEQ ID NO: 20 Aminoacyl tRNA synthetase for the
incorporation of p-azido-phenylalanine RS (AzPheRS-6)
[0569] The transformation of E. coli with plasmids containing the
modified hGH gene and the orthogonal aminoacyl tRNA synthetase/tRNA
pair (specific for the desired non-naturally encoded amino acid)
allows the site-specific incorporation of non-naturally encoded
amino acid into the hGH polypeptide. The transformed E. coli, grown
at 37.degree. C. in media containing between 0.01-100 mM of the
particular non-naturally encoded amino acid, expresses modified hGH
with high fidelity and efficiency. The His-tagged hGH containing a
non-naturally encoded amino acid is produced by the E. coli host
cells as inclusion bodies or aggregates. The aggregates are
solubilized and affinity purified under denaturing conditions in 6M
guanidine HCl. Refolding is performed by dialysis at 4.degree. C.
overnight in 50 mM TRIS-HCl, pH 8.0, 40 .mu.M CuSO.sub.4, and 2%
(w/v) Sarkosyl. The material is then dialyzed against 20 mM
TRIS-HCl, pH 8.0, 100 mM NaCl, 2 mM CaCl.sub.2, followed by removal
of the His-tag. See Boissel et al., (1993) 268:15983-93. Methods
for purification of hGH are well known in the art and are confirmed
by SDS-PAGE, Western Blot analyses, or electrospray-ionization ion
trap mass spectrometry and the like.
[0570] The His-tagged mutant hGH proteins were purified using the
ProBond Nickel-Chelating Resin (Invitrogen, Carlsbad, Calif.) via
the standard His-tagged protein purification procedures provided by
the manufacturer, followed by an anion exchange column prior to
loading on the gel. To further assess the biological activity of
modified hGH polypeptides, an assay measuring a downstream marker
of hGH's interaction with its receptor was used. The interaction of
hGH with its endogenously produced receptor leads to the tyrosine
phosphorylation of a signal transducer and activator of
transcription family member, STAT5, in the human IM-9 lymphocyte
cell line. Two forms of STAT5, STAT5A and STAT5B were identified
from an IM-9 cDNA library. See, e.g., Silva et al., Mol.
Endocrinol. (1996) 10(5):508-518. The human growth hormone receptor
on IM-9 cells is selective for human growth hormone as neither rat
growth hormone nor human prolactin resulted in detectable STAT5
phosphorylation. Importantly, rat GHR (L43R) extra cellular domain
and the G120R bearing hGH compete effectively against hGH
stimulated pSTAT5 phoshorylation.
[0571] IM-9 cells were stimulated with hGH polypeptides of the
present invention. The human IM-9 lymphocytes were purchased from
ATCC (Manassas, Va.) and grown in RPMI 1640 supplemented with
sodium pyruvate, penicillin, streptomycin (Invitrogen, Carlsbad,
San Diego) and 10% heat inactivated fetal calf serum (Hyclone,
Logan, Utah). The IM-9 cells were starved overnight in assay media
(phenol-red free RPMI, 10 mM Hepes, 1% heat inactivated
charcoal/dextran treated FBS, sodium pyruvate, penicillin and
streptomycin) before stimulation with a 12-point dose range of hGH
polypeptides for 10 min at 37.degree. C. Stimulated cells were
fixed with 1% formaldehyde before permeabilization with 90%
ice-cold methanol for 1 hour on ice. The level of STAT5
phosphorylation was detected by intra-cellular staining with a
primary phospho-STAT5 antibody (Cell Signaling Technology, Beverly,
Mass.) at room temperature for 30 min followed by a PE-conjugated
secondary antibody. Sample acquisition was performed on the FACS
Array with acquired data analyzed on the Flowjo software (Tree Star
Inc., Ashland, Oreg.). EC.sub.50 values were derived from dose
response curves plotted with mean fluorescent intensity (MFI)
against protein concentration utilizing SigmaPlot.
[0572] Table 2 below summarizes the IM-9 data generated with mutant
hGH polypeptides. Various hGH polypeptides with a non-natural amino
acid substitution at different positions were tested with human
IM-9 cells as described. Substitutions shown were made with
p-acetyl phenylalanine at the positions indicated. The same assay
was used to assess biological activity of hGH polypeptides
comprising a non-natural amino acid that is PEGylated. From the
data shown in the table, it is apparent that there are differences
in receptor binding activity depending upon the position in which
the non-naturally encoded amino acid was substituted for a
naturally encoded amino acid.
