U.S. patent number RE42,931 [Application Number 12/284,010] was granted by the patent office on 2011-11-15 for covalent tethering of functional groups to proteins.
This patent grant is currently assigned to Promega Corporation. Invention is credited to Robert F. Bulleit, Dieter Klaubert, Georgyi V. Los, Mark McDougall, Keith V. Wood, Chad Zimprich.
United States Patent |
RE42,931 |
Wood , et al. |
November 15, 2011 |
Covalent tethering of functional groups to proteins
Abstract
A mutant hydrolase optionally fused to a protein of interest is
provided. The mutant hydrolase is capable of forming a bond with a
substrate for the corresponding nonmutant (wild-type) hydrolase
which is more stable than the bond formed between the wild-type
hydrolase and the substrate. Substrates for hydrolases comprising
one or more functional groups are also provided, as well as methods
of using the mutant hydrolase and the substrates of the invention.
Also provided is a fusion protein capable of forming a stable bond
with a substrate and cells which express the fusion protein.
Inventors: |
Wood; Keith V. (Mt. Horeb,
WI), Klaubert; Dieter (Arroyo Grande, CA), Los; Georgyi
V. (Madison, WI), Bulleit; Robert F. (Verona, WI),
McDougall; Mark (Arroyo Grande, CA), Zimprich; Chad
(Stoughton, WI) |
Assignee: |
Promega Corporation (Madison,
WI)
|
Family
ID: |
32871922 |
Appl.
No.: |
12/284,010 |
Filed: |
September 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
60444094 |
Jan 31, 2003 |
|
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|
|
60474659 |
May 30, 2003 |
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Reissue of: |
10768976 |
Jan 30, 2004 |
7238842 |
Jul 3, 2007 |
|
|
Current U.S.
Class: |
570/101;
548/304.1; 523/500; 435/18 |
Current CPC
Class: |
G01N
33/5005 (20130101); C07D 493/10 (20130101); C07D
311/78 (20130101); C07D 209/48 (20130101); C07D
311/82 (20130101); C12N 9/86 (20130101); C07C
271/16 (20130101); G01N 33/58 (20130101); C12Y
308/01005 (20130101); C07D 495/04 (20130101); C12Q
1/34 (20130101); C07C 217/08 (20130101); C07F
5/022 (20130101); C07D 405/06 (20130101); C12N
9/14 (20130101); C07D 233/64 (20130101); Y10S
436/80 (20130101) |
Current International
Class: |
C07C
22/00 (20060101); C08L 67/00 (20060101); C12Q
1/34 (20060101); C07D 235/02 (20060101) |
Field of
Search: |
;570/101 ;523/500
;548/304.1 ;435/18 |
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|
Primary Examiner: Kosson; Rosanne
Attorney, Agent or Firm: Casimir Jones, S.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
.Iadd.This application is a reissue of U.S. Pat. No. 7,238,842,
issued on Jul. 3, 2007, filed as application Ser. No. 10/768,976 on
Jan. 30, 2004. .Iaddend.This application claims the benefit of the
filing date of U.S. application Ser. No. 60/444,094, filed Jan. 31,
2003, and U.S. application Ser. No. 60/474,659, filed May 30, 2003,
under 35 U.S.C. .sctn. 119(e), and incorporates those applications
by reference herein.
Claims
What is claimed is:
.[.1. A method for preparing a compound of the formula
biotin-Linker-A--X comprising coupling a compound of formula
biotin-Y with a compound of formula Z-Linker-A--X, wherein Y and Z
are groups that can react to link biotin to Linker-A--X, wherein
the linker is a branched or unbranched carbon chain comprising from
2 to 30 carbon atoms, which chain optionally includes one or more
double or triple bonds, and which chain is optionally substituted
with one or more hydroxy or oxo (.dbd.O) groups, wherein one or
more of the carbon atoms in the chain is optionally replaced with a
non-peroxide --O--, --S--, or --NH--, wherein the linker-A
separates biotin and X by at least 11 atoms, wherein A--X is a
substrate for a dehalogenase, wherein A is (CH.sub.2).sub.n and
n=2-10, wherein X is a halogen, wherein biotin is a functional
group is capable of being coupled through its carboxy terminus to
the linker, and wherein biotin-Y is an activated ester of biotin
and wherein Z is an amine suitable to react with the activated
ester to form an amide bond..].
.[.2. A method for preparing a compound of the formula
biotin-Linker-A--X wherein the Linker comprises an amide bond
comprising coupling a corresponding activated ester with a
corresponding amine to provide the compound of formula biotin
Linker-A--X, wherein biotin is a functional group, wherein the
linker is a branched or unbranched carbon chain comprising from 2
to 30 carbon atoms, which chain optionally includes one or more
double or triple bonds, and which chain is optionally substituted
with one or more hydroxy or oxo (.dbd.O) groups, wherein one or
more of the carbon atoms in the chain is optionally replaced with a
non-peroxide --O--, --S--, or --NH--, wherein A--X is a substrate
for a dehalogenase, wherein A is (CH.sub.2).sub.n and n=2-10, and
wherein X is a halogen..].
.[.3. A compound of formula (I): biotin-linker-A--X, wherein biotin
is a functional group, wherein the linker is a branched or
unbranched carbon chain comprising from 2 to 30 carbon atoms, which
chain optionally includes one or more double or triple bonds, and
which chain is optionally substituted with one or more hydroxy or
oxo (.dbd.O) groups, wherein one or more of the carbon atoms in the
chain is optionally replaced with a non-peroxide --O--, --S-- or
--NH--, wherein the linker-A separates biotin and X by at least 11
atoms, wherein A is (CH.sub.2).sub.n and n=4-10, wherein A--X is a
substrate for a dehalogenase, and wherein X is a halogen, wherein
the biotin functional group is coupled through its carboxy terminus
to the linker..].
.[.4. The compound of claim 3 which is a substrate for a
Rhodococcus dehalogenase..].
.[.5. The compound of claim 3 wherein X is Cl or Br..].
.[.6. The compound of claim 3 wherein the linker comprises 3 to 30
atoms..].
.[.7. The compound of claim 3 wherein the linker has 11 to 30
atoms..].
.[.8. The compound of claim 3 which is
N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-biotin-amide..].
.[.9. The compound of claim 3 wherein biotin is separated from A--X
by up to 100 angstroms..].
.[.10. The compound of claim 3 wherein biotin is separated from
A--X by up to 500 angstroms..].
.[.11. The compound of claim 3 wherein the chain comprises
(CH.sub.2CH.sub.2O).sub.y and y=2-8..].
.[.12. A compound prepared by the method of claim 1 wherein the
compound is: ##STR00020## .].
.[.13. A compound of formula (I): biotin-linker-A--X, wherein
biotin is a functional group, wherein the linker is a branched or
unbranched carbon chain comprising from 2 to 30 carbon atoms, which
chain optionally includes one or more double or triple bonds, and
which chain is optionally substituted with one or more hydroxy or
oxo (.dbd.O) groups, wherein one or more of the carbon atoms in the
chain is optionally replaced with a non-peroxide --O--, --S-- or
--NH--, wherein the linker-A separates biotin and X by at least 11
atoms, wherein A is (CH.sub.2).sub.n and n=2-10, wherein A--X is a
substrate for a dehalogenase, wherein X is a halogen, and wherein
the biotin functional group is coupled through its carboxy terminus
to the linker..].
14. A compound of formula (II): ##STR00021##
.[.15. A compound prepared by the method of claim 1..].
16. A method to detect or determine the presence or amount of a
mutant hydrolase, comprising: a) contacting a mutant hydrolase with
a hydrolase substrate which comprises one or more biotin functional
groups, wherein the mutant hydrolase comprises at least one amino
acid substitution relative to a corresponding wild-type hydrolase,
wherein the at least one amino acid substitution results in the
mutant hydrolase forming a bond with the substrate which is more
stable than the bond formed between the corresponding wild-type
hydrolase and the substrate, wherein the at least one amino acid
substitution in the mutant hydrolase is a substitution at an amino
acid residue in the corresponding wild-type hydrolase that is
associated with activating a water molecule which cleaves the bond
formed between the corresponding wild-type hydrolase and the
substrate or at an amino acid residue in the corresponding
wild-type hydrolase that forms an ester intermediate with the
substrate, wherein the wild-type hydrolase is a dehalogenase,
wherein the mutant hydrolase is a mutant dehalogenase, and wherein
the substrate is a compound of formula (I): biotin-linker-A--X,
wherein the linker is a branched or unbranched carbon chain
comprising from 2 to 30 carbon atoms; which chain optionally
includes one or more double or triple bonds, and which chain is
optionally substituted with one or more hydroxy or oxo (.dbd.O)
groups, wherein one or more of the carbon atoms in the chain is
optionally replaced with a non-peroxide --O--, --S-- or --NH--,
wherein the linker-A separates biotin and X by at least 11 atoms,
wherein A--X is a substrate for a dehalogenase, wherein A is
(CH.sub.2).sub.n and .[.n=2-10.]. .Iadd.n=6-10.Iaddend., and
wherein X is a halogen, and wherein the biotin functional group is
coupled through its carboxy terminus to the linker; and b)
detecting or determining the presence or amount of the
.[.functional group.]. .Iadd.biotin.Iaddend., thereby detecting or
determining the presence or amount of the mutant dehalogenase.
17. The method of claim 16 wherein the substitution is at a residue
in the wild-type dehalogenase that activates the water
molecule.
18. The method of claim 17 wherein the residue in the wild-type
dehalogenase that activates the water molecule is histidine.
19. The method of claim 16 wherein the substitution is at a residue
in the wild-type dehalogenase that forms an ester intermediate with
the substrate.
20. The method of claim 19 wherein the residue in the wild-type
dehalogenase that forms an ester intermediate with the substrate is
aspartate.
21. A method of labeling a cell, comprising: a) contacting a cell
comprising a mutant hydrolase with a hydrolase substrate which
comprises one or more biotin functional groups, wherein the mutant
hydrolase comprises at least one amino acid substitution relative
to a corresponding wild-type hydrolase, wherein the at least one
amino acid substitution results in the mutant hydrolase forming a
bond with the substrate which is more stable than the bond formed
between the corresponding wild-type hydrolase and the substrate,
wherein the at least one amino acid substitution in the mutant
hydrolase is a substitution at an amino acid residue in the
corresponding wild-type hydrolase that is associated with
activating a water molecule which cleaves a bond formed between the
corresponding wild-type hydrolase and the substrate or at an amino
acid residue in the corresponding wild-type hydrolase that forms an
ester intermediate with the substrate, wherein the wild-type
hydrolase is a dehalogenase, wherein the mutant hydrolase is a
mutant dehalogenase, and wherein the substrate is a compound of
formula (I): biotin-linker-A--X, wherein the linker is a branched
or unbranched carbon chain comprising from 2 to 30 carbon atoms,
which chain optionally includes one or more double or triple bonds,
and which chain is optionally substituted with one or more hydroxy
or oxo (.dbd.O) groups, wherein one or more of the carbon atoms in
the chain is optionally replaced with a non-peroxide --O--, --S--
or --NH--, wherein the linker-A separates biotin and X by at least
11 atoms, wherein A--X is a substrate for a dehalogenase, wherein A
is (CH.sub.2).sub.n and .[.n=2-10.]. .Iadd.n=6-10.Iaddend., wherein
X is a halogen, and wherein the biotin functional group is coupled
through its carboxy terminus to the linker; and b) detecting or
determining the presence or amount of the functional group.
22. The method of claim 21 wherein the substitution is at a residue
in the wild-type dehalogenase that activates the water
molecule.
23. The method of claim 21 wherein the residue in the wild-type
dehalogenase that activates the water molecule is histidine.
24. The method of claim 21 wherein the substitution is at a residue
in the wild-type dehalogenase that forms an ester intermediate with
the substrate.
25. The method of claim 24 wherein the residue in the wild-type
dehalogenase that forms an ester intermediate with the substrate is
aspartate.
26. The method of claim 16 or 21 wherein the linker comprises
(CH.sub.2CH.sub.2).sub.y and y=2-8.
27. The method of claim 16 or 21 wherein the linker separates
biotin and A by at least 12 atoms.
28. The method of any one of claim 16 or 21 wherein the mutant
dehalogenase is present in a cell or on the surface of a cell.
29. The method of any one of claims 16 or 21 wherein the presence
of at least one biotin functional group in a cell is correlated to
the subcellular location of the mutant dehalogenase.
30. The method of any one of claim 16 or 21 wherein the mutant
dehalogenase forms a fusion protein with a protein of interest.
31. The method of claim 30 wherein the protein of interest is a
selectable marker protein, membrane protein, cytosolic protein,
nuclear protein, structural protein, an enzyme, an enzyme
substrate, a receptor protein, a transporter protein, a
transcription factor, a channel protein, a phospho-protein, a
kinase, a signaling protein, a metabolic protein, a mitochondrial
protein, a receptor associated protein, a nucleic acid binding
protein, an extracellular matrix protein, a secreted protein, a
receptor ligand, a serum protein, an immunogenic protein, a
fluorescent protein, or a protein with reactive cysteine.
32. The method of claim 21 wherein the mutant dehalogenase further
comprises a selectable marker protein.
33. The method of claim 32 wherein the mutant dehalogenase forms an
ester bond with the substrate.
34. The method of claim 32 wherein the mutant dehalogenase forms a
thioester bond with the substrate.
35. The method of claim 21 further comprising contacting the cell
with a fixative prior to or after contacting the cell with the
substrate.
36. The method of claim 21 further comprising contacting the cell
with a fixative concurrently with contacting the cell with the
substrate.
37. The method of claim 35 or 36 wherein the cell is fixed with
methanol, acetone and/or paraformaldehyde.
38. The method of claim 32 further comprising contacting the cell
with a fixative prior to or after contacting the cell with the
substrate.
39. The method of claim 32 further comprising contacting the cell
with a fixative concurrently contacting the cell with the
substrate.
40. The method of claim 38 or 39 wherein the cell is fixed with
methanol, acetone and/or paraformaldehyde.
Description
FIELD OF THE INVENTION
This invention relates to the field of biochemical assays and
reagents. More specifically, this invention relates to mutant
proteins covalently linked (tethered) to one or more functional
groups and to methods for their use.
BACKGROUND OF THE INVENTION
The specific detection of molecules is a keystone in understanding
the role of that molecule in the cell. Labels, e.g., those that are
covalently linked to a molecule of interest, permit the ready
detection of that molecule in a complex mixture. The label may be
one that is added by chemical synthesis in vitro or attached in
vivo, e.g., via recombinant techniques. For instance, the
attachment of fluorescent or other labels onto proteins has
traditionally been accomplished by in vitro chemical modification
after protein purification (Hermanson, 1996). For in vivo
attachment of a label, green fluorescent protein (GFP) from the
jellyfish Aequorea Victoria can be genetically fused with many host
proteins to produce fluorescent chimeras in situ (Tsien, 1998;
Chalfie et al., 1998). However, while GFP-based indicators are
currently employed in a variety of assays, e.g., measuring pH
(Kneen et al., 1998; Llopis et al., 1998; Miesenbock et al., 1998),
Ca.sup.2+ (Miyawaki et al., 1997; Rosomer et al., 1997), and
membrane potential (Siegel et al., 1997), the fluorescence of
intrinsically labeled proteins such as GFP is limited by the
properties of protein structure, e.g., a limited range of
fluorescent colors and relatively low intrinsic brightness (Cubitt
et al., 1995; Ormo et al., 1996), and
To address the deficiencies of GFP labeling in situ, Griffen et al.
(1998) synthesized a tight-binding pair of molecular components: a
small receptor domain composed of as few as six natural amino acids
and a small (<700 dalton), synthetic ligand that could be linked
to various spectroscopic probes or crosslinks. The receptor domain
included four cysteines at the i, i+1, i+4, and i+5 positions of an
.alpha. helix and the ligand was 4',
5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FLASH). Griffen et al.
disclose that the ligand had relatively few binding sites in
nontransfected mammalian cells, was membrane-permeant and was
nonfluorescent until it bound with high affinity and specificity to
a tetracysteine domain in a recombinant protein, resulting in cells
being fluorescently labeled ("FLASH" labeled) with a nanomolar or
lower dissociation constant. However, with respect to background
binding in cells, Stroffekova et al. (2001) disclose that
FLASH-EDT.sub.2 binds non-specifically to endogenous cysteine-rich
proteins. Furthermore, labeling proteins by FLASH is limited by the
range of fluorophores that may be used.
Receptor-mediated targeting methods use genetically encoded
targeting sequences to localize fluorophores to virtually any
cellular site, provided that the targeted protein is able to fold
properly. For example, Farinas et al. (1999) disclose that cDNA
transfection was used to target a single-chain antibody (sFv) to a
specified site in a cell. Farinas et al. disclose that conjugates
of a hapten (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and
a fluorescent probe (e.g., BODIPY F1, tetramethylrhodamine, and
fluorescein) were bound with high affinity (about 5 nM) to the
subcellular site for the sFv in living Chinese hamster ovary cells,
indicating that the targeted antibody functioned as a high affinity
receptor for the cell-permeable hapten-fluorophore conjugates.
Nevertheless, functional sFv expression may be relatively poor in
reducing environments.
Thus, what is needed is an improved method to label a desired
protein.
SUMMARY OF THE INVENTION
The invention provides methods, compositions and kits for tethering
(linking), e.g., via a covalent or otherwise stable bond, one or
more functional groups to a protein of the invention or to a fusion
protein (chimera) which includes a protein of the invention. A
protein of the invention is structurally related to a wild-type
(native) hydrolase but comprises at least one amino acid
substitution relative to the corresponding wild-type hydrolase and
binds a substrate of the corresponding wild-type hydrolase but
lacks or has reduced catalytic activity relative to the
corresponding wild-type hydrolase (which mutant protein is referred
to herein as a mutant hydrolase). The aforementioned tethering
occurs, for instance, in solution or suspension, in a cell, on a
solid support or at solution/surface interfaces, by employing a
substrate for a hydrolase which includes a reactive group and which
has been modified to include one or more functional groups. As used
herein, a "substrate" includes a substrate having a reactive group
and optionally one or more functional groups. A substrate which
includes one or more functional groups is generally referred to
herein as a substrate of the invention. As used herein, a
"functional group" is a molecule which is detectable or is capable
of detection (e.g., a chromophore, fluorophore or luminophore), or
can be bound or attached to a second molecule (e.g., biotin,
hapten, or a cross-linking group) or includes one or more amino
acids, e.g., a peptide or polypeptide including an antibody or
receptor, one or more nucleotides, lipids including lipid bilayers,
a solid support, e.g., a sedimental particle, and the like. A
functional group may have more than one property such as being
capable of detection and being bound to another molecule. As used
herein a "reactive group" is the minimum number of atoms in a
substrate which are specifically recognized by a particular
wild-type or mutant hydrolase of the invention. The interaction of
a reactive group in a substrate and a wild-type hydrolase results
in a product and the regeneration of the wild-type hydrolase. A
substrate, e.g., a substrate of the invention, may also optionally
include a linker, e.g., a cleavable linker.
A substrate useful in the invention is one which is specifically
bound by a mutant hydrolase, and preferably results in a bond
formed with an amino acid, e.g., the reactive residue, of the
mutant hydrolase which bond is more stable than the bond formed
between the substrate and the corresponding amino acid of the
wild-type hydrolase. While the mutant hydrolase specifically binds
substrates which may be specifically bound by the corresponding
wild-type hydrolase, no product or substantially less product,
e.g., 2-, 10-, 100-, or 1000-fold less, is formed from the
interaction between the mutant hydrolase and the substrate under
conditions which result in product formation by a reaction between
the corresponding wild-type hydrolase and substrate. The lack of,
or reduced amounts of, product formation by the mutant hydrolase is
due to at least one substitution in the mutant hydrolase, which
substitution results in the mutant hydrolase forming a bond with
the substrate which is more stable than the bond formed between the
corresponding wild-type hydrolase and the substrate. Preferably,
the bond formed between a mutant hydrolase and a substrate of the
invention has a half-life (i.e., t.sub.1/2) that is at least
2-fold, and more preferably at least 4- or even 10-fold, and up to
100-, 1000- or 10,000-fold, greater than the t.sub.1/2 of the bond
formed between a corresponding wild-type hydrolase and the
substrate under conditions which result in product formation by the
corresponding wild-type hydrolase. Preferably, the bond formed
between the mutant hydrolase and the substrate has a t.sub.1/2 of
at least 30 minutes and preferably at least 4 hours, and up to at
least 10 hours, and is resistant to disruption by washing, protein
denaturants, and/or high temperatures, e.g., the bond is stable to
boiling in SDS.
In one embodiment, the substrate is a substrate for a dehalogenase,
e.g., a haloalkane dehalogenase or a dehalogenase that cleaves
carbon-halogen bonds in an aliphatic or aromatic halogenated
substrate, such as a substrate for Rhodococcus, Staphylococcus,
Pseudomonas, Burkholderia, Agrobacterium or Xanthobacter
dehalogenase, or a substrate for a serine beta-lactamase. In one
embodiment, a substrate of the invention optionally includes a
linker which physically separates one or more functional groups
from the reactive group in the substrate. For instance, for some
mutant hydrolases, i.e., those with deep catalytic pockets, a
substrate of the invention can include a linker of sufficient
length and structure so that the one or more functional groups of
the substrate of the invention do not disturb the 3-D structure of
the hydrolase (wild-type or mutant). For example, one example of a
substrate of the invention for a dehalogenase includes a reactive
group such as (CH.sub.2).sub.2-3X where X is a halide and a
functional group such as tetramethylrhodamine (TAMRA), e.g.,
TAMRA-C.sub.14H.sub.24O.sub.4--Cl.
In one embodiment, a linker is preferably 12 to 30 atoms in length.
The linker may not always be present in a substrate of the
invention, however, in some embodiments, the physical separation of
the reactive group and the functional group may be needed so that
the reactive group can interact with the reactive residue in the
mutant hydrolase to form a covalent bond. Preferably, when present,
the linker does not substantially alter, e.g., impair, the
specificity or reactivity of a substrate having the linker with the
wild-type or mutant hydrolase relative to the specificity or
reactivity of a corresponding substrate which lacks the linker with
the wild-type or mutant hydrolase. Further, the presence of the
linker preferably does not substantially alter, e.g., impair, one
or more properties, e.g., the function, of the functional
group.
Thus, the invention provides a compound of formula (I):
R-linker-A--X, wherein R is one or more functional groups, wherein
the linker is a multiatom straight or branched chain including C,
N, S, or O, wherein A--X is a substrate for a dehalogenase, and
wherein X is a halogen. In one embodiment, an alkylhalide is
covalently attached to a linker, L, which is a group or groups that
covalently attach one or more functional groups to form a substrate
for a dehalogenase. As described herein, a mutant dehalogenase,
DhaA.H272F, was bound to substrates for DhaA which included 5-(and
6-) carboxy fluorescein (FAM), e.g.,
FAM-C.sub.14H.sub.24O.sub.4--Cl, TAMRA, e.g.,
TAMRA-C.sub.14H.sub.24O.sub.4--Cl, and biotin, e.g.,
biotin-C.sub.18H.sub.32O.sub.4--Cl, and there was no significant
quenching effect of this binding on FAM or TAMRA fluorescence or on
biotin binding to streptavidin. As also described herein, a mutant
dehalogenase, e.g., DhaA.D106C and DhaA.D106E as well as
DhaA.D106C: H272F and DhaA.D106E:H272F, bound
FAM-C.sub.14H.sub.24O.sub.4--Cl and/or
TAMRA-C.sub.14H.sub.24O.sub.4--Cl. In one embodiment, the substrate
is
R--(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.6Cl,
wherein R is a functional group. To prepare such a substrate, a
functional group may be reacted with a molecule such as
NH(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.6Cl.
In one embodiment, substrates of the invention are permeable to the
plasma membranes of cells. For instance, as described herein the
plasma membranes of prokaryotic (E. coli) and eukaryotic (CHO-K1)
cells were permeable to TAMRA-C.sub.14H.sub.24O.sub.4--Cl and
biotin-C.sub.18H.sub.32O.sub.4--Cl and, these substrates were
rapidly and efficiently loaded into and washed out of cells in the
absence of a mutant hydrolase. In the presence of a mutant
hydrolase, at least a portion of the substrate was prevented from
being washed out of the cells. Thus, the bound portion of the
substrate can serve as a marker or as a means to capture the mutant
hydrolase or a fusion thereof.
The invention further provides methods for preparing a substrate
for a hydrolase which substrate is modified to include one or more
functional groups. Exemplary functional groups for use in the
invention include, but are not limited to, an amino acid, protein,
e.g., enzyme, antibody or other immunogenic protein, a
radionuclide, a nucleic acid molecule, a drug, a lipid, biotin,
avidin, streptavidin, a magnetic bead, a solid support, an electron
opaque molecule, chromophore, MRI contrast agent, a dye, e.g., a
xanthene dye, a calcium sensitive dye, e.g.,
1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxy]-2-(2'-am-
ino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (Fluo-3), a
sodium sensitive dye, e.g., 1,3-benzenedicarboxylic acid,
4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-
-6,2-benzofurandiyl)]bis (PBFI), a NO sensitive dye, e.g.,
4-amino-5-methylamino-2',7'-difluorescein, or other fluorophore. In
one embodiment, the functional group is an immunogenic molecule,
i.e., one which is bound by antibodies specific for that molecule.
In one embodiment, the functional group is not a radionuclide.
The invention also includes a mutant hydrolase which comprises at
least one amino acid substitution relative to a corresponding
wild-type hydrolase, which substitution(s) renders the mutant
hydrolase capable of forming a bond, e.g., a covalent bond with a
substrate for the corresponding hydrolase, e.g., a substrate of the
invention, which is more stable than the bond formed between a
corresponding wild-type hydrolase and the substrate.