TABLE-US-00002 TABLE 2 GH EC.sub.50 (nM) WHO WT 0.4 .+-. 0.1 (n =
8) N-6His WT 0.6 .+-. 0.3 (n = 3) rat GH WT >200,000 Y35pAF 0.7
.+-. 0.2 (n = 4) E88pAF 0.9 Q91pAF 2.0 .+-. 0.6 (n = 2) F92pAF 0.8
.+-. 0.4 (n = 9) R94pAF 0.7 S95pAF 16.7 .+-. 1.0 (n = 2) N99pAF 8.5
Y103pAF 130,000 Y111pAF 1.0 G120R >200,000 G120pAF >200,000
G131pAF 0.8 .+-. 0.5 (n = 3) P133pAF 1.0 R134pAF 0.9 .+-. 0.3 (n =
4) T135pAF 0.9 G136pAF 1.4 F139pAF 3.3 K140pAF 2.7 .+-. 0.9 (n = 2)
Y143pAF 0.8 .+-. 0.3 (n = 3) K145pAF 0.6 .+-. 0.2 (n = 3) A155pAF
1.3
Example 4
[0573] This example details cloning and expression of a modified
hIFN polypeptide in E. coli.
[0574] This example demonstrates how a hIFN polypeptide including a
non-naturally encoded amino acid can be expressed in E. coli. See
Nagata et. al., Nature, vol. 284, 316-320 (1980) and U.S. Pat. No.
4,364,863. cDNA encoding the full length hIFN and the mature form
of hIFN lacking the N-terminal signal sequence are shown in SEQ ID
NO: 23 and SEQ ID NO: 24, respectively. The full length and mature
hIFN encoding cDNA is inserted into the pBAD HISc, pET20b, and
pET19b expression vectors following optimization of the sequence
for cloning and expression without altering amino acid
sequence.
[0575] An introduced translation system that comprises an
orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA
synthetase (O-RS) is used to express hGH containing a non-naturally
encoded amino acid. The O-RS preferentially aminoacylates the
O-tRNA with a non-naturally encoded amino acid. In turn the
translation system inserts the non-naturally encoded amino acid
into hGH, in response to an encoded selector codon.
[0576] O-RS and O-tRNA sequences suitable for use with Interferon
expression include those shown in Example 3. The transformation of
E. coli with plasmids containing the modified hIFN gene and the
orthogonal aminoacyl tRNA synthetase/tRNA pair (specific for the
desired non-naturally encoded amino acid) allows the site-specific
incorporation of non-naturally encoded amino acid into the hIFN
polypeptide. The transformed E. coli, grown at 37.degree. C. in
media containing between 0.01-100 mM of the particular
non-naturally encoded amino acid, expresses modified hIFN with high
fidelity and efficiency. The His-tagged hIFN containing a
non-naturally encoded amino acid is produced by the E. coli host
cells and are affinity purified. Methods for purification of hIFN
are well known in the art and are confirmed by SDS-PAGE, Western
Blot analyses, or electrospray-ionization ion trap mass
spectrometry and the like.
Binding Assays.
[0577] The hIFN receptor was prepared as described in U.S. Pat.
Nos. 6,566,132; 5,889,151; 5,861,258; 5,731,169; 5,578,707, which
are incorporated by reference herein. For a non-PEGylated
polypeptide comprising a non-natural amino acid, the affinity of
the hormone for its receptor was measured by using a BLAcore.TM.
biosensor (Pharmacia) technique, which is known in the art. BIAcore
biosensor assays were used to measure the binding characteristics
of hIFN molecules that comprised a non-naturally encoded amino acid
substituted at the positions shown in Table 3, along with the
receptor binding data. From the data shown in the table, it is
apparent that there are differences in receptor binding activity
depending upon the position in which the non-naturally encoded
amino acid was substituted for a naturally encoded amino acid.