In one embodiment, the mutant hydrolase of the invention comprises
at least one amino acid substitution in a residue which, in the
wild-type hydrolase, is associated with activating a water
molecule, e.g., a residue in a catalytic triad or an auxiliary
residue, wherein the activated water molecule cleaves the bond
formed between a catalytic residue in the wild-type hydrolase and a
substrate of the hydrolase. As used herein, an "auxiliary residue"
is a residue which alters the activity of another residue, e.g., it
enhances the activity of a residue that activates a water molecule.
Residues which activate water within the scope of the invention
include but are not limited to those involved in acid-base
catalysis, for instance, histidine, aspartic acid and glutamic
acid. In another embodiment, the mutant hydrolase of the invention
comprises at least one amino acid substitution in a residue which,
in the wild-type hydrolase, forms an ester intermediate by
nucleophilic attack of a substrate for the hydrolase.
For example, wild-type dehalogenase DhaA cleaves carbon-halogen
bonds in halogenated hydrocarbons (HaloC.sub.3-HaloC.sub.10). The
catalytic center of DhaA is a classic catalytic triad including a
nucleophile, an acid and a histidine residue. The amino acids in
the triad are located deep inside the catalytic pocket of DhaA
(about 10 .ANG. long and about 20 .ANG..sup.2 in cross section).
The halogen atom in a halogenated substrate for DhaA, for instance,
the chlorine atom of a Cl-alkane substrate, is positioned in close
proximity to the catalytic center of DhaA. DhaA binds the
substrate, likely forms an ES complex, and an ester intermediate is
formed by nucleophilic attack of the substrate by Asp106 (the
numbering is based on the protein sequence of DhaA) of DhaA (FIG.
1). His272 of DhaA then activates water and the activated water
hydrolyzes the intermediate, releasing product from the catalytic
center. As described herein, mutant DhaAs, e.g., a DhaA.H272F
mutant, which likely retains the 3-D structure based on a computer
modeling study and basic physico-chemical characteristics of
wild-type DhaA (DhaA.WT), were not capable of hydrolyzing one or
more substrates of the wild-type enzyme, e.g., for Cl-alkanes,
releasing the corresponding alcohol released by the wild-type
enzyme. As further described herein, mutant serine beta-lactamases,
e.g., a blaZ.E166D mutant, a blaZ.N170Q mutant and a
blaZ.E166D:N170Q mutant, were not capable of hydrolyzing one or
more substrates of a wild-type serine beta-lactamase.
Thus, in one embodiment of the invention, a mutant hydrolase is a
mutant dehalogenase comprising at least one amino acid substitution
in a residue which, in the wild-type dehalogenase, is associated
with activating a water molecule, e.g., a residue in a catalytic
triad or an auxiliary residue, wherein the activated water molecule
cleaves the bond formed between a catalytic residue in the
wild-type dehalogenase and a substrate of the dehalogenase. In one
embodiment, at least one substitution is in a residue corresponding
to residue 272 in DhaA from Rhodococcus rhodochrous. A
"corresponding residue" is a residue which has the same activity
(function) in one wild-type protein relative to a reference
wild-type protein and optionally is in the same relative position
when the primary sequences of the two proteins are aligned. For
example, a residue which forms part of a catalytic triad and
activates a water molecule in one enzyme may be residue 272 in that
enzyme, which residue 272 corresponds to residue 73 in another
enzyme, wherein residue 73 forms part of a catalytic triad and
activates a water molecule. Thus, in one embodiment, a mutant
dehalogenase of the invention has a phenylalanine residue at a
position corresponding to residue 272 in DhaA from Rhodococcus
rhodochrous. In another embodiment of the invention, a mutant
hydrolase is a mutant dehalogenase comprising at least one amino
acid substitution in a residue corresponding to residue 106 in DhaA
from Rhodococcus rhodochrous. For example, a mutant dehalogenase of
the invention has a cysteine or a glutamate residue at a position
corresponding to residue 106 in DhaA from Rhodococcus rhodochrous.
In a further embodiment, the mutant hydrolase is a mutant
dehalogenase comprising at least two amino acid substitutions, one
in a residue corresponding to residue 106 and one in a residue
corresponding to residue 272 in DhaA from Rhodococcus rhodochrous.
In yet a further embodiment, the mutant hydrolase is a mutant
serine beta-lactamase comprising at least one amino acid
substitution in a residue corresponding to residue 166 or residue
170 in a serine beta-lactamase of Staphylococcus aureus PCl.
The mutant hydrolase may be a fusion protein, e.g., a fusion
protein expressed from a recombinant DNA which encodes the mutant
hydrolase and at least one protein of interest or a fusion protein
formed by chemical synthesis. For instance, the fusion protein may
comprise a mutant hydrolase and an enzyme of interest, e.g.,
luciferase, RNasin or RNase, and/or a channel protein, a receptor,
a membrane protein, a cytosolic protein, a nuclear protein, a
structural protein, a phosphoprotein, a kinase, a signaling
protein, a metabolic protein, a mitochondrial protein, a receptor
associated protein, a fluorescent protein, an enzyme substrate, a
transcription factor, a transporter protein and/or a targeting
sequence, e.g., a myristilation sequence, a mitochondrial
localization sequence, or a nuclear localization sequence, that
directs the mutant hydrolase, for example, a fusion protein, to a
particular location. The protein of interest may be fused to the
N-terminus or the C-terminus of the mutant hydrolase. In one
embodiment, the fusion protein comprises a protein of interest at
the N-terminus, and another protein, e.g., a different protein, at
the C-terminus, of the mutant hydrolase. For example, the protein
of interest may be a fluorescent protein or an antibody.
Optionally, the proteins in the fusion are separated by a connector
sequence, e.g., preferably one having at least 2 amino acid
residues, such as one having 13 to 17 amino acid residues. The
presence of a connector sequence in a fusion protein of the
invention does not substantially alter the function of either
protein in the fusion relative to the function of each individual
protein. Thus, for a fusion of a mutant dehalogenase and Renilla
luciferase, the presence of a connector sequence does not
substantially alter the stability of the bond formed between the
mutant dehalogenase and a substrate therefor or the activity of the
luciferase. For any particular combination of proteins in a fusion,
a wide variety of connector sequences may be employed. In one
embodiment, the connector sequence is a sequence recognized by an
enzyme, e.g., a cleavable sequence. For instance, the connector
sequence may be one recognized by a caspase, e.g., DEVD (SEQ ID
NO:64), or is a photocleavable sequence.
In one embodiment, the fusion protein may comprise a protein of
interest at the N-terminus and, preferably, a different protein of
interest at the C-terminus of the mutant hydrolase. As described
herein, fusions of a mutant DhaA with GST (at the N-terminus), a
Flag sequence (at the C-terminus) and Renilla luciferase (at the
N-terminus or C-terminus) had no detectable effect on bond
formation between the mutant DhaA and a substrate for wild-type
DhaA which includes a functional group. Moreover, a fusion of a
Flag sequence and DhaA.H272F could be attached to a solid support
via a streptavidin-biotin-C.sub.18H.sub.32O.sub.4-DhaA.H272F bridge
(an SFlag-ELISA experiment). Further, a fusion of Renilla
luciferase (R.Luc) and DhaA.H272F could be attached to Magnesil.TM.
particles coated with a substrate for wild-type DhaA which includes
a functional group. In addition, the attached fusion comprising
R.Luc was shown to be enzymatically active.
Exemplary proteins of interest include, but are not limited to, an
immunogenic protein, fluorescent protein, selectable marker
protein, membrane protein, cytosolic protein, nuclear protein,
structural protein, enzyme, e.g., RNase, enzyme substrate, receptor
protein, transporter protein, transcription factor, channel
protein, e.g., ion channel protein, phospho-protein, kinase,
signaling protein, metabolic protein, mitochondrial protein,
receptor associated protein, nucleic acid binding protein,
extracellular matrix protein, secreted protein, receptor ligand,
serum protein, or a protein with reactive cysteines.
The invention also includes compositions and kits comprising a
substrate for a hydrolase which includes a linker, a substrate for
a hydrolase which includes one or more functional groups and
optionally a linker, a linker which includes one or more functional
groups, a substrate for a hydrolase which lacks one or more
functional groups and optionally includes a linker, a linker, or a
mutant hydrolase, or any combination thereof. For example, the
invention includes a solid support comprising a substrate of the
invention, a kit comprising a substrate of the invention, a kit
comprising a vector encoding a dehalogenase of the invention, or a
kit comprising a vector encoding a serine beta-lactamase of the
invention.
Also provided is an isolated nucleic acid molecule (polynucleotide)
comprising a nucleic acid sequence encoding a hydrolase. In one
embodiment, the isolated nucleic acid molecule comprises a nucleic
acid sequence which is optimized for expression in at least one
selected host. Optimized sequences include sequences which are
codon optimized, i.e., codons which are employed more frequently in
one organism relative to another organism, e.g., a distantly
related organism, as well as modifications to add or modify Kozak
sequences and/or introns, and/or to remove undesirable sequences,
for instance, potential transcription factor binding sites. In one
embodiment, the polynucleotide includes a nucleic acid sequence
encoding a dehalogenase, which nucleic acid sequence is optimized
for expression is a selected host cell. In one embodiment, the
optimized polynucleotide no longer hybridizes to the corresponding
non-optimized sequence, e.g., does not hybridize to the
non-optimized sequence under medium or high stringency conditions.
In another embodiment, the polynucleotide has less than 90%, e.g.,
less than 80%, nucleic acid sequence identity to the corresponding
non-optimized sequence and optionally encodes a polypeptide having
at least 80%, e.g., at least 85%, 90% or more, amino acid sequence
identity with the polypeptide encoded by the non-optimized
sequence. Constructs, e.g., expression cassettes, and vectors
comprising the isolated nucleic acid molecule, as well as kits
comprising the isolated nucleic acid molecule, construct or vector
are also provided.
Further provided is a method of expressing a mutant hydrolase of
the invention. The method comprises introducing to a host cell a
recombinant nucleic acid molecule encoding a mutant hydrolase of
the invention so as to express the mutant hydrolase. In one
embodiment, the mutant hydrolase may be isolated from the cell. The
mutant hydrolase may be expressed transiently or stably,
constitutively or under tissue-specific or drug-regulated
promoters, and the like. Also provided is an isolated host cell
comprising a recombinant nucleic acid molecule encoding a mutant
hydrolase of the invention.
In one embodiment, the invention provides a method to detect or
determine the presence or amount of a mutant hydrolase. The method
includes contacting a mutant hydrolase with a hydrolase substrate
which comprises one or more functional groups. The mutant hydrolase
comprises at least one amino acid substitution relative to a
corresponding wild-type hydrolase, wherein the at least one amino
acid substitution results in the mutant hydrolase forming a bond
with the substrate which is more stable than the bond formed
between the corresponding wild-type hydrolase and the substrate,
and wherein the at least one amino acid substitution in the mutant
hydrolase is a substitution at an amino acid residue in the
corresponding wild-type hydrolase that is associated with
activating a water molecule which cleaves the bond formed between
the corresponding wild-type hydrolase and the substrate or at an
amino acid residue in the corresponding wild-type hydrolase that
forms an ester intermediate with the substrate. The presence or
amount of the functional group is detected or determined, thereby
detecting or determining the presence or amount of the mutant
hydrolase. In one embodiment, the mutant hydrolase is in or on the
surface of a cell. In another embodiment, the mutant hydrolase is
in a cell lysate.
Also provided are methods of using a mutant hydrolase and a
substrate for a corresponding hydrolase which includes one or more
functional groups, e.g., to isolate a molecule or to detect or
determine the presence or amount of, location, e.g., intracellular,
subcellular or extracellular location, or movement of certain
molecules in cells. In one embodiment, a method to isolate a
molecule of interest in a sample is provided. The method includes
contacting a sample with a fusion protein comprising a mutant
hydrolase and a protein which binds a molecule of interest with a
hydrolase substrate which comprises one or more functional groups.
The mutant hydrolase comprises at least one amino acid substitution
relative to a corresponding wild-type hydrolase, wherein the at
least one amino acid substitution results in the mutant hydrolase
forming a bond with the substrate which is more stable than the
bond formed between the corresponding wild-type hydrolase and the
substrate, and wherein the at least one amino acid substitution in
the mutant hydrolase is a substitution at an amino acid residue in
the corresponding wild-type hydrolase that is associated with
activating a water molecule which cleaves the bond formed between
the corresponding wild-type hydrolase and the substrate or at an
amino acid residue in the corresponding wild-type hydrolase that
forms an ester intermediate with the substrate. In one embodiment,
at least one functional group is a solid support or a molecule
which binds to a solid support. In one embodiment, the sample
contains intact cells while in another embodiment, the sample is a
cell lysate or subcellular fraction. Then the molecule of interest
is isolated.
For example, the invention includes method to isolate a protein of
interest. The method includes contacting a fusion protein
comprising a mutant hydrolase and a protein of interest with a
hydrolase substrate which comprises at least one functional group.
The mutant hydrolase comprises at least one amino acid substitution
relative to a corresponding wild-type hydrolase, wherein the at
least one amino acid substitution results in the mutant hydrolase
forming a bond with the substrate which is more stable than the
bond formed between the wild-type hydrolase and the substrate, and
wherein the at least one amino acid substitution in the mutant
hydrolase is a substitution at an amino acid residue in the
wild-type hydrolase that is associated with activating a water
molecule which cleaves a bond formed between the wild-type
hydrolase and the substrate or at an amino acid residue in the
wild-type hydrolase that forms an ester intermediate with the
substrate. In one embodiment, at least one functional group is a
solid support or a molecule which binds to a solid support. Then
the protein of interest is isolated.
In another embodiment, the invention includes a method to identify
an agent that alters the interaction of a protein of interest with
a molecule suspected of interacting with the protein of interest.
The method includes contacting at least one agent with the molecule
suspected of interacting with the protein of interest, a fusion
protein comprising mutant hydrolase and the protein of interest,
and a hydrolase substrate which comprises one or more functional
groups. The mutant hydrolase comprises at least one amino acid
substitution relative to a corresponding wild-type hydrolase,
wherein the at least one amino acid substitution results in the
mutant hydrolase forming a bond with the substrate which is more
stable than the bond formed between the corresponding wild-type
hydrolase and the substrate, and wherein the at least one amino
acid substitution in the mutant hydrolase is a substitution at an
amino acid residue in the corresponding wild-type hydrolase that is
associated with activating a water molecule which cleaves a bond
formed between the corresponding wild-type hydrolase and the
substrate at an amino acid residue in the wild-type hydrolase that
forms an ester intermediate with the substrate. In one embodiment
at least one functional group is a solid support or a molecule
which binds to a solid support. Then it is determined whether the
agent alters the interaction between the protein of interest and
the molecule suspected of interacting with the protein of
interest.
Moreover, a substrate of the invention bound to a solid support or
a mutant hydrolase bound to a solid support may be used to generate
protein arrays, cell arrays, vesicle/organelle arrays and cell
membrane arrays.
The invention thus provides methods to monitor the expression,
location and/or movement (trafficking) of proteins in a cell as
well as to monitor changes in microenvironments within a cell. In
one embodiment, the use of a mutant hydrolase and a substrate of
the invention permits functional analysis of proteins, e.g., ion
channels. In another embodiment, the use of two pairs of a mutant
hydrolase/substrate permits multiplexing, simultaneous detection,
and FRET- or BRET-based assays. For example, mutant dehalogenases
with substitutions at different residues of a catalytic triad may
each preferentially bind certain substrates of the invention but
not others or a mutant dehalogenase and a mutant beta-lactamase may
be employed with their respective substrates, thus permitting
multiplexing. Other applications include capturing the stable
complex which results from contacting the mutant hydrolase with a
corresponding substrate of the invention, on a solid substrate for
analytical or industrial purposes (e.g., to study kinetic
parameters of the tethered enzyme, to generate enzyme
chains/arrays, to metabolize industrial components, and the like),
to detect protein-protein interactions, to determine the effect of
different compounds/drugs on an interaction between a fusion
protein comprising a protein of interest and a mutant hydrolase
with other molecules, to isolate or purify molecules which bind to
a protein of interest fused to the mutant hydrolase, or to isolate
or purify cells, organelles or fragments thereof. For example, a
protein of interest may be fused to a mutant hydrolase and then
linked to a solid support via the specific interaction of a
functional group which is a ligand for an acceptor group and is
present in a substrate of the invention, with an acceptor group
present on the solid support. Such a substrate may be contacted
with the fusion protein prior to contact with the solid support,
contacted with the solid support prior to contact with the fusion
protein, or simultaneously contacted with the fusion protein and
the solid support. Such a system permits the resulting complex to
be employed to detect or isolate molecules which bind to the
protein of interest. The binding molecule may be a protein, e.g., a
fusion of the binding protein and a functional group, e.g., GFP,
luciferase, an antibody, e.g., one conjugated to horseradish
peroxidase (HRP), alkaline phosphatase (AP) or a fluorophore.
To isolate, sort or purify cells, the mutant hydrolase may be
expressed on the outside surface of cells (e.g., via a fusion with
a plasma membrane protein). To isolate, purify or separate
organelles, the mutant hydrolase is expressed on the cytosolic
surface of the organelle of interest. In another embodiment, to
create an optimal platform for growing different cells, the mutant
hydrolase is fused with an extracellular matrix component or an
outer membrane protein and tethered to a three-dimensional cell
culture or a platform for tissue engineering. As an example,
primary neurons or embryonic stem cells may be grown on the
platform to form a feeder layer.
Other applications include detecting or labeling cells. Thus, the
use of a mutant hydrolase and a corresponding substrate of the
invention permits the detection of cells, for instance, to detect
cell migration in vitro or in vivo after implantation or injection
into animals (e.g., angiogenesis/chemotaxis assays, migration of
implanted neurons, normal, malignant, or recombinantly modified
cells implanted/injected into animals, and the like), and live cell
imaging followed by immunocytochemistry. In another embodiment, the
invention provides a method to label newly synthesized proteins.
For example, cells comprising a vector which expresses a mutant
hydrolase of the invention or a fusion thereof, are contacted with
a substrate for the hydrolase which lacks a functional group. Cells
are then contacted with an agent, e.g., an inducer of gene
expression, and a substrate for the hydrolase which contains one or
more functional groups. The presence, amount or location of the
mutant hydrolase or fusion thereof is then detected or determined.
The presence, amount or location of the mutant hydrolase or fusion
thereof is due to newly synthesized mutant hydrolase or a fusion
thereof. Alternatively, cells comprising a vector which expresses a
mutant hydrolase of the invention or a fusion thereof, are
contacted with a substrate for the hydrolase having a functional
group, e.g., a green fluorophore, then contacted with an agent and
a substrate having a different functional group, e.g., a red
fluorophore. In one embodiment, the mutant hydrolase is fused to a
membrane localization signal and so can be employed to monitor
events in or near the membrane.
Accordingly, the invention provides a method to label a cell. The
method includes contacting a cell comprising a mutant hydrolase
with a hydrolase substrate which comprises one or more functional
groups. The mutant hydrolase comprises at least one amino acid
substitution relative to a corresponding wild-type hydrolase,
wherein the at least one amino acid substitution results in the
mutant hydrolase forming a bond with the substrate which is more
stable than the bond formed between the corresponding wild-type
hydrolase and the substrate, and wherein the at least one amino
acid substitution in the mutant hydrolase is a substitution at an
amino acid residue in the corresponding wild-type hydrolase that is
associated with activating a water molecule which cleaves a bond
formed between the corresponding wild-type hydrolase and the
substrate or at an amino acid residue in the corresponding
wild-type hydrolase that forms an ester intermediate with the
substrate. Then the presence or amount of the functional group is
detected or determined.
Cells expressing selectable marker proteins, such as ones encoding
resistance to neomycin, hygromycin, or puromycin, are used to
stably transform cells with foreign DNA. It may be desirable to
observe which cells contain selectable marker proteins as well as
fluorescently labeled molecules. For instance, it may be preferable
to label the selectable marker protein with a fluorescent molecule
that is added exogenously to living cells. By this method, the
selectable marker protein becomes visible when only when needed by
addition of the fluorophore, and the fluorescence will subsequently
be lost when selectable marker proteins are naturally regenerated
through cellular metabolism. Thus, in one embodiment, the invention
provides a method for labeling a cell which expresses a selectable
marker protein. The method includes providing a cell comprising an
expression cassette comprising a nucleic acid sequence encoding a
fusion protein. The fusion protein comprises a selectable marker
protein, e.g., one which confers resistance to at least one
antibiotic, and a second protein that is capable of stably and
optionally irreversibly binding a substrate or a portion thereof
which includes an optically detectable molecule. For instance, the
protein may be an alkyl transferase which irreversibly transfers an
alkyl group and an optically detectable molecule from a substrate
to itself, thereby labeling the alkyl transferase, e.g., an alkyl
transferase such as O.sup.6-alkylguanine DNA alkyltransferase.
Exemplary proteins useful in this embodiment of the invention
include, but are not limited to, alkyl transferases, peptidyl
glycine-alpha-amidating monoxygenases, type I topoisomerases,
hydrolases, e.g., serine and epoxide hydrolases as well as the
mutant hydrolases described herein, aminotransferases, cytochrome
P450 monooxygenases, acetyl transferases, decarboxylases, oxidases,
e.g., monoamine oxidases, reductases, e.g., ribonucleotide
reductase, synthetases, e.g., cyclic ADP ribose synthetase or
thymidylate synthetase, dehydrogenases, e.g., aldehyde
dehydrogenase, synthases, e.g., nitric oxide synthase (NOS),
lactamases, cystathionine gamma-lyases, peptidases, e.g.,
carboxypeptidase A, aromatase, proteases, e.g., serine protease,
xylanases, glucosidases, mannosidases, and demethylases and other
proteins, including wild-type proteins, which form an irreversible
or otherwise stable bond with one or more substrates, e.g., enzymes
which are capable of mechanism-based inactivation. Thus, in this
embodiment, a stable bond, i.e., one which is formed between a
substrate and a wild-type or mutant enzyme, has a t.sub.1/2 of at
least 30 minutes and preferably at least 4 hours, and up to at
least 10 hours, and is resistant to disruption by washing, protein
denaturants, and/or high temperatures, e.g., the bond is stable to
boiling in SDS.
The cell which expresses the fusion protein is contacted with the
substrate so as to label the cell. In one embodiment, the cell is
fixed prior to contact with the substrate. In another embodiment,
the substrate and fixative are contacted with the cell at the same
time. In yet another embodiment, the fixative is added to the cell
after the cell is contacted with the substrate. In one embodiment,
the fusion protein forms an ester bond with the substrate. In
another embodiment, the fusion protein forms a thioester bond with
the substrate. Also provided is a fusion gene encoding the fusion
protein, and a cell which expresses the fusion protein.
When performing image analysis on a cell, it may be desirable to
fix the cell with a preservative (fixative) such as
paraformaldehyde, acetone or methanol which generally maintains
most features of cellular structure. Such fixed cells are then
often analyzed by adding fluorescent stains or fluorescently
labeled antibodies to reveal specific structures within the cells.
Another method to fluorescently label cells is to express a
fluorescent protein, e.g., GFP, in cells prior to fixation.
Unfortunately, the efficient fluorescence of these proteins is
dependent on protein structure, which can be disrupted by
preservatives, thus decreasing the efficiency of imaging in those
cells.
Accordingly, the invention provides a method for labeling a cell
with a functional group, e.g., fluorophore. The method includes
providing a cell which expresses a mutant hydrolase of the
invention or a fusion thereof, and contacting the cell with a
hydrolase substrate which includes at least one functional group.
In one embodiment, the cell is fixed prior to contact with the
substrate. In another embodiment, the substrate and fixative are
contacted with the cell at the same time. In yet another
embodiment, the fixative is added to the cell after the cell is
contacted with the substrate. Then the presence or location of the
mutant hydrolase, or fusion thereof, in the cell is detected or
determined. In one embodiment, the mutant hydrolase forms an ester
bond with the substrate, while in another embodiment, the mutant
hydrolase forms a thioester bond with the substrate.
The invention also provides processes and intermediates disclosed
herein that are useful for preparing compounds, compositions,
nucleic acids, proteins, or other materials of the invention.
BRIEF DESCRIPTION OF THE FIGURES
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
FIG. 1 is a schematic of a reaction in the catalytic triad of
Rhodococcus rhodochrous dehalogenase with an alkylhalide
substrate.
FIG. 2 shows a three-dimensional model of a wild-type DhaA
Rhodococcus rhodochrous dehalogenase and four mutant DhaAs (H283Q,
G, A or F). A cyan ribbon is a 3-D model of the DhaA.WT based on
the crystal structure of this protein (Newman et al., 1999) (panel
A). The purple ribbon is a 3-D model of the H272Q, H272G and H272A
mutants (panel A), or a 3-D model of the H272F mutant (panel B).
Three-dimensional models were generated by calculating a Molecular
Probability Density Function followed by several optimization steps
including Restrained Stimulated Annealing Molecular Dynamics (MD)
scheme. 3-D modeling was done on Silicon Graphics computer-station
using software InsightII (USA).
FIG. 3 shows the purification of wild-type and mutant DhaA
proteins. GST-DhaA.WT-Flag (odd numbered lanes) and
GST-DhaA.H272F-Flag (even numbered lanes) fusion proteins were
found to be soluble and efficiently purified on GSS-Sepharose 4FF
(lanes 3 and 4-crude E. coli supernatant; lanes 5 and 6-washes;
lanes 7 through 10-purified proteins). Treatment of the fusion
proteins with Factor Xa led to the formation of two proteins, GST
and DhaA (WT or mutant; lanes 11 and 12, respectively). Moreover,
GST was efficiently removed on GSS-Sepharose 4FF (WT or mutant;
lanes 13 and 14, respectively). All proteins had the predicted
molecular weight.