TABLE-US-00003 TABLE 3 IFN.alpha.2A Variants Kd (nM) Sigma
IFN.alpha.A 11 6His-IFN.alpha.2A 6 C1S IFNa2A 11 C1S E107pAF 9
6His-F36S 1300 6His-F38L 18 6His-F38S 42 6His-L9pAF 14 6His-R12pAF
8 6His-R13pAF 14 6His-M16pAF 18 6His-I24pAF 5 6His-F27pAF 8
6His-K31pAF 52 6His-H34pAF 4 6His-G37pAF 12 6His-P39pAF 17
6His-E41pAF 16 6His-N45pAF 7 6His-Q48pAF 17 6His-K49pAF 10
6His-Q61pAF 21 6His-N65pAF 7 6His-E78pAF 7 6His-Y89pAF 9
6His-E96pAF 12 6His-I100pAF 10 6His-G102pAF 27 6His-V103pAF 14
6His-T106pAF 8 6His-E107pAF 5 6His-P109pAF 17 6His-L110pAF 13
6His-E113pAF 19 6His-L117pAF 8 6His-R120pAF 4 6HisY122S 300
6His-R125pAF 19 6His-K134pAF 10 6His-R149pAF 75 6His-E159pAF
3.5
Example 5
[0578] Conjugates and complexes between proteins and
oligonucleotides have wide applications in diagnosis and
therapeutic, such as immunoPCR, gene therapeutic and more recently
targeted delivery of RNAi. Site-specific conjugation enables
production of specifically designed molecules and nano structures
that have novel functions. Currently, the site-specific
conjugations have been achieved mainly through maleimide chemistry,
in which an engineered protein surface cysteine selectively reacts
with maleimide to form a thioether. The development of
site-specific incorporation of unnatural amino acids into
polypeptides has enabled a large array of chemistries for
conjugation of molecules to proteins. Over 30 non-naturally encoded
amino acids have been incorporated site-specifically into proteins.
In this example using the unnatural amino acid described below as a
handle, oligo nucleotides were conjugated to proteins
site-specifically. Furthermore, using single strand DNA as
template, the conjugated proteins were assembled in one dimension
in a defined manner.
[0579] Protein used in this experiment was human growth hormone Y35
mutant, in which the tyrosine 35 was replaced by the non-naturally
encoded amino acid 9.2 (Scheme 1). The single strand DNAs were
stored as 25 mM solutions in water at -80.degree. C. The sequence
of ssDNA FTam27 is 5'-CAG CCA GCG TGC ACG (SEQ ID NO:21). The 5' of
FTam27 was modified with hydrazide. The sequence for the templates
are FTam28-d1: 5'-CGT GCA CGC TGG CTG CGT GCA CGC TGG CTG (SEQ ID
NO:21); FTam-d2: 5'-CGT GCA CGC TGG CTG T CGT GCA CGC TGG CTG (SEQ
ID NO:22); FTam28-d3: 5'-CGT GCA CGC TGG CTG TT CGT GCA CGC TGG
CTG; FTam28-t1 (SEQ ID NO:23); 5'-CGT GCA CGC TGG CTG CGT GCA CGC
TGG CTG CGT GCA CGC TGG CTG (SEQ ID NO:24); FTam28-t2: 5'-CGT GCA
CGC TGG CTG T CGT GCA CGC TGG T CTG CGT GCA CGC TGG CTG (SEQ ID
NO:25); FTam28-t3: 5'-CGT GCA CGC TGG CTG TT CGT GCA CGC TGG TT CTG
CGT GCA CGC TGG CTG (SEQ ID NO:26).
[0580] Protein-single strand DNA conjugation:
[0581] Protein (1 mg) was buffer exchanged into reaction buffer
(150 mM NaCl, 20 mM NaOAc, 400 mM Arg, 5 mM EDTA, pH 4.0) using PD
10 gel filtration columns. The protein solution was concentrated to
90 .mu.l using 10 kD MWCO CENTROCON (Vivascience). Five .mu.l of
the water solution of 25 mM ssDNA FTam27, which has a 5'
modification of hydrazide, was dispensed in 40 .mu.l of reaction
buffer. The ssDNA solution was added slowly into the protein
solution. Precipitation appeared initially, but dissolved. 20 hours
after incubation at 28.degree. C., 5 mM sodium cyanoboronhydride
was added. The reaction mixture was incubated for another 20 hours
and subjected to analysis and purification.