FIG. 4 illustrates the hydrolysis of 1-Cl-butane by wild-type DhaA
and mutant DhaAs.
FIG. 5 shows precipitation of DhaA.WT and DhaA.H272F/A/G/Q mutants
with various concentrations of (NH.sub.4).sub.2SO.sub.4. Lanes 1,
5, and 9, 0% (NH.sub.4).sub.2SO.sub.4; lanes 2, 6, and 10, 10%
(NH.sub.4).sub.2SO.sub.4; lanes 3, 7, and 11, 10-45%
(NH.sub.4).sub.2SO.sub.4; and lanes 4, 8, and 12, 45-70%
(NH.sub.4).sub.2SO.sub.4. Panel A: lanes 1-4, DhaA.WT; lanes 5-8,
DhaA.H272G; and lanes 9-12, DhaA.H272Q. Panel B: lanes 1-4,
DhaA.WT; lanes 5-8, DhaA.H272F; and lanes 9-12, DhaA.H272A.
FIG. 6 depicts the substrate specificity of wild-type DhaA. Using a
phenol red-based assay (E.sub.558), the initial rate of the
reaction was determined during the first 60 seconds after enzyme
addition by four 15 second readings.
FIG. 7 shows substrates for DhaA which include a functional group
(e.g., 5-(and 6-)-carboxyfluorescein (FAM), Anth (anthracene) or
biotin) and a linker.
FIG. 8A shows a HPLC separation of products of
FAM-C.sub.14H.sub.24O.sub.4--Cl hydrolysis by wild-type DhaA.
FIG. 8B shows a HPLC analysis of product (as a percent of
substrate) produced by wild-type DhaA hydrolysis of
FAM-C.sub.14H.sub.24O.sub.4--Cl over time.
FIG. 9 shows SDS-PAGE analysis of the binding of wild-type DhaA
(lanes 1, 3, and 5 in panel A and lanes 1-8 in panel B) and mutant
DhaA (DhaA.H272F); (lanes 2, 4, and 6 in panel A and lanes 9-14 in
panel B), to TAMRA-C.sub.14H.sub.24O.sub.4--Cl (lanes 1 and 2 in
panel A); ROX-C.sub.14H.sub.24O.sub.4--Cl (lanes 3 and 4 in panel
A); FAM-C.sub.14H.sub.24O.sub.4--Cl (lanes 5 and 6 in panel A); or
biotin-C.sub.18H.sub.32O.sub.4--Cl (panel B). The concentration of
biotin-C.sub.18H.sub.32O.sub.4--Cl in panel B as: 0 .mu.M (lanes 1
and 8), 125 .mu.M (lanes 2 and 9) 25 .mu.M (lanes 3 and 10), 5
.mu.M (lanes 4 and 11), 1 .mu.M (lanes 5 and 12), 0.2 .mu.M (lanes
6 and 13), and 0.04 .mu.M (lanes 7 and 14).
FIG. 10 illustrates that pretreatment of a mutant DhaA with a
substrate, biotin-C.sub.18H.sub.32O.sub.4--Cl, blocks binding of
another substrate. DhaA.WT-lanes 1 and 2; DhaA.H272 is mutants: F,
lanes 3 and 4; G, lanes 5 and 6; A, lanes 7 and 8; and Q, lanes 9
and 10. Samples 2, 4, 6, 8, and 10 were pretreated with
biotin-C.sub.18H.sub.32O.sub.4--Cl.
FIG. 11 shows MALDI-TOF analysis of enzyme substrate complexes.
Mass spectra of GST-DhaA.WT or GST-DhaA.H272F incubated with
FAM-C.sub.14H.sub.24O.sub.4--Cl.
FIG. 12 illustrates SDS-PAGE analysis of the binding properties of
DhaA mutants with substitutions at residue 106, and DhaA mutants
with substitutions at residue 106 and residue 272, to
TAMRA-C.sub.14H.sub.24O.sub.4--Cl. 2 .mu.g of protein and 25 .mu.M
TAMRA-C.sub.14H.sub.24O.sub.4--Cl in 32 .mu.l were incubated for
one hour at room temperature. 10 .mu.l of each reaction was loaded
per lane. Lane 1-DhaA.D106C; lane 2-DhaA.D106C: H272F; lane
3-DhaA.D106E; lane 4-DhaA.D106E:H272F; lane 5-DhaA.D106Q; lane
6-DhaA.D106Q:H272F; lane 7-DhaA.WT; and lane 8-DhaA.H272F. The gel
was imaged with a 570 nm filter.
FIG. 13 depicts analysis of Renilla luciferase activity in samples
having a fusion of luciferase and a mutant DhaA tethered to a solid
support (a streptavidin coated plate). Capture of the fusion was
accomplished using a substrate of DhaA (i.e.,
biotin-C.sub.18H.sub.32O.sub.4--Cl). No activity was found in
fractions with a fusion of Renilla luciferase and wild-type
DhaA.
FIG. 14 shows SDS-PAGE analysis of two-fold serial dilutions of E.
coli expressing either wild-type DhaA (DhaA.WT-Flag, lanes 1-4 of
each panel) or mutant DhaA.H272F (DhaA.H272F-Flag, lanes 5-7 of
each panel) treated with biotin-C.sub.18H.sub.32O.sub.4--Cl (panel
A) or TAMRA-C.sub.12H.sub.24O.sub.4--Cl (panel B) in vivo. Arrows
mark proteins with M.sub.r corresponding to M.sub.r of
DhaA-Flag.
FIG. 15 shows the binding of TAMRA-C.sub.12H.sub.24O.sub.4--Cl to
eukaryotic cell proteins in vivo. Two-fold serial dilutions of
proteins from CHO-K1 cells expressing either DhaA.WT-Flag (lanes
1-4) or DhaA.H272F-Flag (lanes 5-8) were treated with
TAMRA-C.sub.12H.sub.24O.sub.4--Cl. Arrows mark proteins with
M.sub.r corresponding to M.sub.r of DhaA-Flag.
FIG. 16 illustrates the permeability of
TAMRA-C.sub.12H.sub.24O.sub.4--Cl to CHO-K1 cells. CHO-K1 cells (A,
bright field image) were treated with
TAMRA-C.sub.12H.sub.28O.sub.4--Cl (25 .mu.M, for 5 minutes at
37.degree. C.) and quickly washed with PBS (panel B). Panel C shows
the cells after the washing procedure.
FIG. 17 shows images of cells transfected with
GFP-connector-DhaA.WT-Flag or GFP-connector-DhaA.H272F-Flag. CHO-K1
cells were transfected with DNA coding GFP-connector-DhaA.WT-Flag
(panels A-C) or GFP-connector-DhaA.H272F-Flag (panels D-F) and
treated with TAMRA-C.sub.12H.sub.28O.sub.4--Cl. Panels A, D-bright
field; panels B, E-GFP filter set; and panels C, F-TAMRA filter
set.
FIG. 18 shows Western blot analysis of proteins from cells
transfected with GFP-connector-DhaA.WT-Flag (lanes 1-4) or
GFP-connector-DhaA.H272F-Flag (lanes 5-8). CHO-K1 cells were
transfected with either GFP-connector-DhaA.WT-Flag or
GFP-connector-DhaA.H272F-Flag and then treated with
TAMRA-C.sub.14H.sub.24O.sub.4--Cl (25 .mu.M) for 0, 5, 15 or 60
minutes, washed with PBS (4.times.1.0 ml), and collected in
SDS-sample buffer. The samples were resolved on SDS-PAGE, and
analyzed on a fluoroimager. Lanes 1-4, GFP-connector-DhaA.WT-Flag
treated for 0, 5, 15, or 60 minutes, respectively. Lanes 5-8,
GFP-connector-DhaA.H272F-Flag treated for 0, 5, 15, 60 minutes,
respectively. Arrows mark proteins with M.sub.r corresponding to
M.sub.r of GFP-connector-DhaA.H272F-Flag.
FIG. 19 illustrates the toxicity of selected substrates (panel A,
TAMRA and panel B, ROX) for CHO-K1 cells.
FIG. 20 illustrates a reaction scheme for a serine beta-lactamase.
The reaction begins with the formation of a precovalent encounter
complex (FIG. 20A), and moves through a high-energy acylation
tetrahedral intermediate (FIG. 20B) to form a transiently stable
acyl-enzyme intermediate, forming an ester through the catalytic
residue Ser70 (FIG. 20C). Subsequently, the acyl-enzyme is attacked
by hydrolytic water (FIG. 20D) to form a high-energy deacylation
intermediate (FIG. 20E) (Minasov et al., 2002), which collapses to
form the hydrolyzed product (FIG. 20F). The product is then
expelled, regenerating free enzyme.
FIG. 21 shows hydrolysis of FAP by GST-blaZ over time.
FIG. 22 shows the binding of bocellin to fusions of GST and
blaZ.E166D, blaZ.N170Q or blaZ.E166D:N170Q. Lane 1-dye/no blaZ;
lane 2-blaZ.WT; lane 3-blaZ.E166D; lane 4-blaZ.N170Q; and lane
5-blaZ.E166D:N170Q.
FIG. 23 shows the binding of CCF2 to fusions of GST and blaZ.E166D,
blaZ.N170Q or blaZ.E166D:N170Q. Lane 1-dye/no blaZ; lane
2-GST-blaZ.WT; lane 3-GST-blaZ.E166D; lane 4-GST-blaZ.N170Q; and
lane 5-GST-blaZ.E166D:N170Q.
FIG. 24 provides fluorescence and DIC images of living CHO-K1 cells
transfected with a construct encoding GFP-connector-DhaA.H272F-NLS3
and stained with TAMRA-C.sub.14H.sub.24O.sub.4--Cl. TAMRA
filter-top left; GFP filter-top right; "A" and "B" overlaid-bottom
left; overlaid image "C" and DIC image of the cell-bottom right.
NLS3=tandem repeat of a nuclear localization sequence from SV40 T
antigen.
FIG. 25 shows fluorescence images of living CHO-K1 cells
transfected with a construct encoding GFP-.beta.-arrestin2 (left)
and a construct encoding DhaA.H272F-.beta.-arrestin2 and stained
with TAMRA-C.sub.14H.sub.24O.sub.4 (right).
FIG. 26 shows an SDS-PAGE analysis of DhaA expression in E. coli.
Lanes: 1, Molecular weight standards; 2, Wild-type DhaA crude
lysate; 3, Wild-type DhaA cell-free lysate; 4, DhaA.H272F crude
lysate; 5, DhaA.H272F cell-free lysate; 6, vector control crude
lysate; 7, vector control cell-free lysate; 8, DhaA.E130Q Cl mutant
crude lysate; 9, DhaA.E130Q Cl mutant cell-free lysate; 10,
DhaA.E130L A5 mutant crude lysate; 11, DhaA.E130L A5 mutant
cell-free lysate; 12, DhaA.E130A A12 mutant crude lysate; 13,
DhaA.E130A A12 mutant cell-free lysate; 14, Molecular weight
standards. The arrow indicates the location of the DhaA protein.
-s, lysate before centrifugation; +s, lysate after
centrifugation.
FIG. 27 shows an immunoblot analysis of DhaA containing lysates.
Lanes: 1, Wild-type DhaA crude lysate; 2, Wild-type DhaA cell-free
lysate; 3, DhaA.H272F crude lysate; 4, DhaA.H272F cell-free lysate;
5, vector control crude lysate; 6, vector control cell-free lysate;
7, Molecular weight standards; 8, DhaA.E130Q Cl mutant crude
lysate; 9, DhaA.E130Q Cl mutant cell-free lysate; 10, DhaA.E130L A5
mutant crude lysate; 11, DhaA.E130L A5 mutant cell-free lysate; 12,
DhaA.E130A A12 mutant crude lysate; 13, DhaA.E130A A12 mutant
cell-free lysate; 14, Molecular weight standards. The arrow
indicates the location of the DhaA protein.
FIG. 28 provides fluoroimage analysis of in vitro covalent
alkyl-enzyme formation. Lanes: 1, Fluorescent molecular weight
standards; 2, DhaA wild-type; 3. DhaA.H272F mutant; 4, DhaA-(vector
only control); 5, DhaA.E130Q mutant; 6, DhaA.E130L mutant; 7,
DhaA.E130A mutant. The arrow indicates the location of the
fluorescent enzyme-alkyl covalent intermediate.
FIG. 29 provides fluoroimage analysis of covalent alkyl-enzyme
formation in whole cells. Lanes: 1, Fluorescent molecular weight
standards; 2, DhaA wild-type; 3, DhaA.H272F mutant; 4, DhaA-(vector
only control); 5, DhaA.E130Q mutant; 6, DhaA.E130L mutant; 7,
DhaA.E130A mutant; 8, Fluorescent molecular weight standards. The
arrow indicates the location of the fluorescent enzyme-alkyl
covalent intermediate.
FIGS. 30A-B show Western blot analyses of DhaA-Flag captured on
streptavidin (SA) coated beads. CHO-K1 cells transiently expressing
DhaA.H272F-Flag were treated with (A) or without (B)
biotin-C.sub.18H.sub.32O.sub.4--Cl (25 .mu.M, 0.1% DMSO, 60
minutes, 37.degree. C.). Excess biotin-C.sub.18H.sub.4--Cl was
washed out, cells were lysed, and 10 .mu.l of cell lysate was
incubated with 5 .mu.l of SA-coated beads (Pierce) for 60 minutes
at room temperature (RT). Cell lysates (lane 1), proteins which
were not bound to beads (lane 2), and proteins which were bound to
beads (lane 3) were resolved on SDS-PAGE, transferred to
nitrocellulose membrane, and probed with anti-Flag antibody
(Sigma).
FIGS. 30C-D illustrate analyses of hR.Luc-DhaA captured on SA
coated beads. CHO-K1 cells transiently expressing
hR.Luc-connector-DhaA.H272F-Flag were treated with or without
biotin-C.sub.18H.sub.32O.sub.4--Cl (25 .mu.M, 0.1% DMSO, 60
minutes, 37.degree. C.). Cells were lysed, and 10 .mu.l of cell
lysate was incubated with 5 .mu.l of SA-coated beads (Pierce) for
60 minutes at room temperature. Unbound material was washed out,
and hR.Luc activity determined using Promega's "Renilla Luciferase
Assay System" (C) or captured hR.Luc analyzed by Western blot (D).
C) Column 1, cells treated with biotin-C.sub.18H.sub.32O.sub.4--Cl,
and excess biotin-C.sub.18H.sub.32O.sub.4--Cl washed out; column 2,
untreated cells; and column 3, cells treated with
biotin-C.sub.18H.sub.32O.sub.4--Cl without washing out excess
biotin-C.sub.18H.sub.32O.sub.4--Cl. D) Cell lysate (lane 1),
proteins which were not bound to beads (lane 2), and proteins which
were bound to beads (lane 3) were resolved on SDS-PAGE, transferred
to nitrocellulose membrane, and probed with anti-R.Luc antibody
(Chemicon).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
A "nucleophile" is a molecule which donates electrons.
A "selectable marker protein" encodes an enzymatic activity that
confers to a cell the ability to grow in medium lacking what would
otherwise be an essential nutrient (e.g., the TRP1 gene in yeast
cells) or in a medium with an antibiotic or other drug, i.e., the
expression of the gene encoding the selectable marker protein in a
cell confers resistance to an antibiotic or drug to that cell
relative to a corresponding cell without the gene. When a host cell
must express a selectable marker to grow in selective medium, the
marker is said to be a positive selectable marker (e.g., antibiotic
resistance genes which confer the ability to grow in the presence
of the appropriate antibiotic). Selectable markers can also be used
to select against host cells containing a particular gene (e.g.,
the sacB gene which, if expressed, kills the bacterial host cells
grown in medium containing 5% sucrose); selectable markers used in
this manner are referred to as negative selectable markers or
counter-selectable markers. Common selectable marker gene sequences
include those for resistance to antibiotics such as ampicillin,
tetracycline, kanamycin, puromycin, bleomycin, streptomycin,
hygromycin, neomycin, Zeocin.TM., and the like. Selectable
auxotrophic gene sequences include, for example, hisD, which allows
growth in histidine free media in the presence of histidinol.
Suitable selectable marker genes include a bleomycin-resistance
gene, a metallothionein gene, a hygromycin B-phosphotransferase
gene, the AURI gene, an adenosine deaminase gene, an aminoglycoside
phosphotransferase gene, a dihydrofolate reductase gene, a
thymidine kinase gene, a xanthine-guanine phosphoribosyltransferase
gene, and the like.
A "nucleic acid", as used herein, is a covalently linked sequence
of nucleotides in which the 3' position of the pentose of one
nucleotide is joined by a phosphodiester group to the 5' position
of the pentose of the next, and in which the nucleotide residues
(bases) are linked in specific sequence, i.e., a linear order of
nucleotides. A "polynucleotide", as used herein, is a nucleic acid
containing a sequence that is greater than about 100 nucleotides in
length. An "oligonucleotide" or "primer", as used herein, is a
short polynucleotide or a portion of a polynucleotide. The term
"oligonucleotide" or "oligo" as used herein is defined as a
molecule comprised of 2 or more deoxyribonucleotides or
ribonucleotides, preferably more than 3, and usually more than 10,
but less than 250, preferably less than 200, deoxyribonucleotides
or ribonucleotides. The oligonucleotide may be generated in any
manner, including chemical synthesis, DNA replication,
amplification, e.g., polymerase chain reaction (PCR), reverse
transcription (RT), or a combination thereof. A "primer" is an
oligonucleotide which is capable of acting as a point of initiation
for nucleic acid synthesis when placed under conditions in which
primer extension is initiated. A primer is selected to have on its
3' end a region that is substantially complementary to a specific
sequence of the target (template). A primer must be sufficiently
complementary to hybridize with a target for primer elongation to
occur. A primer sequence need not reflect the exact sequence of the
target. For example, a non-complementary nucleotide fragment may be
attached to the 5' end of the primer, with the remainder of the
primer sequence being substantially complementary to the target.
Non-complementary bases or longer sequences can be interspersed
into the primer provided that the primer sequence has sufficient
complementarity with the sequence of the target to hybridize and
thereby form a complex for synthesis of the extension product of
the primer. Primers matching or complementary to a gene sequence
may be used in amplification reactions, RT-PCR and the like.
Nucleic acid molecules are said to have a "5'-terminus" (5' end)
and a "3'-terminus" (3' end) because nucleic acid phosphodiester
linkages occur to the 5' carbon and 3' carbon of the pentose ring
of the substituent mononucleotides. The end of a polynucleotide at
which a new linkage would be to a 5' carbon is its 5' terminal
nucleotide. The end of a polynucleotide at which a new linkage
would be to a 3' carbon is its 3' terminal nucleotide. A terminal
nucleotide, as used herein, is the nucleotide at the end position
of the 3'- or 5'-terminus.
DNA molecules are said to have "5' ends" and "3' ends" because
mononucleotides are reacted to make oligonucleotides in a manner
such that the 5' phosphate of one mononucleotide pentose ring is
attached to the 3' oxygen of its neighbor in one direction via a
phosphodiester linkage. Therefore, an end of an oligonucleotides
referred to as the "5' end" if its 5' phosphate is not linked to
the 3' oxygen of a mononucleotide pentose ring and as the "3' end"
if its 3' oxygen is not linked to a 5' phosphate of a subsequent
mononucleotide pentose ring.
As used herein, a nucleic acid sequence, even if internal to a
larger oligonucleotide or polynucleotide, also may be said to have
5' and 3' ends. In either a linear or circular DNA molecule,
discrete elements are referred to as being "upstream" or 5' of the
"downstream" or 3' elements. This terminology reflects the fact
that transcription proceeds in a 5' to 3' fashion along the DNA
strand. Typically, promoter and enhancer elements that direct
transcription of a linked gene (e.g., open reading frame or coding
region) are generally located 5' or upstream of the coding region.
However, enhancer elements can exert their effect even when located
3' of the promoter element and the coding region. Transcription
termination and polyadenylation signals are located 3' or
downstream of the coding region.
The term "codon" as used herein, is a basic genetic coding unit,
consisting of a sequence of three nucleotides that specify a
particular amino acid to be incorporation into a polypeptide chain,
or a start or stop signal. The term "coding region" when used in
reference to structural gene refers to the nucleotide sequences
that encode the amino acids found in the nascent polypeptide as a
result of translation of a mRNA molecule. Typically, the coding
region is bounded on the 5' side by the nucleotide triplet "ATG"
which encodes the initiator methionine and on the 3' side by a stop
codon (e.g., TAA, TAG, TGA). In some cases the coding region is
also known to initiate by a nucleotide triplet "TTG".
As used herein, the terms "isolated and/or purified" refer to in
vitro preparation, isolation and/or purification of a nucleic acid
molecule, a polypeptide, peptide or protein, so that it is not
associated with in vivo substances. Thus, the term "isolated" when
used in relation to a nucleic acid, as in "isolated
oligonucleotide" or "isolated polynucleotide" refers to a nucleic
acid sequence that is identified and separated from at least one
contaminant with which it is ordinarily associated in its source.
An isolated nucleic acid is present in a form or setting that is
different from that in which it is found in nature. In contrast,
non-isolated nucleic acids (e.g., DNA and RNA) are found in the
state they exist in nature. For example, a given DNA sequence
(e.g., a gene) is found on the host cell chromosome in proximity to
neighboring genes; RNA sequences (e.g., a specific mRNA sequence
encoding a specific protein), are found in the cell as a mixture
with numerous other mRNAs that encode a multitude of proteins.
Hence, with respect to an "isolated nucleic acid molecule", which
includes a polynucleotide of genomic, cDNA, or synthetic origin or
some combination thereof, the "isolated nucleic acid molecule" (1)
is not associated with all or a portion of a polynucleotide in
which the "isolated nucleic acid molecule" is found in nature, (2)
is operably linked to a polynucleotide which it is not linked to in
nature, or (3) does not occur in nature as part of a larger
sequence. The isolated nucleic acid molecule may be present in
single-stranded or double-stranded form. When a nucleic acid
molecule is to be utilized to express a protein, the nucleic acid
contains at a minimum, the sense or coding strand (i.e., the
nucleic acid may be single-stranded), but may contain both the
sense and anti-sense strands (i.e., the nucleic acid may be
double-stranded).
The term "wild-type" as used herein, refers to a gene or gene
product that has the characteristics of that gene or gene product
isolated from a naturally occurring source. A wild-type gene is
that which is most frequently observed in a population and is thus
arbitrarily designated the "wild-type" form of the gene. In
contrast, the term "mutant" refers to a gene or gene product that
displays modifications in sequence and/or functional properties
(i.e., altered characteristics) when compared to the wild-type gene
or gene product. It is noted that naturally-occurring mutants can
be isolated; these are identified by the fact that they have
altered characteristics when compared to the wild-type gene or gene
product.
The term "recombinant DNA molecule" means a hybrid DNA sequence
comprising at least two nucleotide sequences not normally found
together in nature.
The term "vector" is used in reference to nucleic acid molecules
into which fragments of DNA may be inserted or cloned and can be
used to transfer DNA segment(s) into a cell and capable of
replication in a cell. Vectors may be derived from plasmids,
bacteriophages, viruses, cosmids, and the like.
The terms "recombinant vector", "expression vector" or "construct"
as used herein refer to DNA or RNA sequences containing a desired
coding sequence and appropriate DNA or RNA sequences necessary for
the expression of the operably linked coding sequence in a
particular host organism. Prokaryotic expression vectors include a
promoter, a ribosome binding site, an origin of replication for
autonomous replication in a host cell and possibly other sequences,
e.g. an optional operator sequence, optional restriction enzyme
sites. A promoter is defined as a DNA sequence that directs RNA
polymerase to bind to DNA and to initiate RNA synthesis. Eukaryotic
expression vectors include a promoter, optionally a polyadenylation
signal and optionally an enhancer sequence.
A polynucleotide having a nucleotide sequence "encoding a peptide,
protein or polypeptide" means a nucleic acid sequence comprising
the coding region of a gene, or a fragment thereof which encodes a
gene product having substantially the same activity as the
corresponding full-length peptide, protein or polypeptide. The
coding region may be present in either a cDNA, genomic DNA or RNA
form. When present in a DNA form, the oligonucleotide may be
single-stranded (i.e., the sense strand) or double-stranded.
Suitable control elements such as enhancers/promoters, splice
junctions, polyadenylation signals, etc. may be placed in close
proximity to the coding region of the gene if needed to permit
proper initiation of transcription and/or correct processing of the
primary RNA transcript. Alternatively, the coding region utilized
in the expression vectors of the present invention may contain
endogenous enhancers/promoters, splice junctions, intervening
sequences, polyadenylation signals, etc. In further embodiments,
the coding region may contain a combination of both endogenous and
exogenous control elements.
The term "transcription regulatory element" or "transcription
regulatory sequence" refers to a genetic element or sequence that
controls some aspect of the expression of nucleic acid sequence(s).
For example, a promoter is a regulatory element that facilitates
the initiation of transcription of an operably linked coding
region. Other regulatory elements include, but are not limited to,
transcription factor binding sites, splicing signals,
polyadenylation signals, termination signals and enhancer
elements.
Transcriptional control signals in eukaryotes comprise "promoter"
and "enhancer" elements. Promoters and enhancers consist of short
arrays of DNA sequences that interact specifically with cellular
proteins involved in transcription. Promoter and enhancer elements
have been isolated from a variety of eukaryotic sources including
genes in yeast, insect and mammalian cells. Promoter and enhancer
elements have also been isolated from viruses and analogous control
elements, such as promoters, are also found in prokaryotes. The
selection of a particular promoter and enhancer depends on the cell
type used to express the protein of interest. Some eukaryotic
promoters and enhancers have a broad host range while others are
functional in a limited subset of cell types. For example, the SV40
early gene enhancer is very active in a wide variety of cell types
from many mammalian species and has been widely used for the
expression of proteins in mammalian cells. Two other examples of
promoter/enhancer elements active in a broad range of mammalian
cell types are those from the human elongation factor 1 gene
(Uetsuki et al., 1989; Kim et al., 1990; and Mizushima and Nagata,
1990) and the long terminal repeats of the Rous sarcoma virus
(Gorman et al., 1982); and the human cytomegalovirus (Boshart et
al., 1985).