Purification of Conjugate:
[0582] A 1 ml phenyl HIC column was employed for the FPLC
purification of the conjugate. Buffer A: 2 M NaCl, 10 mM Tris.HCl,
pH 7.0; Buffer B: 10 mM Tris.HCl, pH 7.0. The gradient used in the
purification was: 10 column volume (CV) 0% B, 5 CV to 50% B, hold
at 50% B for 5 CV, then 30 CV to 100% B. Purified conjugate was
concentrated, buffer-exchanged to storage buffer (200 mM NaCl, 50
mM Tris.HCl, 1 mM EDTA, pH 8.0) and subjected to PAGE analysis
using 4-12% SDS gel, at 200 V in MES buffer.
Hybridization:
[0583] Five .mu.l of protein-ssDNA conjugate was added to the
complementary ssDNA in storage buffer (200 mM NaCl, 50 mM Tris.HCl,
1 mM EDTA, pH 8.0). The mixtures were supplemented with storage
buffer to give a final volume of 20 .mu.l and heated at 42.degree.
C. for 30 seconds, then cooled to room temperature. The final
products were analyzed by native TRIS-glycine gel electrophoresis
at 125 V, 4.degree. C. for 3 to 5 hours.
##STR00006##
[0584] Non-naturally encoded amino acid 9.2, which has a 1,3
diketone moiety, was incorporated into human growth hormone (hGH)
at amino acid position 35, and used as a handle for the conjugation
with a 15 mer single strand DNA, FTam27, modified at the 5' with
hydrazide functional group (Scheme 1). This conjugation resulted in
a hydrazone initially, which is further reduced with sodium
cyanoborohydride to give an irreversible covalent linkage. With
five fold excess of ssDNA, a 70% yield was obtained (FIG. 17). The
conjugate was purified to about 90% pure using HIC column and
subject to hybridization.
[0585] The conjugate was designed to hybridize with ssDNAs that
have two (d) or three (t) tandem complementary sequence (FTam28)
repeats with zero (1), one (2) and two (3) bases T between them as
spacers (FIG. 18). To determine the relative concentration of
hGH-DNA conjugate, 5 .mu.l of hGH-ssDNA conjugate was mixed with a
series concentration of FTam28-d3, a single strand DNA that has two
repeating sequences complementary to FTam27 and two T bases as a
spacer between them. The result was analyzed with 14% native
glycine gel electrophoresis, 125 V, 3 hr at 4.degree. C. (FIG. 19).
The most complete hybridization was with 5 .mu.l hGH-ssDNA mixed
with 4 .mu.l of 10 .mu.M FTam28-d3 which gave a conjugate
concentration of about 16 .mu.M. According to the gel, hGH-ssDNA
and hGH-ssDNA hybrid monomer with FTam28-d3, were more mobile than
hGH itself, presumably due to the large number of negative charges
on the DNA backbone.
[0586] These phenomena were also demontrated in a control
experiment (FIG. 20). When hGH was mixed with 1 .mu.l of 100 .mu.M
FTam28-d3, no hybridization was observed (lane 4). On the other
hand, when 1 .mu.l of 100 .mu.M FTam28-d3 mixed with hGH-ssDNA
conjugate, hGH dimer is formed through hybridization. There is no
non-specific interaction between hGH and the DNA. The dimerization
of conjugated hGH was the result of specific DNA hybridization.
When a large excess of FTam28-d3 was added, more hybrid monomer and
less hybrid dimer were formed. There was a substantial amount of
hybrid dimer present when 80 pico mole of hGH-ssDNA conjugate was
mixed with 10 equivalents of FTam28-d3 (lane 3). This indicated
that the hybrid dimer was more stable than the hybrid monomer
thermodynamically.
[0587] To demonstrate assembly of protein-ssDNA in a well-defined
manner (FIG. 21), six one dimension structures of hGH using single
strand DNA as templates were assembled. These structures varied by
different valency and spacers between each hGH molecule. hGH-ssDNA
conjugate was mixed with one equivalent of each of the DNA
templates. The mixtures were incubated at 50.degree. C. for 5
minutes, cooled to room temperature and analyzed on a native
glycine gel. These 1-D structures were assembled highly
efficiently. Lane 1 to lane 3 show the results of dimer formation
with spacers of zero, one and two, respectively, T bases between
the DNA sequence repeats. Lane 4 to lane 6 show the assembly
results of trimer formation with spacers of zero, one and two T
bases as spacer.