The term "promoter/enhancer" denotes a segment of DNA containing
sequences capable of providing both promoter and enhancer functions
(i.e., the functions provided by a promoter element and an enhancer
element as described above). For example, the long terminal repeats
of retroviruses contain both promoter and enhancer functions. The
enhancer/promoter may be "endogenous" or "exogenous" or
"heterologous." An "endogenous" enhancer/promoter is one that is
naturally linked with a given gene in the genome. An "exogenous" or
"heterologous" enhancer/promoter is one that is placed in
juxtaposition to a gene by means of genetic manipulation (i.e.,
molecular biological techniques) such that transcription of the
gene is directed by the linked enhancer/promoter.
The presence of "splicing signals" on an expression vector often
results in higher levels of expression of the recombinant
transcript in eukaryotic host cells. Splicing signals mediate the
removal of introns from the primary RNA transcript and consist of a
splice donor and acceptor site (Sambrook et al., 1989). A commonly
used splice donor and acceptor site is the splice junction from the
16S RNA of SV40.
Efficient expression of recombinant DNA sequences in eukaryotic
cells requires expression of signals directing the efficient
termination and polyadenylation of the resulting transcript.
Transcription termination signals are generally found downstream of
the polyadenylation signal and are a few hundred nucleotides in
length. The term "poly(A) site" or "poly(A) sequence" as used
herein denotes a DNA sequence which directs both the termination
and polyadenylation of the nascent RNA transcript. Efficient
polyadenylation of the recombinant transcript is desirable, as
transcripts lacking a poly(A) tail are unstable and are rapidly
degraded. The poly(A) signal utilized in an expression vector may
be "heterologous" or "endogenous." An endogenous poly(A) signal is
one that is found naturally at the 3' end of the coding region of a
given gene in the genome. A heterologous poly(A) signal is one
which has been isolated from one gene and positioned 3' to another
gene. A commonly used heterologous poly(A) signal is the SV40 poly
(A) signal. The SV40 poly(A) signal is contained on a 237 bp BamH
1/Bcl 1 restriction fragment and directs both termination and
polyadenylation (Sambrook et al., 1989).
Eukaryotic expression vectors may also contain "viral replicons" or
"viral origins of replication." Viral replicons are viral DNA
sequences which allow for the extrachromosomal replication of a
vector in a host cell expressing the appropriate replication
factors. Vectors containing either the SV40 or polyoma virus origin
of replication replicate to high copy number (up to 10.sup.4
copies/cell) in cells that express the appropriate viral T antigen.
In contrast, vectors containing the replicons from bovine
papillomavirus or Epstein-Barr virus replicate extrachromosomally
at low copy number (about 100 copies/cell).
The term "in vitro" refers to an artificial environment and to
processes or reactions that occur within an artificial environment.
In vitro environments include, but are not limited to, test tubes
and cell lysates. The term "in situ" refers to cell culture. The
term "in vivo" refers to the natural environment (e.g., an animal
or a cell) and to processes or reaction that occur within a natural
environment.
The term "expression system" refers to any assay or system for
determining (e.g., detecting) the expression of a gene of interest.
Those skilled in the field of molecular biology will understand
that any of a wide variety of expression systems may be used. A
wide range of suitable mammalian cells are available from a wide
range of sources (e.g., the American Type Culture Collection,
Rockland, Md.). The method of transformation or transfection and
the choice of expression vehicle will depend on the host system
selected. Transformation and transfection methods are described,
e.g., in Sambrook et al., 1989. Expression systems include in vitro
gene expression assays where a gene of interest (e.g., a reporter
gene) is linked to a regulatory sequence and the expression of the
gene is monitored following treatment with an agent that inhibits
or induces expression of the gene. Detection of gene expression can
be through any suitable means including, but not limited to,
detection of expressed mRNA or protein (e.g., a detectable product
of a reporter gene) or through a detectable change in the phenotype
of a cell expressing the gene of interest. Expression systems may
also comprise assays where a cleavage event or other nucleic acid
or cellular change is detected.
The term "gene" refers to a DNA sequence that comprises coding
sequences and optionally control sequences necessary for the
production of a polypeptide from the DNA sequence. The polypeptide
can be encoded by a full-length coding sequence or by any portion
of the coding sequence so long as the portion encodes a gene
product with substantially the same activity as the full-length
polypeptide.
Nucleic acids are known to contain different types of mutations. A
"point" mutation refers to an alteration in the sequence of a
nucleotide at a single base position from the wild-type sequence.
Mutations may also refer to insertion or deletion of one or more
bases, so that the nucleic acid sequence differs from a reference,
e.g., a wild-type, sequence.
As used herein, the terms "hybridize" and "hybridization" refer to
the annealing of a complementary sequence to the target nucleic
acid, i.e., the ability of two polymers of nucleic acid
(polynucleotides) containing complementary sequences to anneal
through base pairing. The terms "annealed" and "hybridized" are
used interchangeably throughout, and are intended to encompass any
specific and reproducible interaction between a complementary
sequence and a target nucleic acid, including binding of regions
having only partial complementarity. Certain bases not commonly
found in natural nucleic acids may be included in the nucleic acids
of the present invention and include, for example, inosine and
7-deazaguanine. Those skilled in the art of nucleic acid technology
can determine duplex stability empirically considering a number of
variables including, for example, the length of the complementary
sequence, base composition and sequence of the oligonucleotide,
ionic strength and incidence of mismatched base pairs. The
stability of a nucleic acid duplex is measured by the melting
temperature, or "T.sub.m". The T.sub.m of a particular nucleic acid
duplex under specified conditions is the temperature at which on
average half of the base pairs have disassociated.
The term "stringency" is used in reference to the conditions of
temperature, ionic strength, and the presence of other compounds,
under which nucleic acid hybridizations are conducted. With "high
stringency" conditions, nucleic acid base pairing will occur only
between nucleic acid fragments that have a high frequency of
complementary base sequences. Thus, conditions of "medium" or "low"
stringency are often required when it is desired that nucleic acids
which are not completely complementary to one another be hybridized
or annealed together. The art knows well that numerous equivalent
conditions can be employed to comprise medium or low stringency
conditions. The choice of hybridization conditions is generally
evident to one skilled in the art and is usually guided by the
purpose of the hybridization, the type of hybridization (DNA-DNA or
DNA-RNA), and the level of desired relatedness between the
sequences (e.g., Sambrook et al., 1989; Nucleic Acid Hybridization,
A Practical Approach, IRL Press, Washington D.C., 1985, for a
general discussion of the methods).
The stability of nucleic acid duplexes is known to decrease with an
increased number of mismatched bases, and further to be decreased
to a greater or lesser degree depending on the relative positions
of mismatches in the hybrid duplexes. Thus, the stringency of
hybridization can be used to maximize or minimize stability of such
duplexes. Hybridization stringency can be altered by: adjusting the
temperature of hybridization; adjusting the percentage of helix
destabilizing agents, such as formamide, in the hybridization mix;
and adjusting the temperature and/or salt concentration of the wash
solutions. For filter hybridizations, the final stringency of
hybridizations often is determined by the salt concentration and/or
temperature used for the post-hybridization washes.
"High stringency conditions" when used in reference to nucleic acid
hybridization include conditions equivalent to binding or
hybridization at 42.degree. C. in a solution consisting of
5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4 H.sub.2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 0.1.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
"Medium stringency conditions" when used in reference to nucleic
acid hybridization include conditions equivalent to binding or
hybridization at 42.degree. C. in a solution consisting of
5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4 H.sub.2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 1.0.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
"Low stringency conditions" include conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4 H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,
5.times.Denhardt's reagent [50.times.Denhardt's contains per 500
ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)]
and 100 g/ml denatured salmon sperm DNA followed by washing in a
solution comprising 5.times.SSPE, 0.1% SDS at 42.degree. C. when a
probe of about 500 nucleotides in length is employed.
By "peptide", "protein" and "polypeptide" is meant any chain of
amino acids, regardless of length or post-translational
modification (e.g., glycosylation or phosphorylation). Unless
otherwise specified, the terms are interchangeable. The nucleic
acid molecules of the invention encode a variant (mutant) of a
naturally-occurring (wild-type) protein or fragment thereof which
has substantially the same activity as the full length mutant
protein. Preferably, such a mutant protein has an amino acid
sequence that is at least 85%, preferably 90%, and most preferably
95% or 99%, identical to the amino acid sequence of a corresponding
wild-type protein.
Polypeptide molecules are said to have an "amino terminus"
(N-terminus) and a "carboxy terminus" (C-terminus) because peptide
linkages occur between the backbone amino group of a first amino
acid residue and the backbone carboxyl group of a second amino acid
residue. The terms "N-terminal" and "C-terminal" in reference to
polypeptide sequences refer to regions of polypeptides including
portions of the N-terminal and C-terminal regions of the
polypeptide, respectively. A sequence that includes a portion of
the N-terminal region of polypeptide includes amino acids
predominantly from the N-terminal half of the polypeptide chain,
but is not limited to such sequences. For example, an N-terminal
sequence may include an interior portion of the polypeptide
sequence including bases from both the N-terminal and C-terminal
halves of the polypeptide. The same applies to C-terminal regions.
N-terminal and C-terminal regions may, but need not, include the
amino acid defining the ultimate N-terminus and C-terminus of the
polypeptide, respectively.
The term "isolated" when used in relation to a polypeptide, as in
"isolated protein" or "isolated polypeptide" refers to a
polypeptide that is identified and separated from at least one
contaminant with which it is ordinarily associated in its source.
Thus, an isolated polypeptide (1) is not associated with proteins
found in nature, (2) is free of other proteins from the same
source, e.g., free of human proteins, (3) is expressed by a cell
from a different species, or (4) does not occur in nature. In
contrast, non-isolated polypeptides (e.g., proteins and enzymes)
are found in the state they exist in nature. The terms "isolated
polypeptide", "isolated peptide" or "isolated protein" include a
polypeptide, peptide or protein encoded by cDNA or recombinant RNA
including one of synthetic origin, or some combination thereof.
The term "recombinant protein" or "recombinant polypeptide" as used
herein refers to a protein molecule expressed from a recombinant
DNA molecule. In contrast, the term "native protein" is used herein
to indicate a protein isolated from a naturally occurring (i.e., a
nonrecombinant) source. Molecular biological techniques may be used
to produce a recombinant form of a protein with identical
properties as compared to the native form of the protein.
The term "fusion polypeptide" as used herein refers to a chimeric
protein containing a protein of interest (e.g., luciferase, an
affinity tag or a targeting sequence) joined to a different
protein, e.g., a mutant hydrolase.
As used herein, the term "antibody" refers to a protein having one
or more polypeptides substantially encoded by immunoglobulin genes
or fragments of immunoglobulin genes. The recognized immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon and
mu constant region genes, as well as the myriad of immunoglobulin
variable region genes. Light chains are classified as either kappa
or lambda. Heavy chains are classified as gamma, mu, alpha, delta,
or epsilon, which in turn define the immunoglobulin classes, IgG,
IgM, IgA, IgD and IgE, respectively.
The basic immunoglobulin (antibody) structural unit is known to
comprise a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
Antibodies may exist as intact immunoglobulins, or as modifications
in a variety of forms including, for example, FabFc.sub.2, Fab, Fv,
Fd, (Fab').sub.2, an Fv fragment containing only the light and
heavy chain variable regions, a Fab or (Fab)'.sub.2 fragment
containing the variable regions and parts of the constant regions,
a single-chain antibody, e.g., scFv, CDR-grafted antibodies and the
like. The heavy and light chain of a Fv may be derived from the
same antibody or different antibodies thereby producing a chimeric
Fv region. The antibody may be of animal (especially mouse or rat)
or human origin or may be chimeric or humanized. As used herein the
term "antibody" includes these various forms.
The terms "cell," "cell line," "host cell," as used herein, are
used interchangeably, and all such designations include progeny or
potential progeny of these designations. By "transformed cell" is
meant a cell into which (or into an ancestor of which) has been
introduced a nucleic acid molecule of the invention. Optionally, a
nucleic acid molecule of the invention may be introduced into a
suitable cell line so as to create a stably transfected cell line
capable of producing the protein or polypeptide encoded by the
nucleic acid molecule. Vectors, cells, and methods for constructing
such cell lines are well known in the art. The words
"transformants" or "transformed cells" include the primary
transformed cells derived from the originally transformed cell
without regard to the number of transfers. All progeny may not be
precisely identical in DNA content, due to deliberate or
inadvertent mutations. Nonetheless, mutant progeny that have the
same functionality as screened for in the originally transformed
cell are included in the definition of transformants.
The term "homology" refers to a degree of complementarity. There
may be partial homology or complete homology (i.e., identity).
Homology is often measured using sequence analysis software (e.g.,
Sequence Analysis Software Package of the Genetics Computer Group.
University of Wisconsin Biotechnology Center. 1710 University
Avenue. Madison, Wis. 53705). Such software matches similar
sequences by assigning degrees of homology to various
substitutions, deletions, insertions, and other modifications.
Conservative substitutions typically include substitutions within
the following groups: glycine, alanine; valine, isoleucine,
leucine; aspartic acid, glutamic acid, asparagine, glutamine;
serine, threonine; lysine, arginine; and phenylalanine,
tyrosine.
The term "purified" or "to purify" means the result of any process
that removes some of a contaminant from the component of interest,
such as a protein or nucleic acid. The percent of a purified
component is thereby increased in the sample.
The term "operably linked" as used herein refer to the linkage of
nucleic acid sequences in such a manner that a nucleic acid
molecule capable of directing the transcription of a given gene
and/or the synthesis of a desired protein molecule is produced. The
term also refers to the linkage of sequences encoding amino acids
in such a manner that a functional (e.g., enzymatically active,
capable of binding to a binding partner, capable of inhibiting,
etc.) protein or polypeptide, or a precursor thereof, e.g., the
pre- or preproform of the protein or polypeptide, is produced.
All amino acid residues identified herein are in the natural
L-configuration. In keeping with standard polypeptide nomenclature,
abbreviations for amino acid residues are as shown in the following
Table of Correspondence.
TABLE-US-00001 TABLE OF CORRESPONDENCE 1-Letter 3-Letter AMINO ACID
Y Tyr L-tyrosine G Gly L-glycine F Phe L-phenylalanine M Met
L-methionine A Ala L-alanine S Ser L-serine I Ile L-isoleucine L
Leu L-leucine T Thr L-threonine V Val L-valine P Pro L-proline K
Lys L-lysine H His L-histidine Q Gln L-glutamine E Glu L-glutamic
acid W Trp L-tryptophan R Arg L-arginine D Asp L-aspartic acid N
Asn L-asparagine C Cys L-cysteine
As used herein, the term "poly-histidine tract" or (His tag) refers
to a molecule comprising two to ten histidine residues, e.g., a
poly-histidine tract of five to ten residues. A polyhistidine tract
allows the affinity purification of a covalently linked molecule on
an immobilized metal, e.g., nickel, zinc, cobalt or copper, chelate
column or through an interaction with another molecule (e.g., an
antibody reactive with the His tag).
As used herein, "pure" means an object species is the predominant
species present (i.e., on a molar basis it is more abundant than
any other individual species in the composition), and preferably a
substantially purified fraction is a composition wherein the object
species comprises at least about 50 percent (on a molar basis) of
all macromolecular species present. Generally, a "substantially
pure" composition will comprise more than about 80 percent of all
macromolecular species present in the composition, more preferably
more than about 85%, about 90%, about 95%, and about 99%. Most
preferably, the object species is purified to essential homogeneity
(contaminant species cannot be detected in the composition by
conventional detection methods) wherein the composition consists
essentially of a single macromolecular species.
I. Mutant Hydrolases and Fusions Thereof
Mutant hydrolases within the scope of the invention include but are
not limited to those prepared via recombinant techniques, e.g.,
site-directed mutagenesis or recursive mutagenesis, and comprise
one or more amino acid substitutions which render the mutant
hydrolase capable of forming a stable, e.g., covalent, bond with a
substrate, such as a substrate modified to contain one or more
functional groups, for a corresponding nonmutant (wild-type)
hydrolase. Hydrolases within the scope of the invention include,
but are not limited to, peptidases, esterases (e.g., cholesterol
esterase), glycosidases (e.g., glucosamylase), phosphatases (e.g.,
alkaline phosphatase) and the like. For instance, hydrolases
include, but are not limited to, enzymes acting on ester bonds such
as carboxylic ester hydrolases, thiolester hydrolases, phosphoric
monoester hydrolases, phosphoric diester hydrolases, triphosphoric
monoester hydrolases, sulfuric ester hydrolases, diphosphoric
monoester hydrolases, phosphoric triester hydrolases,
exodeoxyribonucleases producing 5'-phosphomonoesters,
exoribonucleases producing 5'-phosphomonoesters, exoribonucleases
producing 3'-phosphomonoesters, exonucleases active with either
ribo- or deoxyribonucleic acid, exonucleases active with either
ribo- or deoxyribonucleic acid, endodeoxyribonucleases producing
5'-phosphomonoesters, endodeoxyribonucleases producing other than
5'-phosphomonoesters, site-specific endodeoxyribonucleases specific
for altered bases, endoribonucleases producing
5'-phosphomonoesters, endoribonucleases producing other than
5'-phosphomonoesters, endoribonucleases active with either ribo- or
deoxyribonucleic, endoribonucleases active with either ribo- or
deoxyribonucleic glycosylases; glycosidases, e.g., enzymes
hydrolyzing O- and S-glycosyl, and hydrolyzing N-glycosyl
compounds; acting on ether bonds such as trialkylsulfonium
hydrolases or ether hydrolases; enzymes acting on peptide bonds
(peptide hydrolases) such as aminopeptidases, dipeptidases,
dipeptidyl-peptidases and tripeptidyl-peptidases,
peptidyl-dipeptidases, serine-type carboxypeptidases,
metallocarboxypeptidases, cysteine-type carboxypeptidases, omega
peptidases, serine endopeptidases, cysteine endopeptidases,
aspartic endopeptidases, metalloendopeptidases, threonine
endopeptidases, and endopeptidases of unknown catalytic mechanism;
enzymes acting on carbon-nitrogen bonds, other than peptide bonds,
such as those in linear amides, in cyclic amides, in linear
amidines, in cyclic amidines, in nitriles, or other compounds;
enzymes acting on acid anhydrides such as those in
phosphorous-containing anhydrides and in sulfonyl-containing
anhydrides; enzymes acting on acid anhydrides (catalyzing
transmembrane movement); enzymes acting on acid anhydrides or
involved in cellular and subcellular movement; enzymes acting on
carbon-carbon bonds (e.g., in ketonic substances); enzymes acting
on halide bonds (e.g., in C-halide compounds), enzymes acting on
phosphorus-nitrogen bonds; enzymes acting on sulfur-nitrogen bonds;
enzymes acting on carbon-phosphorus bonds; and enzymes acting on
sulfur-sulfur bonds. Exemplary hydrolases acting on halide bonds
include, but are not limited to, alkylhalidase, 2-haloacid
dehalogenase, haloacetate dehalogenase, thyroxine deiodinase,
haloalkane dehalogenase, 4-chlorobenzoate dehalogenase,
4-chlorobenzoyl-CoA dehalogenase, and atrazine chlorohydrolase.
Exemplary hydrolases that act on carbon-nitrogen bonds in cyclic
amides include, but are not limited to, barbiturase,
dihydropyrimidinase, dihydroorotase, carboxymethylhydantoinase,
allantoinase, .beta.-lactamase, imidazolonepropionase,
5-oxoprolinase (ATP-hydrolysing), creatininase, L-lysine-lactamase,
6-aminohexanoate-cyclic-dimer hydrolase, 2,5-dioxopiperazine
hydrolase, N-methylhydantoinase (ATP-hydrolysing), cyanuric acid
amidohydrolase, maleimide hydrolase. "Beta-lactamase" as used
herein includes Class A, Class C and Class D beta-lactamases as
well as D-ala carboxypeptidase/transpeptidase, esterase EstB,
penicillin binding protein 2.times., penicillin binding protein 5,
and D-amino peptidase. Preferably, the beta-lactamase is a serine
beta-lactamase, e.g., one having a catalytic serine residue at a
position corresponding to residue 70 in the serine beta-lactamase
of S. aureus PC1, and a glutamic acid residue at a position
corresponding to residue 166 in the serine beta-lactamase of S.
aureus PC1, optionally having a lysine residue at a position
corresponding to residue 73, and also optionally having a lysine
residue at a position corresponding to residue 234, in the
beta-lactamase of S. aureus PC1.
In one embodiment, the mutant hydrolase is a haloalkane
dehalogenase, e.g., such as those found in Gram-negative (Keuning
et al., 1985) and Gram-positive haloalkane-utilizing bacteria
(Keuning et al., 1985; Yokota et al., 1987; Scholtz et al., 1987;
Sallis et al., 1990). Haloalkane dehalogenases, including DhlA from
Xanthobacter autotrophicus GJ10(Janssen et al., 1988, 1989) and
DhaA from Rhodococcus rhodochrous, are enzymes which catalyze
hydrolytic dehalogenation of corresponding hydrocarbons.
Halogenated aliphatic hydrocarbons subject to conversion include
C.sub.2-C.sub.10 saturated aliphatic hydrocarbons which have one or
more halogen groups attached, wherein at least two of the halogens
are on adjacent carbon atoms. Such aliphatic hydrocarbons include
volatile chlorinated aliphatic (VCA) hydrocarbons. VCA's include,
for example, aliphatic hydrocarbons such as dichloroethane,
1,2-dichloro-propane, 1,2-dichlorobutane and
1,2,3-trichloropropane. The term "halogenated hydrocarbon" as used
herein means a halogenated aliphatic hydrocarbon. As used herein
the term "halogen" includes chlorine, bromine, iodine, fluorine,
astatine and the like. A preferred halogen is chlorine.
As described herein, the invention includes a fusion protein
comprising a mutant hydrolase and amino acid sequences for a
protein of interest, e.g., sequences for a marker protein or
affinity tag, e.g., luciferase, GFP, or a polyhistidine sequence, a
nucleic acid binding protein, an extracellular matrix protein, a
secreted protein, a receptor ligand, a serum protein, an
immunogenic protein, a fluorescent protein, a protein with reactive
cysteines, a receptor protein, e.g., NMDA receptor, a channel
protein, e.g., a sodium-, potassium- or a calcium-sensitive channel
protein including a HERG channel protein, or a transporter protein,
e.g., EAAT1-4 glutamate transporter, as well as targeting signals,
e.g., a plastid targeting signal, a nuclear localization signal or
a myristilation sequence.
II. Optimized Hydrolase Sequences and Vectors and Host Cells
Encoding the Hydrolase
A nucleic acid molecule comprising a nucleic acid sequence encoding
a hydrolase or a fusion thereof is optionally optimized for
expression in a particular host cell and also optionally operably
linked to transcription regulatory sequences, e.g., one or more
enhancers, a promoter, a transcription termination sequence or a
combination thereof, to form an expression cassette.
In one embodiment, a nucleic acid sequence encoding a hydrolase or
a fusion thereof is optimized by replacing codons in a wild-type or
mutant hydrolase sequence with codons which are preferentially
employed in a particular (selected) cell. Preferred codons have a
relatively high codon usage frequency in a selected cell, and
preferably their introduction results in the introduction of
relatively few transcription factor binding sites for transcription
factors present in the selected host cell, and relatively few other
undesirable structural attributes. Thus, the optimized nucleic acid
product has an improved level of expression due to improved codon
usage frequency, and a reduced risk of inappropriate
transcriptional behavior due to a reduced number of undesirable
transcription regulatory sequences.
An isolated and optimized nucleic acid molecule of the invention
may have a codon composition that differs from that of the
corresponding wild-type nucleic acid sequence at more than 30%,
35%, 40% or more than 45%, e.g., 50%, 55%, 60% or more of the
codons. Preferred codons for use in the invention are those which
are employed more frequently than at least one other codon for the
same amino acid in a particular organism and, more preferably, are
also not low-usage codons in that organism and are not low-usage
codons in the organism used to clone or screen for the expression
of the nucleic acid molecule. Moreover, preferred codons for
certain amino acids (i.e., those amino acids that have three or
more codons), may include two or more codons that are employed more
frequently than the other (non-preferred) codon(s). The presence of
codons in the nucleic acid molecule that are employed more
frequently in one organism than in another organism results in a
nucleic acid molecule which, when introduced into the cells of the
organism that employs those codons more frequently, is expressed in
those cells at a level that is greater than the expression of the
wild-type or parent nucleic acid sequence in those cells.
In one embodiment of the invention, the codons that are different
are those employed more frequently in a mammal, while in another
embodiment the codons that are different are those employed more
frequently in a plant. Preferred codons for different organisms are
known to the art, e.g., see www.kazusa.or.jp./codon/. A particular
type of mammal, e.g., a human, may have a different set of
preferred codons than another type of mammal. Likewise, a
particular type of plant may have a different set of preferred
codons than another type of plant. In one embodiment of the
invention, the majority of the codons that differ are ones that are
preferred codons in a desired host cell. Preferred codons for
organisms including mammals (e.g., humans) and plants are known to
the art (e.g., Wada et al., 1990; Ausubel et al., 1997). For
example, preferred human codons include, but are not limited to,
CGC (Arg), CTG (Leu), TCT (Ser), AGC (Ser), ACC (Thr), CCA (Pro),
CCT (Pro), GCC (Ala), GGC (Gly), GTG (Val), ATC (Ile), ATT (Ile),
AAG (Lys), AAC (Asn), CAG (Gln), CAC (His), GAG (Glu), GAC (Asp),
TAC (Tyr), TGC (Cys) and TTC (Phe) (Wada et al., 1990). Thus, in
one embodiment, synthetic nucleic acid molecules of the invention
have a codon composition which differs from a wild type nucleic
acid sequence by having an increased number of the preferred human
codons, e.g., CGC, CTG, TCT, AGC, ACC, CCA, CCT, GCC, GGC, GTG,
ATC, ATT, AAG, AAC, CAG, CAC, GAG, GAC, TAC, TGC, TTC, or any
combination thereof. For example, the nucleic acid molecule of the
invention may have an increased number of CTG or TTG
leucine-encoding codons, GTG or GTC valine-encoding codons, GGC or
GGT glycine-encoding codons, ATC or ATT isoleucine-encoding codons,
CCA or CCT proline-encoding codons, CGC or CGT arginine-encoding
codons, AGC or TCT serine-encoding codons, ACC or ACT
threonine-encoding codon, GCC or GCT alanine-encoding codons, or
any combination thereof, relative to the wild-type nucleic acid
sequence. In another embodiment, preferred C. elegans codons
include, but are not limited, to UUC (Phe), UUU (Phe), CUU (Leu),
UUG (Leu), AUU (Ile), GUU (Val), GUG (Val), UCA (Ser), UCU (Ser),
CCA (Pro), ACA (Thr), ACU (Thr), GCU (Ala), GCA (Ala), UAU (Tyr),
CAU (His), CAA (Gln), AAU (Asn), AAA (Lys), GAU (Asp), GAA (Glu),
UGU (Cys), AGA (Arg), CGA (Arg), CGU (Arg), GGA (Gly), or any
combination thereof. In yet another embodiment, preferred
Drosophilia codons include, but are not limited to, UUC (Phe), CUG
(Leu), CUC (Leu), AUC (Ile), AUU (Ile), GUG (Val), GUC (Val), AGC
(Ser), UCC (Ser), CCC (Pro), CCG (Pro), ACC (Thr), ACG (Thr), GCC
(Ala), GCU (Ala), UAC (Tyr), CAC (His), CAG (Gln), AAC (Asn), AAG
(Lys), GAU (Asp), GAG (Glu), UGC (Cys), CGC (Arg), GGC (Gly), GGA
(gly), or any combination thereof. Preferred yeast codons include
but are not limited to UUU (Phe), UUG (Leu), UUA (Leu), CCU (Leu),
AUU (Ile), GUU (Val), UCU (Ser), UCA (Ser), CCA (Pro), CCU (Pro),
ACU (Thr), ACA (Thr), GCU (Ala), GCA (Ala), UAU (Tyr), UAC (Tyr),
CAU (His), CAA (Gln), AAU (Asn), AAC (Asn), AAA (Lys), AAG (Lys),
GAU (Asp), GAA (Glu), GAG (Glu), UGU (Cys), CGU (Trp), AGA (Arg),
CGU (Arg), GGU (Gly), GGA (Gly), or any combination thereof.
Similarly, nucleic acid molecules having an increased number of
codons that are employed more frequently in plants, have a codon
composition which differs from a wild-type or parent nucleic acid
sequence by having an increased number of the plant codons
including, but not limited to, CGC (Arg), CTT (Leu), TCT (Ser), TCC
(Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCT (Ser), GGA (Gly), GTG
(Val), ATC (Ile), ATT (Ile), AAG (Lys), AAC (Asn), CAA (Gln), CAC
(His), GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys), TTC (Phe), or
any combination thereof (Murray et al., 1989). Preferred codons may
differ for different types of plants (Wada et al., 1990).
In one embodiment, an optimized nucleic acid sequence encoding a
hydrolase or fusion thereof has less than 100%, e.g., less than 90%
or less than 80%, nucleic acid sequence identity relative to a
non-optimized nucleic acid sequence encoding a corresponding
hydrolase or fusion thereof. For instance, an optimized nucleic
acid sequence encoding DhaA has less than about 80% nucleic acid
sequence identity relative to non-optimized (wild-type) nucleic
acid sequence encoding a corresponding DhaA, and the DhaA encoded
by the optimized nucleic acid sequence optionally has at least 85%
amino acid sequence identity to a corresponding wild-type DhaA. In
one embodiment, the activity of a DhaA encoded by the optimized
nucleic acid sequence is at least 10%, e.g., 50% or more, of the
activity of a DhaA encoded by the non-optimized sequence, e.g., a
mutant DhaA encoded by the optimized nucleic acid sequence binds a
substrate with substantially the same efficiency, i.e., at least
50%, 80%, 100% or more, as the mutant DhaA encoded by the
non-optimized nucleic acid sequence binds the same substrate.
The nucleic acid molecule or expression cassette may be introduced
to a vector, e.g., a plasmid or viral vector, which optionally
includes a selectable marker gene, and the vector introduced to a
cell of interest, for example, a prokaryotic cell such as E.coli,
Streptomyces spp., Bacillus spp. Staphylococcus spp. and the like,
as well as eukaryotic cells including a plant (dicot or monocot),
fungus, yeast, e.g., Pichia, Saccharomyces or Schizosaccharomyces,
or mammalian cell. Preferred mammalian cells include bovine,
caprine, ovine, canine, feline, non-human primate, e.g., simian,
and human cells. Preferred mammalian cell lines include, but are
not limited to, CHO, COS, 293, Hela, CV-1, SH-SY5Y (human
neuroblastoma cells), HEK293, and NIH3T3 cells.
The expression of the encoded mutant hydrolase may be controlled by
any promoter capable of expression in prokaryotic cells or
eukaryotic cells. Preferred prokaryotic promoters include, but are
not limited to, SP6, T7, T5, tac, bla, trp, gal, lac or maltose
promoters. Preferred eukaryotic promoters include, but are not
limited to, constitutive promoters, e.g., viral promoters such as
CMV, SV40 and RSV promoters, as well as regulatable promoters,
e.g., an inducible or repressible promoter such as the tet
promoter, the hsp70 promoter and a synthetic promoter regulated by
CRE. Preferred vectors for bacterial expression include
pGEX5.times.-3, and for eukaryotic expression include
pClneo-CMV.
The nucleic acid molecule, expression cassette and/or vector of the
invention may be introduced to a cell by any method including, but
not limited to, calcium-mediated transformation, electroporation,
microinjection, lipofection, particle bombardment and the like.
III. Functional Groups
Functional groups useful in the substrates and methods of the
invention are molecules that are detectable or capable of
detection. A functional group within the scope of the invention is
capable of being covalently linked to one reactive substituent of a
bifunctional linker or a substrate for a hydrolase, and, as part of
a substrate of the invention, has substantially the same activity
as a functional group which is not linked to a substrate found in
nature and is capable of forming a stable complex with a mutant
hydrolase. Functional groups thus have one or more properties that
facilitate detection, and optionally the isolation, of stable
complexes between a substrate having that functional group and a
mutant hydrolase. For instance, functional groups include those
with a characteristic electromagnetic spectral property such as
emission or absorbance, magnetism, electron spin resonance,
electrical capacitance, dielectric constant or electrical
conductivity as well as functional groups which are ferromagnetic,
paramagnetic, diamagnetic, luminescent, electrochemiluminescent,
fluorescent, phosphorescent, chromatic, antigenic, or have a
distinctive mass. A functional group includes, but is not limited
to, a nucleic acid molecule, i.e., DNA or RNA, e.g., an
oligonucleotide or nucleotide, a protein, e.g., a luminescent
protein, a peptide, for instance, an epitope recognized by a
ligand, e.g., biotin or streptavidin, a hapten, an amino acid, a
lipid, a lipid bilayer, a solid support, a fluorophore, a
chromophore, a reporter molecule, a radionuclide, an electron
opaque molecule, a MRI contrast agent, e.g., manganese, gadolinium
(III) or iron-oxide particles, and the like. Methods to detect a
particular functional group are known to the art. For example, a
nucleic acid molecule can be detected by hybridization,
amplification, binding to a nucleic acid binding protein specific
for the nucleic acid molecule, enzymatic assays (e.g., if the
nucleic acid molecule is a ribozyme), or, if the nucleic acid
molecule itself comprises a molecule which is detectable or capable
of detection, for instance, a radiolabel or biotin, it can be
detected by an assay suitable for that molecule.
Exemplary functional groups include haptens, e.g., molecules useful
to enhance immunogenicity such as keyhole limpet hemacyanin (KLH),
cleavable labels, for instance, photocleavable biotin, and
fluorescent labels, e.g., N-hydroxysuccinimide (NHS) modified
coumarin and succinimide or sulfonosuccinimide modified BODIPY
(which can be detected by UV and/or visible excited fluorescence
detection), rhodamine, e.g., R110, rhodols, CRG6, Texas Methyl Red
(TAMRA), Rox5, FAM, or fluoroscein, coumarin derivatives, e.g., 7
aminocoumarin, and 7-hydroxycoumarin, 2-amino-4-methoxynapthalene,
1-hydroxypyrene, resorufin, phenalenones or benzphenalenones (U.S.
Pat. No. 4,812,409), acridinones (U.S. Pat. No. 4,810,636),
anthracenes, and derivatives of .alpha.- and .beta.-napthol,
fluorinated xanthene derivatives including fluorinated fluoresceins
and rhodols (e.g., U.S. Pat. No. 6,162,931), and bioluminescent
molecules, e.g., luciferase or GFP. A fluorescent (or
bioluminescent) functional group linked to a mutant hydrolase by
virtue of being linked to a substrate for a corresponding wild-type
hydrolase, may be used to sense changes in a system, like
phosphorylation, in real time. Moreover, a fluorescent molecule,
such as a chemosensor of metal ions, e.g., a 9-carbonylanthracene
modified glycyl-histidyl-lysine (GHK) for Cu.sup.2+, in a substrate
of the invention may be employed to label proteins which bind the
substrate. A bioluminescent or fluorescent functional group such as
BODIPY, rhodamine green, GFP, or infrared dyes, also finds use as a
functional group and may, for instance, be employed in interaction
studies, e.g., using BRET, FRET, LRET or electrophoresis.
Another class of functional group is a molecule that selectively
interacts with molecules containing acceptor groups (an "affinity"
molecule). Thus, a substrate for a hydrolase which includes an
affinity molecule can facilitate the separation of complexes having
such a substrate and a mutant hydrolase, because of the selective
interaction of the affinity molecule with another molecule, e.g.,
an acceptor molecule, that may be biological or non-biological in
origin. For example, the specific molecule with which the affinity
molecule interacts (referred to as the acceptor molecule) could be
a small organic molecule, a chemical group such as a sulfhydryl
group (--SH) or a large biomolecule such as an antibody or other
naturally occurring ligand for the affinity molecule. The binding
is nominally chemical in nature and may involve the formation of
covalent or non-covalent bonds or interactions such as ionic or
hydrogen bonding. The acceptor molecule might be free in solution
or itself bound to a solid or semi-solid surface, a polymer matrix,
or reside on the surface of a solid or semi-solid substrate. The
interaction may also be triggered by an external agent such as
light, temperature, pressure or the addition of a chemical or
biological molecule that acts as a catalyst. The detection and/or
separation of the complex from the reaction mixture occurs because
of the interaction, normally a type of binding, between the
affinity molecule and the acceptor molecule.
Examples of affinity molecules include molecules such as
immunogenic molecules, e.g., epitopes of proteins, peptides,
carbohydrates or lipids, i.e., any molecule which is useful to
prepare antibodies specific for that molecule; biotin, avidin,
streptavidin, and derivatives thereof; metal binding molecules; and
fragments and combinations of these molecules. Exemplary affinity
molecules include His5 (HHHHH) (SEQ ID NO:19), HisX6 (HHHHHH) (SEQ
ID NO:20), C-myc (EQKLISEEDL) (SEQ ID NO:21), Flag (DYKDDDDK) (SEQ
ID NO:22), SteptTag (WSHPQFEK) (SEQ ID NO:23), HA Tag (YPYDVPDYA)
(SEQ ID NO:24), thioredoxin, cellulose binding domain, chitin
binding domain, S-peptide, T7 peptide, calmodulin binding peptide,
C-end RNA tag, metal binding domains, metal binding reactive
groups, amino acid reactive groups, inteins, biotin, streptavidin,
and maltose binding protein. For example, a substrate for a
hydrolase which includes biotin is contacted with a mutant
hydrolase. The presence of the biotin in a complex between the
mutant hydrolase and the substrate permits selective binding of the
complex to avidin molecules, e.g., streptavidin molecules coated
onto a surface, e.g., beads, microwells, nitrocellulose and the
like. Suitable surfaces include resins for chromatographic
separation, plastics such as tissue culture surfaces or binding
plates, microliter dishes and beads, ceramics and glasses,
particles including magnetic particles, polymers and other
matrices. The treated surface is washed with, for example,
phosphate buffered saline (PBS), to remove molecules that lack
biotin and the biotin-containing complexes isolated. In some case
these materials may be part of biomolecular sensing devices such as
optical fibers, chemfets, and plasmon detectors.
Another example of an affinity molecule is dansyllysine. Antibodies
which interact with the dansyl ring are commercially available
(Sigma Chemical; St. Louis, Mo.) or can be prepared using known
protocols such as described in Antibodies: A Laboratory Manual
(Harlow and Lane, 1988). For example, the anti-dansyl antibody is
immobilized onto the packing material of a chromatographic column.
This method, affinity column chromatography, accomplishes
separation by causing the complex between a mutant hydrolase and a
substrate of the invention to be retained on the column due to its
interaction with the immobilized antibody, while other molecules
pass through the column. The complex may then be released by
disrupting the antibody-antigen interaction. Specific
chromatographic column materials such as ion-exchange or affinity
Sepharose, Sephacryl, Sephadex and other chromatography resins are
commercially available (Sigma Chemical; St. Louis, Mo.; Pharmacia
Biotech; Piscataway, N.J.). Dansyllysine may conveniently be
detected because of its fluorescent properties.
When employing an antibody as an acceptor molecule, separation can
also be performed through other biochemical separation methods such
as immunoprecipitation and immobilization of antibodies on filters
or other surfaces such as beads, plates or resins. For example,
complexes of a mutant hydrolase and a substrate of the invention
may be isolated by coating magnetic beads with an affinity
molecule-specific or a hydrolase-specific antibody. Beads are
oftentimes separated from the mixture using magnetic fields.
Another class of functional molecules includes molecules detectable
using electromagnetic radiation and includes but is not limited to
xanthene fluorophores, dansyl fluorophores, coumarins and coumarin
derivatives, fluorescent acridinium moieties, benzopyrene based
fluorophores, as well as 7-nitrobenz-2-oxa-1,3-diazole, and
3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diamino-propionic acid.
Preferably, the fluorescent molecule has a high quantum yield of
fluorescence at a wavelength different from native amino acids and
more preferably has high quantum yield of fluorescence that can be
excited in the visible, or in both the UV and visible, portion of
the spectrum. Upon excitation at a preselected wavelength, the
molecule is detectable at low concentrations either visually or
using conventional fluorescence detection methods.
Electrochemiluminescent molecules such as ruthenium chelates and
its derivatives or nitroxide amino acids and their derivatives are
detectable at femtomolar ranges and below.
In addition to fluorescent molecules, a variety of molecules with
physical properties based on the interaction and response of the
molecule to electromagnetic fields and radiation can be used to
detect complexes between a mutant hydrolase and a substrate of the
invention. These properties include absorption in the UV, visible
and infrared regions of the electromagnetic spectrum, presence of
chromophores which are Raman active, and can be further enhanced by
resonance Raman spectroscopy, electron spin resonance activity and
nuclear magnetic resonances and molecular mass, e.g., via a mass
spectrometer.
Methods to detect and/or isolate complexes having affinity
molecules include chromatographic techniques including gel
filtration, fast-pressure or high-pressure liquid chromatography,
reverse-phase chromatography, affinity chromatography and ion
exchange chromatography. Other methods of protein separation are
also useful for detection and subsequent isolation of complexes
between a mutant hydrolase and a substrate of the invention, for
example, electrophoresis, isoelectric focusing and mass
spectrometry.
IV. Linkers
The term "linker", which is also identified by the symbol `L`,
refers to a group or groups that covalently attach one or more
functional groups to a substrate which includes a reactive group or
to a reactive group. A linker, as used herein, is not a single
covalent bond. The structure of the linker is not crucial, provided
it yields a substrate that can be bound by its target enzyme. In
one embodiment, the linker can be a divalent group that separates a
functional group (R) and the reactive group by about 5 angstroms to
about 1000 angstroms, inclusive, in length. Other suitable linkers
include linkers that separate R and the reactive group by about 5
angstroms to about 100 angstroms, as well as linkers that separate
R and the substrate by about 5 angstroms to about 50 angstroms, by
about 5 angstroms to about 25 angstroms, by about 5 angstroms to
about 500 angstroms, or by about 30 angstroms to about 100
angstroms.
In one embodiment the linker is an amino acid.
In another embodiment, the linker is a peptide.
In another embodiment, the linker is a divalent branched or
unbranched carbon chain comprising from about 2 to about 30 carbon
atoms, which chain optionally includes one or more (e.g., 1, 2, 3,
or 4) double or triple bonds, and which chain is optionally
substituted with one or more (e.g., 2, 3, or 4) hydroxy or oxo
(.dbd.O) groups, wherein one or more (e.g., 1, 2, 3, or 4) of the
carbon atoms in the chain is optionally replaced with a
non-peroxide --O--, --S-- or --NH--.
In another embodiment, the linker is a divalent group of the
formula --W--F--W-- wherein F is (C.sub.1-C.sub.30)alkyl,
(C.sub.2-C.sub.30)alkenyl, (C.sub.2-C.sub.30)alkynyl,
(C.sub.3-C.sub.8)cycloalkyl, or (C.sub.6-C.sub.10)aryl, wherein W
is --N(Q)C(.dbd.O)--, --C(.dbd.O)N (Q)--, --OC(.dbd.O)O--,
--C(.dbd.O)O--, --O--, --S--, --S(O)--, --S(O).sub.2--, --N(Q)--,
--C(.dbd.O)--, or a direct bond; wherein each Q is independently H
or (C.sub.1-C.sub.6)alkyl
In another embodiment, the linker is a divalent branched or
unbranched carbon chain comprising from about 2 to about 30 carbon
atoms, which chain optionally includes one or more (e.g., 1, 2, 3,
or 4) double or triple bonds, and which chain is optionally
substituted with one or more (e.g., 2, 3, or 4) hydroxy or oxo
(.dbd.O) groups.
In another embodiment, the linker is a divalent branched or
unbranched carbon chain comprising from about 2 to about 30 carbon
atoms, which chain optionally includes one or more (e.g., 1, 2, 3,
or 4) double or triple bonds.
In another embodiment, the linker is a divalent branched or
unbranched carbon chain comprising from about 2 to about 30 carbon
atoms.
In another embodiment, the linker is a divalent branched or
unbranched carbon chain comprising from about 2 to about 20 carbon
atoms, which chain optionally includes one or more (e.g., 1, 2, 3,
or 4) double or triple bonds, and which chain is optionally
substituted with one or more (e.g., 2, 3, or 4) hydroxy or oxo
(.dbd.O) groups.
In another embodiment, the linker is a divalent branched or
unbranched carbon chain comprising from about 2 to about 20 carbon
atoms, which chain optionally includes one or more (e.g., 1, 2, 3,
or 4) double or triple bonds.
In another embodiment, the linker is a divalent branched or
unbranched carbon chain comprising from about 2 to about 20 carbon
atoms.
In another embodiment, the linker is
--(CH.sub.2CH.sub.2O)--.sub.1-10.
In another embodiment, the linker is --C(.dbd.O )NH
(CH.sub.2).sub.3;
--C(.dbd.O)NH(CH.sub.2).sub.5C(.dbd.O)NH(CH.sub.2)--;
--CH.sub.2OC(.dbd.O)NH(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2)--;
--C(.dbd.O) NH(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.3--;
--CH.sub.2OC(.dbd.O)NH(CH.sub.2).sub.2 O(CH.sub.2).sub.3--;
--(CH.sub.2).sub.4C(.dbd.O)NH(CH.sub.2).sub.2O(CH.sub.2).sub.2O
(CH.sub.2).sub.3--;
--C(.dbd.O)NH(CH.sub.2).sub.5C(.dbd.O)NH(CH.sub.2).sub.2O(CH.sub.2).sub.2
O(CH.sub.2).sub.3--;
Specifically, (C.sub.1-C.sub.30)alkyl can be methyl, ethyl, propyl,
isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl,
heptyl, octyl, nonyl, or decyl; (C.sub.3-C.sub.8)cycloalkyl can be
cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
(C.sub.2-C.sub.30)alkenyl can be vinyl, allyl, 1-propenyl,
2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl,
2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl,
3-hexenyl, 4-hexenyl, 5-hexenyl, heptenyl, octenyl, nonenyl, or
decenyl; (C.sub.2-C.sub.30)alkynyl can be ethynyl, 1-propynyl,
2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl,
2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl,
3-hexynyl, 4-hexynyl, 5-hexynyl, heptynyl, octynyl, nonynyl, or
decynyl; and (C.sub.6-C.sub.10)aryl can be phenyl, indenyl, or
naphthyl
The term "amino acid," when used with reference to a linker,
comprises the residues of the natural amino acids (e.g., Ala, Arg,
Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as
unnatural amino acids (e.g., phosphoserine, phosphothreonine,
phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric
acid, octahydroindole-2-carboxylic acid, statine,
1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,
ornithine, citruline, .alpha.-methyl-alanine,
para-benzoylphenylalanine, phenylglycine, propargylglycine,
sarcosine, and tert-butylglycine). The term also includes natural
and unnatural amino acids bearing a conventional amino protecting
group (e.g., acetyl or benzyloxycarbonyl), as well as natural and
unnatural amino acids protected at the carboxy terminus (e.g. as a
(C.sub.1-C.sub.6)alkyl, phenyl or benzyl ester or amide). Other
suitable amino and carboxy protecting groups are known to those
skilled in the art (see for example, Greene, Protecting Groups In
Organic Synthesis; Wiley: New York, 1981, and references cited
therein). An amino acid can be linked to another molecule through
the carboxy terminus, the amino terminus, or through any other
convenient point of attachment, such as, for example, through the
sulfur of cysteine.
The term "peptide" when used with reference to a linker, describes
a sequence of 2 to 25 amino acids (e.g. as defined hereinabove) or
peptidyl residues. The sequence may be linear or cyclic. For
example, a cyclic peptide can be prepared or may result from the
formation of disulfide bridges between two cysteine residues in a
sequence. A peptide can be linked to another molecule through the
carboxy terminus, the amino terminus, or through any other
convenient point of attachment, such as, for example, through the
sulfur of a cysteine. Preferably a peptide comprises 3 to 25, or 5
to 21 amino acids. Peptide derivatives can be prepared as disclosed
in U.S. Pat. Nos. 4,612,302; 4,853,371; and 4,684,620. Peptide
sequences specifically recited herein are written with the amino
terminus on the left and the carboxy terminus on the right.
In one embodiment, a substrate of the invention for a dehalogenase
which has a linker has the formula (I): R-linker-A--X (1) wherein R
is one or more functional groups (such as a fluorophore, biotin,
luminophore, or a fluorogenic or luminogenic molecule, or is a
solid support, including microspheres, membranes, glass beads, and
the like), wherein the linker is a multiatom straight or branched
chain including C, N, S, or O, wherein A--X is a substrate for a
dehalogenase, and wherein X is a halogen. In one embodiment, A--X
is a haloaliphatic or haloaromatic substrate for a dehalogenase. In
one embodiment, the linker is a divalent branched or unbranched
carbon chain comprising from about 12 to about 30 carbon atoms,
which chain optionally includes one or more (e.g., 1, 2, 3, or 4)
double or triple bonds, and which chain is optionally substituted
with one or more (e.g., 2, 3, or 4) hydroxy or oxo (.dbd.O) groups,
wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in
the chain is optionally replaced with a non-peroxide --O--, --S--
or --NH--.In one embodiment, A is CH.sub.2CH.sub.2 or
CH.sub.2CH.sub.2CH.sub.2. In one embodiment, a linker in a
substrate for a dehalogenase such as a Rhodococcus dehalogenase, is
a multiatom straight or branched chain including C, N, S, or O, and
preferably 11-30 atoms when the functional group R includes an
aromatic ring system or is a solid support.
In another embodiment, a substrate of the invention for a
dehalogenase which has a linker has formula (II):
R-linker-CH.sub.2--CH.sub.2--CH.sub.2--X (II) where X is a halogen,
preferably chloride. In one embodiment, R is one or more functional
groups, such as a fluorophore, biotin, luminophore, or a
fluorogenic or luminogenic molecule, or is a solid support,
including microspheres, membranes, glass beads, and the like. When
R is a radiolabel, or a small detectable atom such as a
spectroscopically active isotope, the linker can be 0-30 atoms. V.
Syntheses for Exemplary Substrates
[2-(2-Hydroxy-ethoxy)-ethyl]-carbamic acid anthracen-9-ylmethyl
ester. To a stirring slurry of 9-anthracenemethanol (10 g, 48 mmol)
and 4-nitrophenyl chloroformate (13.6 g, 67.5 mmol) in 200 ml
CH.sub.2Cl.sub.2 was added triethylamine (6.7 ml, 0.19 mol). The
resulting gold colored solution was allowed to stir 16 hrs at room
temperature. At this point, 2-(2-aminoethoxy)ethanol (14.4 ml,
0.144 mol) was added and stirring continued for another 24 hours.
The CH.sub.2Cl.sub.2 reaction mixture was then washed with a 2%
sodium hydroxide (w/w) solution until no p-nitrophenol was observed
in the organic layer. The dichloromethane was dried with sodium
sulfate, filtered, and evaporated under reduced pressure.