[0588] Using non-naturally encoded amino acids as a chemical
handle, single strand DNA was conjugated to the protein surface
site-specifically. This single strand DNA-protein conjugate can be
used to assemble protein 1-D structures highly efficiently using
DNA as a template. Site-specific oligonucleotide conjugation can
also be used to assemble well defined 3-D structures creating novel
nano structures with novel functions. Moreover, the protein-oligo
nucleotide conjugation technology may be applied to create protein
drug "plug and play" libraries. In this case, the oligonucleotide
may be used as both a linkage and a "name tag" to encode the
individual small molecule and/or protein. The protein-oligo
nucleotide conjugate may be used in immunoPCR for diagnostic
applications. This technology can also be used to create protein
RNA or PNA conjugates which can be used in targeted RNAi
therapeutics.
[0589] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
those of ordinary skill in the art and are to be included within
the spirit and purview of this application and scope of the
appended claims. While preferred embodiments of the present
invention have been shown and described herein, it will be obvious
to those skilled in the art that such embodiments are provided by
way of example only. Numerous variations, changes, and
substitutions will now occur to those skilled in the art without
departing from the invention. It should be understood that various
alternatives to the embodiments of the invention described herein
may be employed in practicing the invention. It is intended that
the following claims define the scope of the invention and that
methods and structures within the scope of these claims and their
equivalents be covered thereby.
Sequence CWU 1
1
301217PRTHomo sapiens 1Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu
Ala Phe Gly Leu Leu1 5 10 15Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala
Phe Pro Thr Ile Pro Leu20 25 30Ser Arg Leu Phe Asp Asn Ala Met Leu
Arg Ala His Arg Leu His Gln35 40 45Leu Ala Phe Asp Thr Tyr Gln Glu
Phe Glu Glu Ala Tyr Ile Pro Lys50 55 60Glu Gln Lys Tyr Ser Phe Leu
Gln Asn Pro Gln Thr Ser Leu Cys Phe65 70 75 80Ser Glu Ser Ile Pro
Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys85 90 95Ser Asn Leu Glu
Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp100 105 110Leu Glu
Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val115 120
125Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu
Glu130 135 140Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly
Ser Pro Arg145 150 155 160Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser
Lys Phe Asp Thr Asn Ser165 170 175His Asn Asp Asp Ala Leu Leu Lys
Asn Tyr Gly Leu Leu Tyr Cys Phe180 185 190Arg Lys Asp Met Asp Lys
Val Glu Thr Phe Leu Arg Ile Val Gln Cys195 200 205Arg Ser Val Glu
Gly Ser Cys Gly Phe210 2152191PRTHomo sapiens 2Phe Pro Thr Ile Pro
Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg1 5 10 15Ala His Arg Leu
His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu20 25 30Glu Ala Tyr
Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro35 40 45Gln Thr
Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg50 55 60Glu
Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu65 70 75
80Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val85
90 95Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr
Asp100 105 110Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met
Gly Arg Leu115 120 125Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe
Lys Gln Thr Tyr Ser130 135 140Lys Phe Asp Thr Asn Ser His Asn Asp
Asp Ala Leu Leu Lys Asn Tyr145 150 155 160Gly Leu Leu Tyr Cys Phe
Arg Lys Asp Met Asp Lys Val Glu Thr Phe165 170 175Leu Arg Ile Val
Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe180 185 1903176PRTHomo
sapiens 3Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met
Leu Arg1 5 10 15Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln
Glu Phe Asn20 25 30Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro
Thr Pro Ser Asn35 40 45Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu
Leu Leu Arg Ile Ser50 55 60Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro
Val Gln Phe Leu Arg Ser65 70 75 80Val Phe Ala Asn Ser Leu Val Tyr
Gly Ala Ser Asp Ser Asn Val Tyr85 90 95Asp Leu Leu Lys Asp Leu Glu
Glu Gly Ile Gln Thr Leu Met Gly Arg100 105 110Leu Glu Asp Gly Ser
Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr115 120 125Ser Lys Phe
Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn130 135 140Tyr
Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr145 150
155 160Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly
Phe165 170 175477DNAMethanococcus jannaschii 4ccggcggtag ttcagcaggg
cagaacggcg gactctaaat ccgcatggcg ctggttcaaa 60tccggcccgc cggacca
77588DNAHalobacterium sp. NRC-1 5cccagggtag ccaagctcgg ccaacggcga
cggactctaa atccgttctc gtaggagttc 60gagggttcga atcccttccc tgggacca
88689DNAHalobacterium sp. NRC-1 6gcgagggtag ccaagctcgg ccaacggcga
cggacttcct aatccgttct cgtaggagtt 60cgagggttcg aatccctccc ctcgcacca
897306PRTMethanococcus jannaschii 7Met Asp Glu Phe Glu Met Ile Lys
Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg Glu Val
Leu Lys Lys Asp Glu Lys Ser Ala Gly20 25 30Ile Gly Phe Glu Pro Ser
Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys Met Ile
Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu Ala Asp
Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75 80Glu Ile
Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85 90 95Gly
Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys100 105
110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu
Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp
Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln
Val Asn Thr Tyr Tyr145 150 155 160Tyr Leu Gly Val Asp Val Ala Val
Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala Arg Glu
Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro Val Leu
Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200 205Lys Gly
Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala210 215
220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn
Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro
Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr
Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys Asn Lys
Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala Glu Glu
Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu3058306PRTMethanococcus jannaschii 8Met Asp Glu Phe Glu Met Ile
Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg Glu
Val Leu Lys Lys Asp Glu Lys Ser Ala Gly20 25 30Ile Gly Phe Glu Pro
Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys Met
Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu Ala
Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75 80Glu
Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85 90
95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Thr Ser His145 150 155 160Tyr Leu Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200
205Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg
Ala210 215 220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu
Gly Asn Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu
Tyr Pro Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp
Leu Thr Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys
Asn Lys Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala
Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu3059305PRTMethanococcus jannaschii 9Met Asp Glu Phe Glu Met Ile
Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg Glu
Val Leu Lys Lys Asp Glu Lys Ser Ala Ala20 25 30Ile Gly Phe Glu Pro
Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys Met
Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu Ala
Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75 80Glu
Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85 90
95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Pro Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Ala Ile Tyr145 150 155 160Leu Ala Val Asp Val Ala
Val Gly Gly Met Glu Gln Arg Lys Ile His165 170 175Met Leu Ala Arg
Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn180 185 190Pro Val
Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys195 200
205Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
Lys210 215 220Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly
Asn Pro Ile225 230 235 240Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr
Pro Leu Thr Ile Lys Arg245 250 255Pro Glu Lys Phe Gly Gly Asp Leu
Thr Val Asn Ser Tyr Glu Glu Leu260 265 270Glu Ser Leu Phe Lys Asn
Lys Glu Leu His Pro Met Asp Leu Lys Asn275 280 285Ala Val Ala Glu
Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg290 295
300Leu30510305PRTMethanococcus jannaschii 10Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Pro Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Ile Pro Tyr145 150 155 160Leu Pro Val Asp Val Ala
Val Gly Gly Met Glu Gln Arg Lys Ile His165 170 175Met Leu Ala Arg
Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn180 185 190Pro Val
Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys195 200
205Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
Lys210 215 220Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly
Asn Pro Ile225 230 235 240Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr
Pro Leu Thr Ile Lys Arg245 250 255Pro Glu Lys Phe Gly Gly Asp Leu
Thr Val Asn Ser Tyr Glu Glu Leu260 265 270Glu Ser Leu Phe Lys Asn
Lys Glu Leu His Pro Met Asp Leu Lys Asn275 280 285Ala Val Ala Glu
Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg290 295
300Leu30511305PRTMethanococcus jannaschii 11Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Lys Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Ala Ile Tyr145 150 155 160Leu Ala Val Asp Val Ala
Val Gly Gly Met Glu Gln Arg Lys Ile His165 170 175Met Leu Ala Arg
Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn180 185 190Pro Val
Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys195 200
205Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
Lys210 215 220Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly
Asn Pro Ile225 230 235 240Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr
Pro Leu Thr Ile Lys Arg245 250 255Pro Glu Lys Phe Gly Gly Asp Leu