The crude product was further purified by column chromatography on
silica gel 60, progressively eluting with 1% to 3% methanol in
dichloromethane. 7.6 g (58% yield) of a yellow solid was isolated:
.sup.1H NMR (CDCl.sub.3) .delta. 8.38 (s, H-10), 8.28 (d, H-1, 8),
7.94 (d, H-4, 5), 7.44 (m, H-2, 3, 6, 7), 6.06 (s, CH.sub.2-anth),
5.47 (t, exchangeable, NH), 3.53 (bs, CH.sub.2--OH) 3.33 (m, three
--CH.sub.2--). Mass spectrum, m/e Calcd for
C.sub.20H.sub.22NO.sub.4.sup.+: 340.15. Found: 340.23. Calcd for
C.sub.20H.sub.21NNaO.sub.4.sup.+: 340.15. Found: 340.23.
##STR00001##
{2-[2-(6-Chloro-hexyloxy)-ethoxy]-ethyl}-carbamic acid
anthracen-9-ylmethyl ester. A 100 ml round bottom flask was charged
with [2-(2-Hydroxy-ethoxy)-ethyl]-carbamic acid
anthracen-9-ylmethyl ester (1.12 g, 3 mmol) and fresh sodium
hydride, 60% dispersion in mineral oil (360 mg, 9 mmol) under inert
atmosphere. 20 ml anhydrous THF was added and the reaction allowed
to stir for 30 minutes. The flask is then cooled to between -10 and
-20.degree. C. by means of an ice/NaCl bath. When the temperature
is reached 1-chloro-6-Iodohexane (1 ml, 6 mmol) is added via
syringe. The reaction is maintained at ice/NaCl temperature for 2
hours, then slowly allowed to warm to room temperature overnight.
At this point silica gel 60 is co-absorbed onto the reaction
mixture with loss of solvent under reduced pressure. Silica gel
chromatography takes place initially with heptane as eluent,
followed by 10%, 20%, and 25% ethyl acetate. A total of 0.57 g (41%
yield) of product is isolated from appropriate fractions: .sup.1H
NMR (CDCl.sub.3) .delta. 8.48 (s, H-10), 8.38 (d, H-1, 8), 8.01 (d,
H-4, 5), 7.52 (dt, H-2, 3, 6, 7), 6.13 (s, CH.sub.2-anth), 5.29
(bs, exchangeable, NH), 3.74 (m, 4H), 3.55-3.15 (m, 8H), 1.84 (m,
4H), 1.61 (m, 1H), 1.43 (m, 1 H), 1.25 (m, 2H). Mass spectrum, m/e
Calcd for C.sub.26H.sub.32ClNO.sub.4H.sub.2O: 475.21(100%),
476.22(29.6%). Found: 475.21, 476.52.
##STR00002## 2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl-ammonium
trifluoro-acetate. To
{2-[2-(6-Chloro-hexyloxy)-ethoxy]ethyl}-carbamic acid
anthracen-9-ylmethyl ester (0.56 g, 1.2 mmol) dissolved in 4 ml
dichloromethane was added 2 drops of anisole. The reaction mixture
is cooled by means of an ice/NaCl bath. After 10 minutes
trifluoroacetic acid (2 ml) is added. The reaction mixture turns
dark brown upon addition and is allowed to stir for 30 minutes. All
volatiles are removed under reduced atmosphere. The residue is
re-dissolved in CH.sub.2Cl.sub.2 and washed twice with water. The
aqueous fractions are frozen and lyophilized overnight. An oily
residue remains and is dissolved in anhydrous DMF to be used as a
stock solution in further reactions. Mass spectrum, m/e Calcd for
C.sub.10H.sub.23ClNO.sub.2.sup.+: 224.14(100%), 226.14(32%). Found:
224.2, 226.2.
##STR00003##
General methodology for reporter group conjugation to
2-[2-(6-chloro-hexyloxy)-ethoxy]-ethylamine. To one equivalent of
the succinimidyl ester of the reporter group in DMF is added 3
equivalence of 2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl-ammonium
trifluoro-acetate stock solution, followed by
diisopropylethylamine. The reaction is stirred from 8 to 16 hours
at room temperature. Purification is accomplished by preparative
scale HPLC or silica gel chromatography.
N-{2-[2-(6-Chlorohexyloxy)-ethoxy]ethyl}-fluorescein-5-amide. The
title compound was prepared using the above methodology.
Purification was accomplished using preparative scale HPLC. Mass
spectrum, m/e Calcd for C.sub.31H.sub.31ClNO.sub.8.sup.-:
580.17(100%), 581.18(32%). Found: 580.18, 581.31.
##STR00004##
N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-biotin-amide. The title
compound was prepared using the above methodology. Purification was
accomplished using silica gel chromatography (2% to 5% methanol in
dichloromethane). Mass spectrum, m/e Calcd for
C.sub.20H.sub.37ClN.sub.3O.sub.4S.sup.+: 450.22(100%), 452.22(32%).
Found: 449.95, 451.89.
##STR00005##
N-{2-[2-(6-Chlorohexyloxy)-ethoxy]ethyl}-tetramethyl-rhodamine-5-(and
-6)-amide. The title compound was prepared using the above
methodology. Purification was accomplished using preparative scale
HPLC. Separation of structural isomers was realized. Mass spectrum,
m/e Calcd for C.sub.35H.sub.43ClN.sub.3O.sub.6.sup.+: 636.28(100%),
637.29(39.8%), 638.28 (32.4%). Found: 636.14, 637.15, 638.14.
##STR00006## N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-rhodamine
R110-5-(and -6)-amide. The title compound was prepared using the
above methodology. Purification was accomplished using preparative
scale HPLC. Separation of structural isomers was realized. Mass
spectrum, m/e Calcd for C.sub.31H.sub.35ClN.sub.3O.sub.6.sup.+:
580.2(100%), 581.2(35.6%), 582.2(32.4%). Found: 580.4, 581.4,
582.2.
##STR00007##
6-({4-[-4,4difluoro-5-(thiophen-2-yl)-4-bora-3a-4a-diaza-s-indacene-3-yl]-
phenoxy}-acetylamino)-hexanoic acid
{2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide. The title compound
was prepared using the above methodology. Purification was
accomplished using silica gel chromatography (3% to 5% methanol in
dichloromethane). Mass spectrum, m/e Calcd for
C.sub.37H.sub.47BClF.sub.2N.sub.4O.sub.5S.sup.+: 743.3(100%).
Found: 743.4.
##STR00008##
6-({4-[4,4difluoro-5-(thiophen-2-yl)-4-bora-3a-4a-diaza-s-indacene-3-yl]s-
tyryloxy}-acetylamino)-hexanoic acid
{2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide. The title compound
was prepared using the above methodology. Purification was
accomplished using silica gel chromatography (3% methanol in
dichloromethane). Mass spectrum, m/e Calcd for
C39H48BClF2N4NaO5S.sup.+: 791.3(100%). Found: 7.91.3.
##STR00009## Triethylammonium
3-[5-[2-(4-tert-Butyl-7-diethylaminochromen-2-ylidene)-ethylidene]-3-(5-{-
2-[2-(6-chlorohexyloxy)-ethoxy]-ethylcarbamoyl}-pentyl)-2,4,6-trioxo-tetra-
hydro-pyrimidin-1-yl]-propane-1-sulfonic acid anion. The title
compound was prepared using the above methodology. Purification was
accomplished using preparative scale HPLC. Mass spectrum, m/e Calcd
for C.sub.42H.sub.62ClN.sub.4O.sub.10S.sup.-: 849.4(100%),
850.4(48.8%), 851.4(36.4%). Found: 849.6, 850.5, 851.5.
##STR00010##
2-tert-Butyl-4-{3-[1-(5-{2-[2-(6-chlorohexyloxy)-ethoxy]ethylcarbamoyl}-p-
entyl)-3,3-dimethyl-5-sulfo-1,3-dihydro-indol-2-ylidenel-propenyl}-7-dieth-
ylamino-chromenylium chloride. The title compound was prepared
using the above methodology. Purification was accomplished using
preparative scale HPLC. Mass spectrum, m/e Calcd for
C.sub.45H.sub.67ClN.sub.3O.sub.7S.sup.-: 840.4(100%), 841.4(54.4%).
Found: 840.5, 841.5.
##STR00011##
N-{2-12-(6-Chlorohexyloxy)-ethoxy]-ethyl}-3-{4-[5-(4-dimethylamino-phenyl-
)-oxazol-2-yl]-benzenesulfonylamino}-propionamide. The title
compound was prepared using the above methodology. Purification was
accomplished using preparative scale HPLC. Mass spectrum, m/e Calcd
for C.sub.30H.sub.40ClN.sub.4O.sub.6S.sup.-: 619.2(100%),
620.2(35%). Found: 619.5, 620.7.
N-{2-[2-(6-Chlorohexyloxy)-ethox]-ethyl}-9'-chloroseminaphthofuorescein-5-
-(and -6)-amide. The title compound was prepared using the above
methodology. Purification was accomplished using preparative scale
HPLC. Separation of structural isomers was realized. Mass spectrum,
m/e Calcd for C.sub.35H.sub.34C.sub.12NO.sub.8.sup.+: 666.17(100%),
668.16(64%), 667.17 (39.8%). Found: 666.46, 668.44, 667.51.
##STR00012##
N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-seminaphthodimethylrhodamine-5--
(and -6)-amide. The title compound was prepared using the above
methodology. Purification was accomplished using preparative scale
HPLC. Mass spectrum, m/e Calcd for
C.sub.37H.sub.38ClN.sub.2O.sub.7.sup.-: 657.24 (100%), 658.24(42%),
659.23(32%). Found: 657.46, 658.47, 659.45.
##STR00013## 6-(3',6'-dipivaloylfluorescein-5-(and-6)-carboxamido)
hexanoic acid {2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide. To a
100 ml round bottom flask containing
6-(3',6'-dipivaloylfluorescein-5-(and-6)-carboxamido) hexanoic acid
succinimidyl ester (0.195 g, 0.26 mmol) was added
2-[2-(6-chlorohexyloxy)-ethoxy]-ethylamine (-0.44 mmol) in 25 ml
Et.sub.2O, followed by 2 ml of pyridine. The reaction mixture was
allowed to stir overnight. After evaporation under reduced
pressure, the residue was subjected to silica gel 60 column
chromatography, progressively using 2% to 5% methanol in
dichloromethane as eluent. The appropriate fractions were collected
and dried under vacuum (0.186 g, 0.216 mmol, and 84% yield). Mass
spectrum, m/e Calcd for C.sub.47H.sub.60ClN.sub.2O.sub.11.sup.+:
863.39(100%), 864.39(54.4%), 865.39(34.6%). Found: 862.94, 864.07,
864.94.
##STR00014## 6-(fluorescein-5-(and-6)-carboxamido) hexanoic acid
{2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide.
6-(3',6'-dipivaloylfluorescein-5-(and-6)-carboxamido) hexanoic acid
{2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide (0.186 g, 0.216 nmol)
was dissolved in 5 ml methanol and 0.5 ml 2M sodium carbonate(aq)
added. The reaction mixture was stirred for 16 hours, then
filtered. Purification was accomplished using preparative scale
HPLC. Separation of structural isomers was realized. Mass spectrum,
m/e Calcd for C.sub.37H.sub.44ClN.sub.2O.sub.9.sup.+: 695.27
(100.0%), 696.28 (42.2%), 697.27 (32.3%). Found:
##STR00015## {2-[2-(4-Chlorobutoxy)-ethoxy]-ethyl}-carbamic acid
anthracen-9-ylmethyl ester. A 50 ml round bottom flask was charged
with [2-(2-Hydroxyethoxy)-ethyl]-carbamic acid anthracen-9-ylmethyl
ester (0.25 g, 0.74 mmol) and fresh sodium hydride, 60% dispersion
in mineral oil (150 mg, 3.75 mmol) under inert atmosphere. 10 ml
anhydrous THF was added and the reaction allowed to stir for 5
minutes. After this point, 1-chloro-4-Iodobutane (180 .mu.l, 1.5
mmol) is added via syringe. The reaction is stirred at room
temperature for 24 hours. Silica gel 60 is co-absorbed onto the
reaction mixture with loss of solvent under reduced pressure.
Silica gel column chromatography takes place initially with heptane
as eluent, followed by 10%, 20%, and 30% ethyl acetate. A total of
0.1 g (32% yield) of product is isolated from appropriate
fractions: .sup.1H NMR (CDCl.sub.3) .delta. 8.50 (s, H-10), 8.40
(d, H-1, 8), 8.03 (d, H-4, 5), 7.53 (dt, H-2, 3, 6, 7), 6.15 (s,
CH2-anth), 5.19 (m, exchangeable, NH), 3.93-3.32 (m, 12H) 1.69-1.25
(m, 4H). Mass spectrum, m/e Calcd for
C.sub.24H.sub.28ClNO.sub.4.H.sub.2O: 447.18 (100.0%), 448.18
(27.1%). Found: 447.17, 448.41.
##STR00016##
2-(2-{2-[2-(2-Chloroethoxy)-ethoxy]-ethoxy}-ethyl)-isoindole-1,3-dione.
2-(2-{2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethoxy}-ethyl)-isoindole-1,3-dione
(0.5 g, 1.55 mmol) was prepared by the method of Nielsen, J. and
Janda, K. D. (Methods: A Companion to Methods in Enzymology 6,
361-371 (1994)). To this reagent was added polystyrene-supported
triphenylphosphine about 3 mmol P/g (0.67 g, 2 mmol) and 6 ml
carbon tetrachloride, into a 25 ml round bottom fitted with a
reflux condenser. The reaction set-up was sparged with argon then
heated to reflux for 2 hours. Upon cooling, more
polystyrene-supported triphenylphosphine (0.1 g, 0.3 mmol) was
added and the reaction refluxed for an additional one hour. The
cooled solution was filtered and the resin washed with additional
carbon tetrachloride. Evaporation of solvent yielded 0.4 g (75.5%
yield) of pure title compound: .sup.1H NMR (CDCl.sub.3) .delta.
7.82 (dd, 2H), 7.69 (dd, 2H), 3.88 (t, 2H), 3.71 (q, 4H), 3.63-3.56
(m, 12H). Mass spectrum, m/e Calcd for
C.sub.16H.sub.21ClNO.sub.5.sup.+: 342.11 (100.0%), 344.11 (32.0%).
Found: 341.65, 343.64.
##STR00017##
2-[2-(2-{2-[2-(2-Chloroethoxy)-ethoxy]ethoxy}-ethoxy)-ethyl]-isoindole-1,-
3-dione. The title compound was prepared according to the previous
example in 89% yield: .sup.1H NMR (CDCl.sub.3) .delta. 7.77 (dd,
2H), .delta. 7.64 (dd, 2H), 3.83 (t, 2H), 3.67 (m, 4H), 3.60-3.52
(m, 14H). Mass spectrum, m/e Calcd for
C.sub.18H.sub.25ClNO.sub.6.sup.+: 386.14 (100.0%), 388.13 (32.0%).
Found: 385.88, 387.83.
##STR00018##
2-{2-[2-(2-{2-[2-(2-Chloroethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethyl}--
isoindole-1,3-dione. The title compound was prepared according to
the synthesis of
2-(2-{2-[2-(2-Chloro-ethoxy)-ethoxy]-ethoxy}-ethyl)-isoindole-1,3-dione
in 92% yield: .sup.1H NMR (CDCl.sub.3) .delta. 7.84 (dd, 2H), 7.71
(dd, 2H), 3.90 (t, 2H), 3.74 (q, 4H), 3.67-3.58 (m, 18H). Mass
spectrum, m/e Calcd for C.sub.20H.sub.29ClNO.sub.7.sup.+: 430.16
(100.0%). Found: 429.85.
##STR00019## VI. Exemplary Methods of Use
The invention provides methods to monitor the expression, location
and/or trafficking of molecules in a cell, as well as to monitor
changes in microenvironments within a cell. In one embodiment, a
mutant hydrolase and a corresponding substrate which includes a
functional group are employed to label a cell, e.g., a cell in an
organism or cell culture, or a cellular component. For instance,
cells are contacted with a vector encoding the mutant hydrolase,
such as one encoding a fusion between the mutant hydrolase and a
nuclear localization signal. The expression of the vector in the
cell may be transient or stable. Then the cell is contacted with a
substrate of the invention recognized by the mutant hydrolase.
Alternatively, cells are concurrently contacted with the vector and
the substrate. Then the presence or location of the functional
group of the substrate in the cell, a lysate thereof, or a
subcellular fraction thereof, is detected or determined.
The substrates of the invention are preferably soluble in an
aqueous or mostly aqueous solution, including water and aqueous
solutions having a pH greater than or equal to about 6. Stock
solutions of substrates of the invention, however, may be dissolved
in organic solvent before diluting into aqueous solution or buffer.
Preferred organic solvents are aprotic polar solvents such as DMSO,
DMF, N-methylpyrrolidone, acetone, acetonitrile, dioxane,
tetrahydrofuran and other nonhydroxylic, completely water-miscible
solvents. In general, the amount of substrate of the invention
employed is the minimum amount required to detect the presence of
the functional group in the sample comprising a mutant hydrolase or
a fusion thereof, within a reasonable time, with minimal background
or undesirable labeling. The exact concentration of a substrate of
the invention and a corresponding mutant hydrolase to be used is
dependent upon the experimental conditions and the desired results.
The concentration of a substrate of the invention typically ranges
from nanomolar to micromolar. The required concentration for the
substrate of the invention with a corresponding mutant hydrolase is
determined by systematic variation in substrate until satisfactory
labeling is accomplished. The starting ranges are readily
determined from methods known in the art.
In one embodiment, a substrate which includes a functional group
with optical properties is employed with a mutant hydrolase to
label a sample. Such a substrate is combined with the sample of
interest comprising the mutant hydrolase for a period of time
sufficient for the mutant hydrolase to bind the substrate, after
which the sample is illuminated at a wavelength selected to elicit
the optical response of the functional group. Optionally, the
sample is washed to remove residual, excess or unbound substrate.
In one embodiment, the labeling is used to determine a specified
characteristic of the sample by further comparing the optical
response with a standard or expected response. For example, the
mutant hydrolase bound substrate is used to monitor specific
components of the sample with respect to their spatial and temporal
distribution in the sample. Alternatively, the mutant hydrolase
bound substrate is employed to determine or detect the presence or
quantity of a certain molecule. In another embodiment, the mutant
hydrolase bound substrate is used to analyze the sample for the
presence of a molecule that responds specifically to the functional
group.
A detectable optical response means a change in, or occurrence of,
a parameter in a test system that is capable of being perceived,
either by direct observation or instrumentally. Such detectable
responses include the change in, or appearance of, color,
fluorescence, reflectance, chemiluminescence, light polarization,
light scattering, or x-ray scattering. Typically the detectable
response is a change in fluorescence, such as a change in the
intensity, excitation or emission wavelength distribution of
fluorescence, fluorescence lifetime, fluorescence polarization, or
a combination thereof. The detectable optical response may occur
throughout the sample comprising a mutant hydrolase or a fusion
thereof or in a localized portion of the sample comprising a mutant
hydrolase or a fusion thereof. Comparison of the degree of optical
response with a standard or expected response can be used to
determine whether and to what degree the sample comprising a mutant
hydrolase or a fusion thereof possesses a given characteristic.
In another embodiment, the functional group is a ligand for an
acceptor molecule. Typically, where the substrate comprises a
functional group that is a member of a specific binding pair (a
ligand), the complementary member (the acceptor) is immobilized on
a solid or semi-solid surface, such as a polymer, polymeric
membrane or polymeric particle (such as a polymeric bead).
Representative specific binding pairs include biotin and avidin (or
streptavidin or anti-biotin), IgG and protein A or protein G, drug
and drug receptor, toxin and toxin receptor, carbohydrate and
lectin or carbohydrate receptor, peptide and peptide receptor,
protein and protein receptor, enzyme substrate and enzyme, sense
DNA or RNA and antisense (complementary) DNA or RNA, hormone and
hormone receptor, and ion and chelator. Ligands for which naturally
occurring receptors exist include natural and synthetic proteins,
including avidin and streptavidin, antibodies, enzymes, and
hormones; nucleotides and natural or synthetic oligonucleotides,
including primers for RNA and single- and double-stranded DNA;
lipids; polysaccharides and carbohydrates; and a variety of drugs,
including therapeutic drugs and drugs of abuse and pesticides.
Where the functional group is a chelator of calcium, sodium,
magnesium, potassium, or another biologically important metal ion,
the substrate comprising such a functional group functions as an
indicator of the ion. Alternatively, such a substrate may act as a
pH indicator. Preferably, the detectable optical response of the
ion indicator is a change in fluorescence.
The sample comprising a mutant hydrolase or a fusion thereof is
typically labeled by passive means, i.e., by incubation with the
substrate. However, any method of introducing the substrate into
the sample comprising a mutant hydrolase or a fusion thereof, such
as microinjection of a substrate into a cell or organelle, can be
used to introduce the substrate into the sample comprising a mutant
hydrolase or a fusion thereof. The substrates of the present
invention are generally non-toxic to living cells and other
biological components, within the concentrations of use.
The sample comprising a mutant hydrolase or a fusion thereof can be
observed immediately after contact with a substrate of the
invention. The sample comprising a mutant hydrolase or a fusion
thereof is optionally combined with other solutions in the course
of labeling, including wash solutions, permeabilization and/or
fixation solutions, and other solutions containing additional
detection reagents. Washing following contact with the substrate
generally improves the detection of the optical response due to the
decrease in non-specific background after washing. Satisfactory
visualization is possible without washing by using lower labeling
concentrations. A number of fixatives and fixation conditions are
known in the art, including formaldehyde, paraformaldehyde,
formalin, glutaraldehyde, cold methanol and 3:1 methanol:acetic
acid. Fixation is typically used to preserve cellular morphology
and to reduce biohazards when working with pathogenic samples.
Selected embodiments of the substrates are well retained in cells.
Fixation is optionally followed or accompanied by permeabilization,
such as with acetone, ethanol, DMSO or various detergents, to allow
bulky substrates of the invention, to cross cell membranes,
according to methods generally known in the art. Optionally, the
use of a substrate may be combined with the use of an additional
detection reagent that produces a detectable response due to the
presence of a specific cell component, intracellular substance, or
cellular condition, in a sample comprising a mutant hydrolase or a
fusion thereof. Where the additional detection reagent has spectral
properties that differ from those of the substrate, multi-color
applications are possible.
At any time after or during contact with the substrate comprising a
functional group with optical properties, the sample comprising a
mutant hydrolase or a fusion thereof is illuminated with a
wavelength of light that results in a detectable optical response,
and observed with a means for detecting the optical response. While
some substrates are detectable calorimetrically, using ambient
light, other substrates are detected by the fluorescence properties
of the parent fluorophore. Upon illumination, such as by an
ultraviolet or visible wavelength emission lamp, an arc lamp, a
laser, or even sunlight or ordinary room light, the substrates,
including substrates bound to the complementary specific binding
pair member, display intense visible absorption as well as
fluorescence emission. Selected equipment that is useful for
illuminating the substrates of the invention includes, but is not
limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon
lamps, argon lasers, laser diodes, and YAG lasers. These
illumination sources are optionally integrated into laser scanners,
fluorescence microplate readers, standard or mini fluorometers, or
chromatographic detectors. This colorimetric absorbance or
fluorescence emission is optionally detected by visual inspection,
or by use of any of the following devices: CCD cameras, video
cameras, photographic film, laser scanning devices, fluorometers,
photodiodes, quantum counters, epifluorescence microscopes,
scanning microscopes, flow cytometers, fluorescence microplate
readers, or by means for amplifying the signal such as
photomultiplier tubes. Where the sample comprising a mutant
hydrolase or a fusion thereof is examined using a flow cytometer, a
fluorescence microscope or a fluorometer, the instrument is
optionally used to distinguish and discriminate between the
substrate comprising a functional group which is a fluorophore and
a second fluorophore with detectably different optical properties,
typically by distinguishing the fluorescence response of the
substrate from that of the second fluorophore. Where the sample
comprising a mutant hydrolase or a fusion thereof is examined using
a flow cytometer, examination of the sample comprising a mutant
hydrolase or a fusion thereof optionally includes isolation of
particles within the sample comprising a mutant hydrolase or a
fusion thereof based on the fluorescence response of the substrate
by using a sorting device.
In one embodiment, intracellular movements may be monitored using a
fusion of the mutant hydrolase of the invention. For example,
beta-arrestin is a regulator of G-protein coupled receptors, that
moves from the cytoplasm to the cell membrane when it is activated.
A cell containing a fusion of a mutant hydrolase and beta-arrestin
and a substrate of the invention allows the detection of the
movement of beta-arrestin from the cytoplasm to the cell membrane
as it associates with activated G-protein coupled receptors.
In another embodiment, FRET may be employed with a fusion of the
mutant hydrolase and a fluorescent protein, e.g., GFP, or a fusion
with a protein that binds fluorescent molecules, e.g.,
O-alkylguanine-DNA alkyltransferase (AGT) (Keppler et al., 2003).
Alternatively, a fusion of a mutant hydrolase and a protein of
interest and a second fusion of a fluorescent protein and a
molecule suspected of interacting with the protein of interest may
be employed to study the interaction of the protein of interest
with the molecule, e.g., using FRET. One cell may contain the
fusion of a mutant hydrolase and a protein of interest while
another cell may contain the second fusion of a fluorescent protein
and a molecule suspected of interacting with the protein of
interest. A population with those two cells may be contacted with a
substrate and an agent, e.g., a drug, after which the cells are
monitored to detect the effect of agent administration on the two
populations.