Thr Val Asn Ser Tyr Glu Glu Leu260 265 270Glu Ser Leu Phe Lys Asn
Lys Glu Leu His Pro Met Asp Leu Lys Asn275 280 285Ala Val Ala Glu
Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg290 295
300Leu30512306PRTMethanococcus jannaschii 12Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Asn Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Pro Leu His145 150 155 160Tyr Gln Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200
205Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg
Ala210 215 220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu
Gly Asn Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu
Tyr Pro Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp
Leu Thr Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys
Asn Lys Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala
Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu30513306PRTMethanococcus jannaschii 13Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu
Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp
Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln
Val Asn Pro Leu His145 150 155 160Tyr Gln Gly Val Asp Val Ala Val
Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala Arg Glu
Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro Val Leu
Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200 205Lys Gly
Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala210 215
220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn
Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro
Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr
Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys Asn Lys
Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala Glu Glu
Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu30514306PRTMethanococcus jannaschii 14Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Pro Val His145 150 155 160Tyr Gln Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200
205Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg
Ala210 215 220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu
Gly Asn Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu
Tyr Pro Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp
Leu Thr Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys
Asn Lys Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala
Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu30515306PRTMethanococcus jannaschii 15Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Pro Ser His145 150 155 160Tyr Gln Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200
205Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg
Ala210 215 220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu
Gly Asn Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu
Tyr Pro Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp
Leu Thr Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys
Asn Lys Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala
Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu30516306PRTMethanococcus jannaschii 16Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Gly Cys His145 150 155 160Tyr Arg Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200
205Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg
Ala210 215 220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu
Gly Asn Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu
Tyr Pro Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp
Leu Thr Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys
Asn Lys Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala
Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu30517306PRTMethanococcus jannaschii 17Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Gly Thr His145 150 155 160Tyr Arg Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200
205Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg
Ala210 215 220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu
Gly Asn Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu
Tyr Pro Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp
Leu Thr Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys
Asn Lys Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala
Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu30518306PRTMethanococcus jannaschii 18Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Gly Gly His145 150 155 160Tyr Leu Gly Val Asp Val
Ile Val Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200
205Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg
Ala210 215 220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu
Gly Asn Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu
Tyr Pro Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp
Leu Thr Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys
Asn Lys Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala
Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu30519306PRTMethanococcus jannaschii 19Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Arg Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Val Ile His145 150 155 160Tyr Asp Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200
205Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg
Ala210 215 220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu
Gly Asn Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu
Tyr Pro Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp
Leu Thr Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys
Asn Lys Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala
Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu30520306PRTMethanococcus jannaschii 20Met Asp Glu Phe Glu Met
Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser1 5 10 15Glu Glu Glu Leu Arg
Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly20 25 30Ile Gly Phe Glu
Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln35 40 45Ile Lys Lys
Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile50 55 60Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp65 70 75
80Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met85
90 95Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp
Lys100 105 110Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys115 120 125Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro130 135 140Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Thr Tyr Tyr145 150 155 160Tyr Leu Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile165 170 175His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His180 185 190Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser195 200
205Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg
Ala210 215 220Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu
Gly Asn Pro225 230 235 240Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu
Tyr Pro Leu Thr Ile Lys245 250 255Arg Pro Glu Lys Phe Gly Gly Asp
Leu Thr Val Asn Ser Tyr Glu Glu260 265 270Leu Glu Ser Leu Phe Lys
Asn Lys Glu Leu His Pro Met Asp Leu Lys275 280 285Asn Ala Val Ala
Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys290 295 300Arg
Leu30521654DNAHomo sapiens 21atggctacag gctcccggac gtccctgctc
ctggcttttg gcctgctctg cctgccctgg 60cttcaagagg gcagtgcctt cccaaccatt
cccttatcca ggctttttga caacgctatg 120ctccgcgccc atcgtctgca
ccagctggcc tttgacacct accaggagtt tgaagaagcc 180tatatcccaa
aggaacagaa gtattcattc ctgcagaacc cccagacctc cctctgtttc
240tcagagtcta ttccgacacc ctccaacagg gaggaaacac aacagaaatc
caacctagag 300ctgctccgca tctccctgct gctcatccag tcgtggctgg
agcccgtgca gttcctcagg 360agtgtcttcg ccaacagcct ggtgtacggc
gcctctgaca gcaacgtcta tgacctccta 420aaggacctag aggaaggcat
ccaaacgctg atggggaggc tggaagatgg cagcccccgg 480actgggcaga
tcttcaagca gacctacagc aagttcgaca caaactcaca caacgatgac
540gcactactca agaactacgg gctgctctac tgcttcagga aggacatgga
caaggtcgag 600acattcctgc gcatcgtgca gtgccgctct gtggagggca
gctgtggctt ctag 65422576DNAHomo sapiens 22ttcccaacca ttcccttatc
caggcttttt gacaacgcta tgctccgcgc ccatcgtctg 60caccagctgg cctttgacac
ctaccaggag tttgaagaag cctatatccc aaaggaacag 120aagtattcat
tcctgcagaa cccccagacc tccctctgtt tctcagagtc tattccgaca
180ccctccaaca gggaggaaac acaacagaaa tccaacctag agctgctccg
catctccctg 240ctgctcatcc agtcgtggct ggagcccgtg cagttcctca
ggagtgtctt cgccaacagc 300ctggtgtacg gcgcctctga cagcaacgtc
tatgacctcc taaaggacct agaggaaggc 360atccaaacgc tgatggggag
gctggaagat ggcagccccc ggactgggca gatcttcaag 420cagacctaca
gcaagttcga cacaaactca cacaacgatg acgcactact caagaactac
480gggctgctct actgcttcag gaaggacatg gacaaggtcg agacattcct
gcgcatcgtg 540cagtgccgct ctgtggaggg cagctgtggc ttctag
57623567DNAHomo sapiens 23atggccttga cctttgcttt actggtggcc
ctcctggtgc tcagctgcaa gtcaagctgc 60tctgtgggct gtgatctgcc tcaaacccac
agcctgggta gcaggaggac cttgatgctc 120ctggcacaga tgaggagaat
ctctcttttc tcctgcttga aggacagaca tgactttgga 180tttccccagg
aggagtttgg caaccagttc caaaaggctg aaaccatccc tgtcctccat
240gagatgatcc agcagatctt caatctcttc agcacaaagg actcatctgc
tgcttgggat 300gagaccctcc tagacaaatt ctacactgaa ctctaccagc
agctgaatga cctggaagcc 360tgtgtgatac agggggtggg ggtgacagag
actcccctga tgaaggagga ctccattctg
420gctgtgagga aatacttcca aagaatcact ctctatctga aagagaagaa
atacagccct 480tgtgcctggg aggttgtcag agcagaaatc atgagatctt
tttctttgtc aacaaacttg 540caagaaagtt taagaagtaa ggaatga
56724498DNAHomo sapiens 24tgtgatctgc ctcaaaccca cagcctgggt
agcaggagga ccttgatgct cctggcacag 60atgaggagaa tctctctttt ctcctgcttg
aaggacagac atgactttgg atttccccag 120gaggagtttg gcaaccagtt
ccaaaaggct gaaaccatcc ctgtcctcca tgagatgatc 180cagcagatct
tcaatctctt cagcacaaag gactcatctg ctgcttggga tgagaccctc
240ctagacaaat tctacactga actctaccag cagctgaatg acctggaagc
ctgtgtgata 300cagggggtgg gggtgacaga gactcccctg atgaaggagg
actccattct ggctgtgagg 360aaatacttcc aaagaatcac tctctatctg
aaagagaaga aatacagccc ttgtgcctgg 420gaggttgtca gagcagaaat
catgagatct ttttctttgt caacaaactt gcaagaaagt 480ttaagaagta aggaatga
4982530DNAArtificial SequencePCR Cloning Primer 25cgtgcacgct
ggctgcgtgc acgctggctg 302631DNAArtificial SequencePCR Cloning
Primer 26cgtgcacgct ggctgtcgtg cacgctggct g 312732DNAArtificial
SequencePCR Cloning Primer 27cgtgcacgct ggctgttcgt gcacgctggc tg
322845DNAArtificial SequencePCR Cloning Primer 28cgtgcacgct
ggctgcgtgc acgctggctg cgtgcacgct ggctg 452947DNAArtificial
SequencePCR Cloning Primer 29cgtgcacgct ggctgtcgtg cacgctggtc
tgcgtgcacg ctggctg 473049DNAArtificial SequencePCR Cloning Primer
30cgtgcacgct ggctgttcgt gcacgctggt tctgcgtgca cgctggctg 49
* * * * *
References