In yet another embodiment, the mutant hydrolase is fused to a
fluorescent protein. The fusion protein can thus be detected in
cells by detecting the fluorescent protein or by contacting the
cells with a substrate of the invention and detecting the
functional group in the substrate. The detection of the fluorescent
protein may be conducted before the detection of the functional
group. Alternatively, the detection of the functional group may be
conducted before the detection of the fluorescent protein.
Moreover, those cells can be contacted with additional substrates,
e.g., those having a different functional group, and the different
functional group in the cell detected, which functional group is
covalently linked to mutant hydrolase not previously bound by the
first substrate.
In yet another embodiment, a fusion of a mutant hydrolase and a
transcription factor may be employed to monitor activation of
transcription activation pathways. For example, a fusion of a
mutant hydrolase to a transcription factor present in the cytoplasm
in an inactive form but which is translocated to the nucleus upon
activation (e.g., NF kappa Beta) can monitor transcription
activation pathways.
In another embodiment, biotin is employed as a functional group in
a substrate and the fusion includes a mutant hydrolase fused to a
protein of interest suspected of interacting with another molecule,
e.g., a protein, in a cell. The use of such reagents permits the
capture of the other molecule which interacts in the cell with the
protein fused to the mutant hydrolase, thereby identifying and/or
capturing (isolating) the interacting molecule(s).
In one embodiment, the mutant hydrolase is fused to a protein that
is secreted. Using that fusion and a substrate of the invention,
the secreted protein may be detected and/or monitored. Similarly,
when the mutant hydrolase is fused to a membrane protein that is
transported between different vesicular compartments, in the
presence of the substrate, protein processing within these
compartments can be detected. In yet another embodiment, when the
mutant hydrolase is fused to an ion channel or transport protein,
or a protein that is closely associated with the channel or
transport protein, the movement of ions across cell or organeie
membranes can be monitored in the presence of a substrate of the
invention which contains an ion sensitive fluorophore. Likewise,
when the mutant hydrolase is fused to proteins associated with
vesicals or cytoskeleton, in the presense of the substrate,
transport of proteins or vesicals along cytoskeletal structures can
be readily detected.
In another embodiment, the functional group is a drug or toxin. By
combining a substrate with such a functional group with a fusion of
a mutant hydrolase and a targeting molecule such as an antibody,
e.g., one which binds to an antigen associated with specific tumor
cells, a drug or toxin can be targeted within a cell or within an
animal. Alternatively, the functional group may be a fluorophore
which, when present in a substrate and combined with a fusion of a
mutant hydrolase and a targeting molecule such as a single chain
antibody, the targeting molecule is labeled, e.g., a labeled
antibody for in vitro applications such as an ELISA.
In yet another embodiment, when fused to a protein expressed on the
cell surface, a mutant hydrolase on the cell surface, when combined
with a substrate of the invention, e.g., one which contains a
fluorophore, may be employed to monitor cell migration (e.g.,
cancer cell migration) in vivo or in vitro. In one embodiment, the
substrate of the invention is one that has low or no permeability
to the cell membrane. Alternatively, such a system can be used to
monitor the effect of different agents, e.g., drugs, on different
pools of cells. In yet another embodiment, the mutant hydrolase is
fused to a HERG channel. Cells expressing such a fusion, in the
presence of a substrate of the invention which includes a
K+-sensitive fluorophore, may be employed to monitor the activity
of the HERG channel, e.g., to monitor drug-toxicity.
In another embodiment, the substrate of the invention includes a
functional group useful to monitor for hydrophobic regions, e.g.,
Nile Red, in a cell or organism.
Thus, the mutant hydrolases and substrates of the invention are
useful in a wide variety of assays, e.g., phage display, panning,
ELISA, Western blot, fluorometric microvolume assay technology
(FMAT), and cell and subcellular staining.
The invention will be further described by the following
non-limiting examples.
EXAMPLE I
General Methodologies
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the field of molecular biology and cellular
signaling and modeling. Generally, the nomenclature used herein and
the laboratory procedures in spectroscopy, drug discovery, cell
culture, molecular genetics, plastic manufacture, polymer
chemistry, diagnostics, amino acid and nucleic acid chemistry, and
alkane chemistry described below are those well known and commonly
employed in the art. Standard techniques are typically used for
preparation of plastics, signal detection, recombinant nucleic acid
methods, polynucleotide synthesis, and microbial culture and
transformation (e.g., electroporation, lipofection).
The techniques and procedures are generally performed according to
conventional methods in the art and various general references (see
generally, Sambrook et. al. Molecular Cloning: A laboratory manual,
2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., and Lakowicz, J. R. Principles of Fluorescence
Spectroscopy, New York: Plenum Press (1983) for fluorescent
techniques, which are incorporated herein by reference) and which
are provided throughout this document. Standard techniques are used
for chemical synthesis, chemical analysis, and biological
assays.
Materials
All oligonucleotides were synthesized, purified and sequenced by
Promega Corporation (Madison, Wis.) or the University of Iowa DNA
Facility (Iowa City, Iowa). Restriction enzymes and DNA modifying
enzymes were obtained from Promega Corporation (Madison, Wis.), New
England Biolabs, Inc. (Beverly, Mass.) or Stratagene Cloning
Systems (La Jolla, Calif.), and were used according to the
manufacturer's protocols. Competent E. coil JM109 were provided by
Promega Corporation or purchased from Stratagene Cloning Systems.
Small-scale plasmid DNA isolations were done using the Qiagen
Plasmid Mini Kit (Qiagen Inc., Chatsworth, Calif.). DNA ligations
were performed with pre-tested reagent kits purchased from
Stratagene Cloning Systems. DNA fragments were purified with
QIAquick Gel Extraction Kits or QIAquick PCR purification Kits
purchased from Qiagen Inc.
The vectors used for generating DhaA mutants and their fusions were
as follows: pET21 (Invitrogen, Carlsbad, Calif.), pRL-null
(Promega, Madison, Wis.), pGEX-5x-3 (Amersham Biosciences;
Piscataway, N.J.), and EGFP and DsRED2 (both from CLONTECH, Palo
Alto, Calif.).
SDS-polyacrylamide gels and associated buffers and stains, as well
as electroblot transfer buffers, were obtained from Bio Whittaker
Molecular Applications (Rockland, Me.). Protein molecular weight
standards were purchased from Invitrogen.
Sigma-Aldrich was the source of Anti Flag.sup.R monoclonal antibody
antibodies (anti FLAG.sup.R M2 monoclonal antibody (mouse)
(F3165)), Anti FLAG.sup.R M2 HRP Conjugate and Anti FLAG.sup.R M2
FITC conjugate (A8592 and F4049, respectively). Chemicon (Temecula,
Calif.) was the source of monoclonal anti-Renilla luciferase
antibody (MAB4410). Promega Corp. was the source of HRP-conjugated
goat anti-mouse IgG and HRP-conjugated streptavidin (W4021 and
G714, respectively).
1-Cl-butane, 1-Cl-hexane, 1-Cl-octane, 1-Cl-decane, 1-Cl-butanol,
1-Cl-hexanol, 1-Cl-octanol, and 1-Cl-decanol were obtained from
Aldrich or from Fluka (USA). All salts, monobasic potassium
phosphate, dibasic potassium phosphate, imidazole, HEPES, sodium
EDTA, ammonium sulfate, and Tris free base were from Fisher
(Biotech Grade).
Glutathione Sepharose 4 FF, glutathione, MonoQ and Sephadex G-25
prepackaged columns were from Amersham Biosciences.
Luria-Broth ("LB") was provided by Promega Corporation.
Methods
PCR reactions. DNA amplification was performed using standard
polymerase chain reaction buffers supplied by Promega Corp.
Typically, 50 .mu.l reactions included 1.times. concentration of
the manufacturer's supplied buffer, 1.5 mM MgCl.sub.2, 125 .mu.M
dATP, 125 .mu.M dCTP, 125 .mu.M dGTP, 125 .mu.M dTTP, 0.10-1.0
.mu.M forward and reverse primers, 5 U AmpliTaq.RTM. DNA Polymerase
and <1 ng target DNA. Unless otherwise indicated, the thermal
profile for amplification of DNA was 35 cycles of 0.5 minutes at
94.degree. C.; 1 minute at 55.degree. C.; and 1 minute at
72.degree. C.
DNA sequencing. All clones were confirmed by DNA sequencing using
the dideoxy-terminal cycle-sequencing method (Sanger et al., 1977)
and a Perkin-Elmer Model 310 DNA sequencer. (Foster City,
Calif.).
SDS-PAGE. Proteins were solubilized in a sample buffer (1% SDS, 10%
glycerol, and 1.0 mM .beta.-mercaptoethanol, pH 6.8; Promega
Corporation), boiled for 5 minutes and resolved on SDS-PAGE (4-20%
gradient gels; Bio Whittaker Molecular Applications). Gels were
stained with Coomassie Blue (Promega Corp.) for Western blot
analysis or were analyzed on a fluoroimager (Hitachi, Japan) at an
E.sub.ex/E.sub.em appropriate for each fluorophore evaluated.
Western blot analysis. Electrophoretic transfer of proteins to a
nitrocellulose membrane (0.2 .mu.m, Scheicher & Schnell,
Germany) was carried out in 25 mM Tris base/188 mM glycine (pH
8.3), 20% (v/v) methanol for 2.0 hours with a constant current of
80 mA (at 4.degree. C.) in Xcell II Blot module (Invitrogen). The
membranes were rinsed with TBST buffer (10 mM Tris-HCl, 150 mM
NaCl, pH 7.6, containing 0.05% Tween 20) and incubated in blocking
solution (3% dry milk or 1% BSA in TBST buffer) for 30 minutes at
room temperature or overnight at 4.degree. C. Then membranes were
washed with 50 ml of TBST buffer and incubated with anti-FLAG.sup.R
monoclonal antibody M2 (dilution 1:5,000), anti-Renilla luciferase
monoclonal antibody (dilution 1:5,000), or HRP-conjugated
streptavidin (dilution 1:10,000) for 45 minutes at room
temperature. Then the membranes were washed with TBST buffer (50
ml, 5 minutes, 3 times). The membranes that had been probed with
antibody were then incubated with HRP-conjugated donkey anti-mouse
IgG (30 minutes, room temperature) and then the washing procedure
was repeated. The proteins were visualized by the enhanced
chemiluminescence (ECL) system (Pharmacia-Amersham) according to
the manufacturer's instructions. Levels of proteins were quantified
using computer-assisted densitometry.
Protein concentration. Protein was measured by the microliter
protocol of the Pierce BCA Protein assay (Pierce, Rockford, Ill.)
using bovine scrum albumin (BSA) as a standard.
Statistic analysis. Data were expressed as mean +/-S.E.M. values
from experiments performed in quadruplicate, representative of at
least 3 independent experiments with similar results. Statistical
significance was assessed by the student's t test and considered
significant when p<0.05.
Bacterial cells. The initial stock of Dh5.alpha. cells containing
pET-3a with Rhodococcus rodochorus (DhaA) was kindly provided by
Dr. Clifford J. Unkefer (Los Alamos National Laboratory, Los
Alamos, N.Mex.) (Schindler et al., 1999; Newman et al., 1999).
Bacteria were cultured in LB using a premixed reagent provided by
Promega Corp. Freezer stocks of E. coli BL21 (.lamda.DE3) pET3a
(stored in 10% glycerol, -80.degree. C.) were used to inoculate
Luria-Bertani agar plates supplemented with ampicillin (50
.mu.g/ml) (Sambrook et al., 1989). Single colonies were selected
and used to inoculate two 10 ml cultures of Luria-Bertani medium
containing 50 .mu.g/ml ampicillin. The cells were cultured for 8
hours at 37.degree. C. with shaking (220 rpm), after which time 2
ml was used to inoculate each of two 50 ml of Luria-Bertani medium
containing 50 .mu.g/ml ampicillin, which were grown overnight at
37.degree. C. with shaking. Ten milliliters of this culture was
used to inoculate each of two 0.5 L Luria-Bertani medium with
ampicillin. When the A.sub.600 of the culture reached 0.6,
isopropyl-1-thio-.beta.-D-galactopyranoside (IPTG) was added to a
final concentration of 0.5 mM, and cultures were maintained for an
additional 4 hours at 30.degree. C. with shaking. The cells were
then harvested by centrifugation and washed with 10 mM
Tris-SO.sub.4, 1 mM EDTA, pH 7.5. The cell pellets were stored at
-70.degree. C. prior to cell lysis.
Mammalian cells. CHO-K1 cells (ATCC-CCL61) were cultured in a 1:1
mixture of Ham's F12 nutrients and Dulbecco's modified minimal
essential medium supplemented with 10% fetal bovine serum (FBS),
100 U/ml penicillin, and 100 mg/ml streptomycin, in an atmosphere
of 95% air and 5% CO.sub.2 at 37.degree. C.
Rat hippocampal (E18) primary neurons were isolated as described
below. Briefly, fragments of embryonic (E 18) rat hippocampus in
Hiberate.TM. E media (GIBCO, Invitrogen, Carlsbad, Calif.),
obtained from Dr. Brewer (Southern Illinois University), were
dissociated and plated on poly-D-lysin coated (0.28 mg/cm.sup.2;
Sigma) glass/plastic-ware and cultured in serum-free Neurobasal.TM.
media with B27 supplement (NB27, GIBCO). All media were changed
every 2-3 days.
Transfection. To study transient expression of different proteins,
cells were plated in 35 mm culture dishes or 24 well plates. At
about 80-90% confluency, the cells were exposed to a mixture of
lipofectamine/DNA/antibiotic free media according to the
manufacturer's (GIBCO) instructions. The following day, media was
replaced with fresh media and cells were allowed to grow for
various periods of time.
Fluorescence. Fluorescence in cells in 96 well plates was measured
on fluorescent plate reader CytoFluorII (Beckman) at an
E.sub.ex/E.sub.em appropriate for particular fluorophores (e.g.,
E.sub.ex/E.sub.em for TAMRA is 540/575 nm).
EXAMPLE II
A DhaA-Based Tethering System
A. Wild-Type and Mutant DhaA Proteins and Fusions Thereof
A halo-alkane dehydrogenase from Rhodococcus rhodochrous is a
product of the DhaA gene (MW about 33 kDa). This enzyme cleaves
carbon-halogen bonds in aliphatic and aromatic halogenated
compounds, e.g., HaloC.sub.3-HaloC.sub.10. The catalytic center of
DhaA is a typical "catalytic triad", comprising a nucleophile, an
acid and a histidine residue. It is likely that substrate binds to
DhaA to form an ES complex, after which nucleophilic attack by
Asp106 forms an ester intermediate, His272 then activates H.sub.2O
that hydrolyzes the intermediate, releasing product from the
catalytic center. To determine whether a point mutation of the
catalytic His272 residue impairs enzymatic activity of the enzyme
so as to enable covalent tethering of a functional group (FG) to
this protein, mutant DhaAs were prepared.
Materials and Methods
To prepare mutant DhaA vectors, Promega's in vitro mutagenesis kit
which is based on four primer overlap-extension method was employed
(Ho et al., 1989) to produce DhaA.H272 to F, A, G, or H mutations.
The external primers were oligonucleotides
5'-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3' (SEQ ID NO:1) and
5'-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3' (SEQ ID NO:2), and the
internal mutagenic primers were as follows: H272F
(5'-CCGGGATTGTTCTACCTCCAGGAAGAC-3'), SEQ ID NO:3), H272A
(5'-CCGGGATTGGCCTACCTCCAGGAAGAC-3'; SEQ ID NO:4), H272G
(5'-CCGGGATTGCAGTACCTCCAGGAAGAC-3'; SEQ ID NO:5), and H272Q
(5'-CCGGGATTGGGCTACCTCCAGGAAGAC-3'; SEQ ID NO:6) (the mutated
codons are underlined). The mutated dehalogenase genes were
subcloned into the pET-3a vector. For overexpression of mutant
dehalogenases, the pET-3a vector was transformed into competent E.
coli BL21 (DE3). The DhaA sequence in clones was confirmed by DNA
sequencing.
GST-DhaA (WT or H272F/A/G/H mutants) fusion cassettes were
constructed by cloning the appropriate DhaA coding regions into
SalI/NotI sites of pGEX5.times.3 vector. Two primers
(5'-ACGCGTCGACGCCGCCATGTCAGAAATCGGTACAGGC-3' and
5'-ATAAGAATGCGGCCGCTCAAGCGCTTCAACCGGTGAGTGCGGGGAGCCA GCGCGC-3'; SEQ
ID NOs:7 and 8, respectively) were designed to add a SalI site and
a Kozak consensus sequence to the 5' coding regions of DhaA, to add
a NotI, EcoR47III, and AgeI restriction site and stop codons to the
3' coding region of DhaA, and to amplify a 897 bp fragment from a
DhaA (WT or mutant) template. The resulting fragments were inserted
into the SalI/NotI site of pGEX-5.times.-3, a vector containing a
glutathione S-transferase (GST) gene, a sequence encoding a Factor
Xa cleavage site, and multiple cloning sites (MCS) followed by a
stop codon.
A Flag coding sequence was then inserted into the AgeI/EcoR47III
restriction sites of the pGEX5.times.-3 vector. In frame with the
six nucleotide AgeI site is a sequence for an 11 amino acid
peptide, the final octapeptide of which corresponds to the Flag
peptide (Kodak Imaging Systems, Rochester, N.Y.). Two complementary
oligonucleotides (5'-CCGGTGACTACAAGGACGATGACGACAAGTGAAGC-3', sense,
SEQ ID NO:9, and 5'-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3', antisense,
SEQ ID NO:10) coding the Flag peptide (Kodak Imaging Systems,
Rochester, N.Y.) were annealed. The annealed DNA had an AgeI site
at the 5' end and an EcoR47III at the 3' end. The annealed DNA was
digested with AgeI and EcoR47III and then subcloned into the
GST-DhaA.WT or GST-DhaA.H272F mutant constructs at the AgeI and
EcoR47III sites. All gene fusion constructs were confirmed by DNA
sequencing.
To generate GST-DhaA fusion proteins, enzyme expression was induced
by the addition of isopropyl-b-D-thiogalactopyranoside (at a final
concentration of 0.5 mM) when the culture reached an optical
density of 0.6 at 600 nm. The cells were harvested in Buffer A (10
mM Tris-SO.sub.4. 1 mM EDTA, 1 mM .beta.-mercaptoethanol, and 10%
glycerol, pH 7.5), and disrupted by sonication using a Vibra
Cell.TM. sonicator (Sones & Materials, Danbury, Conn., USA).
Cell debris was removed by centrifugation at 19,800.times.g for 1
hour. The crude extract was further purified on a GSS-Sepharose 4
fast flow column (Amersham Biosciences; Piscataway, NJ.) according
to the manufacturer's instructions. The elution fractions
containing GST-DhaA fusion protein were pooled, dialyzed against a
10 mM Tris-SO.sub.4 buffer (containing 20 mM Na.sub.2SO.sub.4 and 1
mM EDTA-Na.sub.2) overnight at 4.degree. C., and stored at
-20.degree. C. until use. To generate DhaA (WT or mutant), GST was
cleaved from the fusion proteins with Factor Xa, and the products
purified on GSS-Sepharose 4 (Amersham Biosciences; Piscataway,
N.J.) according to the manufacturer's instructions. Homogeneity of
the proteins was verified by SDS-PAGE. In some experiments, the
cell free extract was fractionated using 45-70% saturated ammonium
sulfate as described by Newman et al. (1999).
Results
FIG. 3 shows robust, IPTG inducible production of GSTDhaA.WT-Flag
(lane 1) and GST-DhaA.H272F-Flag (lane 2) fusion proteins.
Moreover, the proteins were soluble and could be efficiently
purified on GSS-Sepharose 4FF (lanes 5-10, odd numbered lanes
correspond to GST-DhaA.WT-Flag and even numbered lanes correspond
to GSTDhaA.H272F-Flag). Treatment of the fusion proteins with
Factor Xa led to the formation of two proteins GST and DhaA (WT or
mutant, lanes 11 and 12, respectively), and GST was efficiently
removed on GSS-Sepharose 4FF (WT or mutant, lanes 13 and 14,
respectively). In addition, all proteins had the predicted
molecular weight.
B. Mutation of H272 Impairs Ability of DhaA to Hydrolyze
Cl-Alkanes.
Inability of an enzyme to release product of the enzymatic reaction
into surrounding media is essential for the tethering system. This
inability can be detected by significant reduction of the
hydrolytic activity of the enzyme.
To study the effect of a point mutation on the activity of DhaA (WT
or mutant) hydrolysis of Cl-alkanes, a pH-indicator dye system as
described by Holloway et al. (1998) was employed.
Materials and Methods
The reaction buffer for a pH-indicator dye system consisted of 1 mM
HEPES-SO.sub.4 (pH 8.2), 20 mM Na.sub.2SO.sub.4, and 1 mM EDTA.
Phenol red was added to a final concentration 25 .mu.g/ml. The
halogenated compounds were added to apparent concentrations that
could insure that the dissolved fraction of the substrate was
sufficient for the maximum velocity of the dehalogenation reaction.
The substrate-buffer solution was vigorously mixed for 30 seconds
by vortexing, capped to prevent significant evaporation of the
substrate and used within 1-2 hours. Prior to each kinetic
determination, the phenol red was titrated with a standardized
solution of HCl to provide an apparent extinction coefficient. The
steady-state kinetic constants for DhaA were determined at 558 nm
at room temperature on a Beckman Du640 spectrophotometer (Beckman
Coulter, Fullerton, Calif.). Kinetic constants were calculated from
initial rates using the computer program SigmaPlot. One unit of
enzyme activity is defined as the amount required to dehalogenate
1.0 mM of substrate/minute under the specific conditions.
Results
As shown in FIG. 4, using 0.1 mg/ml of enzyme and 10 mM substrate
at pH 7.0-8.2, no catalytic activity was found with any of four
mutants. Under these conditions, the wild-type enzyme had an
activity with 1-Cl-butane of 5 units/mg of protein. Thus, the
activity of the mutants was reduced by at least 700-fold.
Aliquots of the supernatant obtained from E. coli expressing DhaA
(WT or one of the mutants) were treated with increasing
concentrations of (NH.sub.4).sub.2SO.sub.4. The proteins were
exposed to each (NH.sub.4).sub.2SO.sub.4 concentration for 2 hours
(4.degree. C.), pelleted by centrifugation, dialyzed overnight
against buffer A, and resolved on SDS-PAGE.
As shown in FIG. 5, a major fraction of DhaA.WT and the DhaA.H272F
mutant was precipitated by 45-70% of (NH.sub.4).sub.2 SO.sub.4. No
precipitation of these proteins was observed at low
(NH.sub.4).sub.2SO.sub.4 concentrations. In contrast, the
DhaA.H272Q, DhaA.H272G and DhaA.H272A mutants could be precipitated
by 10% (NH.sub.4).sub.2SO.sub.4. This is a strong indication of the
significant change of the physico-chemical characteristics of the
DhaA.H272Q, DhaA.H272G and DhaA.H272A mutants. At the same time,
the DhaA.H272F mutation had no significant effect on these
parameters. These data are in good agreement with results of
computer modeling of the effect of mutations on the 3-D structure
of DhaA, indicating that among all tested mutants, only the
DhaA.H272F mutation had no significant effect on the predicted
3-dimensional model (see FIG. 2). Based on these results,
DhaA.H272F was chosen for further experiments.
To form a covalent adduct, the chlorine atom of Cl-alkane is likely
positioned in close proximity to the catalytic amino acids of DhaA
(WT or mutant) (FIG. 2). The crystal structure of DhaA (Newman et
al., 1999) indicates that these amino acids are located deep inside
of the catalytic pocket of DhaA (approximately 10 .ANG. long and
about 20 .ANG..sup.2 in cross section). To permit entry of the
reactive group in a substrate for DhaA which includes a functional
group into the catalytic pocket of DhaA, a linker was designed to
connect the Cl-containing substrate with a functional group so that
the functional group is located outside of the catalytic pocket,
i.e., so as not to disturb/destroy the 3-D structure of DhaA.
To determine if DhaA is capable of hydrolyzing Cl-alkanes with a
long hydrophobic carbon chain, DhaA.WT was contacted with various
Cl-alkane alcohols. As shown in FIG. 6, DhaA.WT can hydrolyze
1-Cl-alkane alcohols with 4-10 carbon atoms. Moreover, the initial
rate of hydrolysis (IRH) of Cl-alkanes had an inverse relationship
to the length of a carbon chain, although poor solubility of
long-chain Cl-alkanes in aqueous buffers may affect the efficiency
of the enzyme-substrate interaction. Indeed, as shown in FIG. 6,
the IRH of 1-Cl-alkane-10-decanol is much higher than the IRH of
1-Cl-decane. More importantly, these data indicate that DhaA can
hydrolyze Cl-alkanes containing relatively polar groups (e.g.,
HO-group).
FAM-modified Cl-alkanes with linkers of different length and/or
hydrophobicity were prepared (FIG. 7). DhaA.WT efficiently
hydrolyzed Cl-alkanes with a relatively bulky functional group
(FAM) if the linker was 12 or more atoms long. No activity of
DhaA.H272F/A/G/Q mutants was detected with any of the tested
Cl-alkanes (data not shown). In addition, modification of the
(CH.sub.2).sub.6 region adjacent to the Cl-atom led to a
significant reduction of the IRH of the 14-atom linker by DhaA.WT.
Nevertheless, if the length and structure of the linker is
compatible with the catalytic site of a hydrolase, the presence of
a linker in a substrate of the invention has substantially no
effect on the reaction.
Some of the samples were analyzed on an automated HPLC
(Hewlett-Packard Model 1050) system. A DAD detector was set to
record UV-visible spectra over the 200-600 nm range. Fluorescence
was detected at an E.sub.ex/E.sub.em equal 480/520 nm and 540/575
nm for FAM- and TAMRA-modified substrates, respectively. Ethanol
extracts of Cl-alkanes or products of Cl-alkane hydrolysis were
analyzed using analytical reverse phase C.sub.18 column
(Adsorbosphere HS, 5.mu., 150.times.4.6 mm; Hewlett-Packard,
Clifton, N.J.) with a linear gradient of 10 mM ammonium acetate (pH
7.0): ACN (acetonitrile) from 25:75 to 1:99 (v/v) applied over 30
minutes at 1.0 ml/minute. Quantitation of the separated compounds
was based on the integrated surface of the collected peaks.
FIG. 8A shows the complete separation of the substrate and the
product of the reaction. FIG. 8B indicates that wild-type DhaA very
efficiently hydrolyzed FAM-C.sub.14H.sub.24O.sub.4--Cl. Similar
results were obtained when TAMRA-C.sub.14H.sub.24O.sub.4--Cl or
ROX.5-C.sub.14H.sub.24O.sub.4--Cl were used as substrates (data not
shown). Taken together these data confirm the results of the
pH-indicator dye-based assay showing complete inactivation of DhaA
by the DhaA.H272F mutation.
C. Covalent Tethering of Functional Groups to DhaA Mutants In Vitro
Materials and Methods
MALDI analysis of proteins was performed at the University of
Wisconsin Biotechnology Center using a matrix assisted laser
desorption/ionization time-of-life (MALDI-TOF) mass spectrometer
Bruker Biflex III (Bruker, USA.). To prepare samples, 100 .mu.g of
purified DhaA (WT or H272F mutant) or GST-DhaA (WT or H272F mutant)
fusion protein (purified to about 90% homogeneity) in 200 .mu.l of
buffer (1 mM HEPES-SO.sub.4 (pH 7.4), 20 mM Na.sub.2SO.sub.4, and 1
mM EDTA) were incubated with or without substrate
(FAM-C.sub.14H.sub.24O.sub.4--Cl, at 1.0 mM, final concentration)
for 15 minutes at room temperature. Then the reaction mixtures were
dialyzed against 20 mM CH.sub.3COONH.sub.4 (pH 7.0) overnight at
4.degree. C. and M/Z values of the proteins and protein-substrate
complexes determined.
Oligonucleotides employed to prepare DhaA.D106 mutants include for
DhaA.D106C: 5'-CTTGGGTTTGGAAGAGGTCGTCCTGGTCATCCACTGCTGGGGC-3' (SEQ
ID NO:13) and 5'-TGAGCCCCAGCAGTGGATGACCAGGACGACCTCTTCCAAACC-3' (SEQ
ID NO:14); for DhaA.D106Q:
5'-CTTGGGTTTGGAAGAGGTCGTCCTGGTCATCCACCAGTGGGGC-3' (SEQ ID NO:34)
and 5'-TGAGCCCCACTGGTGGATGACCAGGACGACCTCTTCCAAACC-3' (SEQ ID
NO:35); for DhaA.D106E:
5'-CTTGGGTTTGGAAGAGGTCGTCCTGGTCATCCACGAATGGGGC-3' (SEQ ID NO:52)
and 5'-TGAGCCCCATTCGTGGATGACCAGGACGACCTCTTCAAACC-3' (SEQ ID NO:53);
and for DhaA.D106Y:
5'-CTTGGGTTTGGAAGAGGTCGTCCTGGTCATCCACTACTGGGGC-3' (SEQ ID NO:54)
and 5'-TGAGCCCCAGTAGTGGATGACCAGGACGACCTCTTCCAAACC-3' (SEQ ID
NO:55). The annealed oligonucleotides contained a StyI site at the
5' end and the BlpI site at the 3' end. The annealed
oligonucleotides were digested with StyI and BlpI and subcloned
into GST-DhaA.WT or GST-DhaA.H272F at StyI and BlpI sites. All
mutants were confirmed by DNA sequencing.
Results
To confirm that DhaA.H272 mutants were capable of binding
Cl-alkanes with functional groups, these mutants or their
GST-fusions, as well as the corresponding wild-type proteins or
fusions, were contacted with FAM-C.sub.14H.sub.24O.sub.4--Cl,
TAMRA-C.sub.14H.sub.24O.sub.4--Cl,
ROX.5-C.sub.14H.sub.24O.sub.4--Cl, or
biotin-C.sub.18H.sub.32O.sub.4--Cl for 15 minutes at room
temperature. Then the proteins were resolved on SDS-PAGE. The gels
containing proteins were incubated with
FAM-C.sub.14H.sub.24O.sub.4--Cl, TAMRA-C.sub.14H.sub.24O.sub.4--Cl,
or ROX.5-C.sub.14H.sub.24O.sub.4--Cl and were analyzed by
fluoroimager (Hitachi, Japan) at an E.sub.m/E.sub.em appropriate
for each fluorophore. Gels containing proteins incubated with
biotin-C.sub.18H.sub.32O.sub.4--Cl were transferred to a
nitrocellulose membrane and probed with HRP conjugated
streptavidin.
As shown in FIG. 9, TAMRA-C.sub.14H.sub.24O.sub.4--Cl (lanes 1 and
2 in panel A). FAM-C.sub.14H.sub.24O.sub.4--Cl (lanes 3 and 4 in
panel A), and ROX.5-C.sub.14H.sub.24O.sub.4--Cl (lanes 5 and 6 in
panel A) bound to DhaA.H272F (lanes 2, 4 and 6 in panel A) but not
to DhaA.WT (lanes 1, 3 and 5 in panel A).
Biotin-C.sub.18H.sub.34O.sub.4--Cl bound to DhaA.H272F (lanes 9-14
in panel B) but not to DhaA. WT (lanes 1-8 in panel B). Moreover,
the binding of biotin-C.sub.18H.sub.34O.sub.4--Cl to DhaA.H272F
(lanes 9-14 in panel B) was dose dependent and could be detected at
0.2 .mu.M. Further, the bond between substrates and DhaA.H272F was
very strong, since boiling with SDS did not break the bond.
All tested DhaA.H272 mutants, i.e. H272F/G/A/Q, bound to
TAMRA-C.sub.14--Cl (FIG. 10). Further, the DhaA.H272 mutants bind
the substrates in a highly specific manner, since pretreatment of
the mutants with one of the substrates
(biotin-C.sub.18H.sub.34O.sub.4--Cl) completely blocked the binding
of another substrate (TAMRA-C.sub.14H.sub.24O.sub.4--Cl) (FIG.
10).
To determine the nature of the bond between Cl-alkanes and the
DhaA.H272F mutant (or the GST-DhaA.H272F mutant fusion protein),
these proteins were incubated with and without
FAM-C.sub.14H.sub.24O.sub.4--Cl, and analyzed by MALDI. As shown in
FIG. 11, the bond between mutant DhaA.H272F and
FAM-C.sub.14H.sub.24O.sub.4--Cl is strong. Moreover, the analysis
of the E*S complex indicated the covalent nature of the bond
between the substrate (e.g., FAM-C.sub.14H.sub.24O.sub.4--Cl) and
DhaA.H272F. The MALDI-TOF analysis also confirms that the
substrate/protein adduct is formed in a 1:1 relationship.
DhaA mutants at another residue in the catalytic triad, residue
106, were prepared. The residue at position 106 in wild-type DhaA
is D, one of the known nucleophilic amino acid residues. D at
residue 106 in DhaA was substituted with nucleophilic amino acid
residues other than D, e.g., C, Y and E, which may form a bond with
a substrate which is more stable than the bond formed between
wild-type DhaA and the substrate. In particular, cysteine is a
known nucleophile in cysteine-based enzymes, and those enzymes are
not known to activate water.
A control mutant, DhaA.D106Q, single mutants DhaA.D106C,
DhaA.D106Y, and DhaA.D106E, as well as double mutants
DhaA.D106C:H272F. DhaA.D106E:H272F, DhaA.D106Q:H272F, and
DhaA.D106Y:H272F were analyzed for binding to
TAMRA-C.sub.14H.sub.24O.sub.4--Cl (FIG. 12). As shown in FIG. 12,
TAMRA-C.sub.14H.sub.24O.sub.4--Cl bound to DhaA.D106C,
DhaA.D106C:H272F, DhaA.D106E, and DhaA.H272F. Thus, the bond formed
between TAMRA-C.sub.14H.sub.24O.sub.4--Cl and cysteine or glutamate
at residue 106 in a mutant DhaA is stable relative to the bond
formed between TAMRA-C.sub.14H.sub.24O.sub.4--Cl and wild-type
DhaA. Other substitutions at position 106 alone or in combination
with substitutions at other residues in DhaA may yield similar
results. Further, certain substitutions at position 106 alone or in
combination with substitutions at other residues in DhaA may result
in a mutant DhaA that forms a bond with only certain
substrates.
EXAMPLE III
Tethering of Luciferase to a Solid Support via a Mutant DhaA and a
Substrate of the Invention
Materials and Methods
phRLuc-linker-DhaA.WT-Flag and phRLuc-linker-DhaA.H272F-Flag fusion
cassettes were constructed by cloning the phRLuc coding region into
the NheI/SalI sites of the pCIneo vector which contains a myristic
acid attachment peptide coding sequence (MAS). Two primers
(5'-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3'; SEQ ID NO:11) and
(5'-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3'; SEQ ID NO:12) were designed
to add NheI and SalI sites to the 5' and 3' coding regions,
respectively, of phRLuc and to amplify a 900 bp fragment from a
phRLuc template (pGL3 vector, Promega). Then, a myristic acid
attachment peptide coding sequence was excised with NheI and SalI
restriction enzymes and the amplified fragment containing phRLuc
was inserted into the NheI/SalI restriction sites of
pCIneo.DhaA.(WT or H272F)-Flag vector. The sequence of each
construct was confirmed by DNA sequencing. Promega's TNT.RTM.
T7Quick system was then used to generate fusion proteins in
vitro.
Results
To demonstrate tethering of proteins to a solid support via
DhaA.H272F-Cl-alkane bridge, vectors encoding a fusion protein of
Renilla luciferase (hRLuc, N-terminus of the fusion), a protein
connector (17 amino acids, see Table I), and DhaA (WT or H272F
mutant) were prepared. The Flag epitope was then fused to the
C-terminus of DhaA.
TABLE-US-00002 TABLE I Peptide Fusion Sequence Connector GST-DhaA
atcgaaggtcgtgggatccccaggaattcccgggtcgacgccgcc iegrgiprnsrvdaa (SEQ
ID NO: 26) (SEQ ID NO: 27) GFP-DhaA
tccggatcaagcttgggcgacgaggtggacggcgggccctctagagccacc sgsslgdevdggp-
srat (SEQ ID NO: 28) (SEQ ID NO: 29) DhaA-Rluc
accggttccggatcaagcttgcggtaccgcgggccctctagagcc tgsgsslryrgpsra (SEQ
ID NO: 30) (SEQ ID NO: 31) Rluc-DhaA
tccggatcaagcttgcggtaccgcgggccctctagagccgtcgacgccgcc sgsslryrgpsr-
avdaa (SEQ ID NO: 32) (SEQ ID NO: 33) DhaA-Flag Accggt Tg
SDS-PAGE followed by Western blot analysis showed that the proteins
had their predicted molecular weights and were recognized by
anti-R.Luc and anti-Flag.sup.R M2 antibodies. In addition, all
fusion proteins had Renilla luciferase activity (as determined by
Promega's Renilla Luciferase Assay System in PBS pH 7.4
buffer).
Tethering of proteins to a solid support via a DhaA.H272F-Cl-alkane
bridge was shown by using biotin-C.sub.18H.sub.32O.sub.4--Cl as a
substrate and streptavidin (SA)-coated 96 well plates (Pierce, USA)
as solid support. Translated proteins were contacted with
biotin-C.sub.18H.sub.32O.sub.4--Cl substrate at 25 .mu.M (final
concentration), for 60 minutes at room temperature. Unbound
biotin-C.sub.18H.sub.32O.sub.4--Cl was removed by gel-filtration on
Sephadex G-25 prepackaged columns (Amersham Biosciences). Collected
fractions of R.Luc-connector-DhaA fusions were placed in SA-coated
96-well plate for 1 hour at room temperature, unbound proteins were
washed out and luciferase activity was measured.
FIG. 13A shows Renilla luciferase activity captured on the plate.
Analysis of these data indicated that only the fusion containing
the mutant DhaA was captured. The efficiency of capturing was very
high (more than 50% of Renilla luciferase activity added to the
plate was captured). In contrast, the efficiency of capturing of
fusions containing wild-type DhaA as well as Renilla luciferase was
negligibly small (<0.1%). Pretreatment of
R.Luc-connector-DhaA.H272F with a non-biotinylated substrate
(TAMRA-C.sub.14H.sub.24O.sub.4--Cl) decreased the efficiency of
capturing by about 80%. Further, there was no effect of
pretreatment with a nonbiotinylated substrate on the capturing of
the R.Luc-connector-DhaA.WT or Renilla luciferase.
Taken together, these data demonstrate that active enzymes (e.g.,
Renilla luciferase) can be tethered to a solid support that forms
part of a substrate of the invention (Cl-alkane-DhaA.H272F-bridge),
and retain enzymatic activity.
EXAMPLE IV
Mutant DhaA and Substrate System In Vivo
A. Covalent Tethering of Functional Groups to DhaA Mutants In Vivo:
in Prokaryotes and Eukaryotes
Materials and Methods
To study the binding of a substrate of the invention to a mutant
hydrolase expressed in prokaryotes, E. coli cells BL21 (.lamda.DE3)
pLys65 were transformed with pGEX-5.times.-3. DhaA.WT-Flag or
pGEX-5.times.-3. DhaA.H272F-Flag, grown in liquid culture, and
induced with IPTG. Either TAMRA-C.sub.14H.sub.24O.sub.4--Cl or
biotin-C.sub.18H.sub.32O.sub.4--Cl was added to the induced cells
(final concentration, 25 .mu.M). After 1 hour, cells were
harvested, washed with cold PBS (pH 7.3), disrupted by sonication,
and fractionated by centrifugation at 19,800.times.g for 1 hour.
Soluble fractions were subjected to SDS-PAGE. Gels with proteins
isolated from cells treated with TAMRA-C.sub.14H.sub.24O.sub.4--Cl
were analyzed on a fluoroimager, while proteins from cells treated
with biotin-C.sub.18H.sub.32O.sub.4--Cl were transferred to a
nitrocellulose membrane and probed with HRP-conjugated
streptavidin.
To study the binding of TAMRA-C.sub.14H.sub.24O.sub.4--Cl in
mammalian cells, DhaA.WT-Flag and DhaA.H272F-Flag coding regions
were excised from pGEX-5.times.-3. DhaA.WT-Flag or pGEX-5.times.-3.
DhaA.H272F-Flag, respectively, gel purified, and inserted into
SalI/NotI restriction sites of pClneo.CMV vector (Promega). The
constructs were confirmed by DNA sequencing.
CHO-K1 cells were plated in 24 well plates (Labsystems) and
transfected with a pCIneo-CMV.DhaA.WT-Flag or
pCIneo-CMV.DhaA.H272F-Flag vector. Twenty-four hours later, media
was replaced with fresh media containing 25 .mu.M
TAMRA-C.sub.14H.sub.24O.sub.4--Cl and the cells were placed into a
CO.sub.2 incubator for 60 minutes. Following this incubation, media
was removed, cells were quickly washed with PBS (pH 7.4; four
consecutive washes: 1.0 ml/cm.sup.2; 5 seconds each) and the cells
were solubilized in a sample buffer (1% SDS, 10% glycerol, and the
like; 250 .mu.l/well). Proteins (10 .mu.l/lane) were resolved on
SDS-PAGE (4-20% gradient gels) and the binding of the
TAMRA-C.sub.14H.sub.24O.sub.4--Cl was detected by a fluoroimager
(Hitachi, Japan) at E.sub.ex/E.sub.em equal 540/575 nm.
Results
FIGS. 14A and B show the binding of
biotin-C.sub.18H.sub.32O.sub.4--Cl (A) and
TAMRA-C.sub.12H.sub.24O.sub.4--Cl (B) to E. coli proteins in vivo.
The low molecular band on FIG. 14A is an E. coli protein
recognizable by HRP-SA, while the fluorescence detected in the
bottom part of Panel B was fluorescence of free
TAMRA-C.sub.12H.sub.24O.sub.4--Cl. FIG. 15 shows the binding of
TAMRA-C.sub.12H.sub.24O.sub.4--Cl to eukaryotic cell proteins in
vivo.
Analysis of FIG. 14 and FIG. 15 showed that the DhaA.H272F-Flag
mutant but not DhaA.WT-Flag binds TAMRA-C.sub.14H.sub.24O.sub.4--Cl
or biotin-C.sub.18H.sub.32O.sub.4--Cl in vivo. Moreover, the bond
between DhaA.H272F-Flag and the substrate was very strong (probably
covalent), since boiling with SDS followed by SDS-PAGE did not
disrupt the bond between the mutant enzyme and the substrate.
B. Permeability of Cell Membrane to Substrates of the Invention
Materials and Methods
CHO-K1 Cells (ATCC-CCL61) were cultured in a 1:1 mixture of Ham's
F12 nutrients and Dulbecco's modified minimal essential medium
supplemented with 10% fetal bovine serum (FBS), 100 U/ml
penicillin, and 100 mg/ml streptomycin, in an atmosphere of 95% air
and 5% CO.sub.2 at 37.degree. C.
To study uptake of different substrates, cells were plated in LT-II
chambers (Nunc) or 96 well plates (Labsystems) at a density of
30,000 cells/cm.sup.2. The following day, media was replaced with
media containing different concentrations of the substrates and
cells were placed back in a CO.sub.2 incubator for 2, 5 or 15
minutes. At the end of the incubation, media containing substrate
was removed and cells were quickly washed with PBS (pH 7.4; four
consecutive washes: 1.0 ml/cm.sup.2; 5 seconds each). Fresh media
was then added to cells, and the cells were returned to the
CO.sub.2 incubator at 37.degree. C. The level of fluorescence in
cells in 96 well plates was measured on fluorescent plate reader
CytoFluor II (Beckman) at E.sub.ex/E.sub.em equal 480/520 nm and
540/575 nm for FAM- and TAMRA-modified substrates, respectively.
Fluorescent images of the cells were taken on inverted
epifluorescent microscope Axiovert-100 (Carl Zeiss) with filter
sets appropriate for detection of FITC and TAMRA.
Results
As shown in FIG. 16, CHO-K1 cells treated with
TAMRA-C.sub.14H.sub.28O.sub.4--Cl (25 .mu.M, 5 minutes at
37.degree. C.) could be quickly and efficiently loaded with
TAMRA-C.sub.14H.sub.28O.sub.4--Cl. Image analysis indicated that
the fluorescent dye crossed the cell membrane. FIG. 16 also shows
that TAMRA-C.sub.14H.sub.28O.sub.4--Cl could be efficiently washed
out of the cells. Taken together these data indicate that the
plasma membrane of CHO-K1 cells is permeable to
TAMRA-C.sub.14H.sub.28O.sub.4--Cl.
In contrast, FAM-C.sub.14H.sub.24O.sub.4--Cl did not cross the
plasma membrane of CHO-K1 cells, even when cells were pre-treated
with FAM-C.sub.14H.sub.24O.sub.4--Cl at high concentrations (i.e.,
100 .mu.M) and for much longer periods of time (60 minutes) (data
not shown). Thus, the different permeabilities of the cell plasma
membrane for various substrates of the invention, e.g.,
TAMRA-C.sub.14H.sub.24O.sub.4--Cl and
FAM-C.sub.14H.sub.24O.sub.4--Cl, provides a unique opportunity to
label proteins expressed on the cell surface and proteins expressed
inside the cell with different fluorophores, thereby allowing
biplexing.
EXAMPLE V
DhaA-based Tethering for Cell Imaging In Vivo
A. Colocalization of GFP and TAMRA-C.sub.12H.sub.24O.sub.4--Cl in
Living Mammalian Cells
Materials and Methods
A GFP-connector-DhaA fusion cassette was constructed by replacing
the Renilla luciferase coding region in Packard's vector coding
GFP-DEVD-Rluc(h) (Packard #6310066) with DhaA.WT-Flag or
DhaA.H272F-Flag coding regions. Two printers
(5'-GGAATGGGCCCTCTAGAGCGACGATGTCA-3'; SEQ ID NO:15, and
5'-CAGTCAGTCACGATGGATCCGCTCAA-3'; SEQ ID NO:16) were designed to
add ApaI and BamHI sites (underlined) to the 5' and 3' coding
regions of DhaA, respectively, and to amplify a 980 bp fragment
from a pGEX-5.times.-3. DhaA.WT-Flag or pGEX5.times.-3.
DhaA.H272F-Flag template. The R.Luc coding region was excised with
ApaI and BamHI restriction enzymes. Then the 980 bp fragment
containing DhaA was inserted into the ApaII/BamHI site of the
GFP-DEVD-Rluc(h) coding vector. The sequence of the gene fusion
constructs was confirmed by DNA sequencing.
Cells transiently expressing GFP-connector-DhaA.WT-Flag or
GFP-connector-DhaA.H272F-Flag fusion proteins were plated in LT-II
chambers (Nunc) at a density of 30,000 cells/cm . The next day,
media was replaced with fresh media containing 25 .mu.M of
TAMRA-C.sub.14H.sub.24O.sub.4--Cl and the cells were placed back
into in a CO.sub.2 incubator for 60 minutes. At the end of the
incubation, media containing substrates was removed, cells were
quickly washed with PBS (pH 7.4; four consecutive washes: 1.0 ml/cm
2; 5 seconds each) and new media was added to the cells. The cells
were placed back into in a CO.sub.2 incubator and after 60 minutes
the cells were quickly washed with PBS (pH 7.4; four consecutive
washes: 1.0 ml/cm.sup.2; 5 seconds each). Fluorescent images of the
cells were taken on inverted epifluorescent microscope Axiovert-100
(Carl Zeiss) with filter sets appropriate for detection of GFP and
TAMRA.
Results
As shown by the images in FIG. 17, cells transfected with either
GFP-connector-DhaA.WT-Flag or GFP-connector-DhaA.H272F-Flag showed
robust expression of the protein(s) with light emitting
characteristics of GFP. Analysis of the images of the same cells
taken with a TAMRA-filter set showed that cells expressing
GFP-connector-DhaA.WT-Flag were dark and could not be distinguished
from cells that do not express this fusion protein. In contrast,
cells expressing GFP-connector-DhaA.H272F-Flag were very bright and
unmistakably recognizable.
Western blot analysis of proteins isolated from CHO-K1 cells
transfected with GFP-connector-DhaA.WT-Flag or
GFP-connector-DhaA.H272F-Flag vectors showed that these cells
expressed proteins that were recognized by an anti-Flag antibody
and had the predicted molecular weight for the fusion proteins
(data not shown). A fluoroscan of the SDS-PAGE gel with these
proteins showed strong/covalent binding of TAMRA to
GFP-connector-DhaA.H272F-Flag and no binding to
GFP-connector-DhaA.WT-Flag (FIG. 18).
B. Fusion Partners of DhaA in DhaA.WT-Flag and DhaA.H272F-Flag are
Functional
To determine whether fusion of two proteins leads to the loss of
the activity of one or both proteins, several DhaA-based fusion
proteins (see Table II) with DhaA at the C- or N-terminus of the
fusion and a connector sequence, e.g., one having 13 to 17 amino
acids, between the two proteins, were prepared. The data showed
that the functional activity of both proteins in the fusion was
preserved.
TABLE-US-00003 TABLE II N-Terminal C-terminal Function of Function
of protein Connector protein protein #1 protein #2 GST + DhaA.H272F
Binding to GSS binding column GFP + DhaA.H272F Green binding
fluorescence R.Luc + DhaA.H272F hydrolysis of binding
coelenterazine DhaA. + R.Luc Binding hydrolysis of H272F
coelenterazine DhaA. + Flag binding Recognized by H272F
antibody
C. Toxicity of Cl-Alkanes Materials and Methods
To study the toxicity of Cl-alkanes, CHO-K1 cells were plated in 96
well plates to a density of 5,000 cells per well. The next day,
media was replaced with fresh media containing 0-100 .mu.M
concentrations of Cl-alkanes and the cells were placed back into a
CO.sub.2 incubator for different periods of time. Viability of the
cells was measured with CellTiter-Glo.TM. Luminescence Cell
Viability Assay (Promega) according to the manufacturer's protocol.
Generally, 100 .mu.l of CellTiter-Glo.TM. reagent was added
directly to the cells and the luminescence was recorded at 10
minutes using a DYNEX MLX microtiter plate luminometer. In some
experiments, in order to prevent fluorescence/luminescence
interference, the media containing fluorescent Cl-alkanes was
removed and the cells were quickly washed with PBS (pH 7.4; four
consecutive washes: 1.0 ml/cm.sup.2; 5 seconds each) before
addition of CellTiter-Glo.TM. reagent. Control experiments
indicated that this procedure had no effect on the sensitivity or
accuracy of the CellTiter-Glo.TM. assay.
Results
As shown in FIG. 19, TAMRA-C.sub.14H.sub.24O.sub.4--Cl showed no
toxicity on CHO-K1 cells even after a 4 hour treatment at a 100
.mu.M concentration the (the highest concentration tested). After a
24 hour treatment, no toxicity was detected at concentrations of
6.25 .mu.M (the "maximum non-toxic concentration"). At
concentrations >6.25 .mu.M, the relative luminescence in CHO-K1
cells was reduced in a dose-dependent manner with an IC.sub.50 of
about 100 .mu.M. No toxicity of biotin-C.sub.18H.sub.34O.sub.4--Cl
was observed even after 24 hours of treatment at 100 .mu.M. In
contrast, ROX5-C.sub.14H.sub.24O.sub.4--Cl had a pronounced toxic
effect as a reduction of the RLU in CHO-K1 cells could be detected
after a 1 hour treatment. The IC.sub.50 value of this effect was
about 75 .mu.M with no apparent ATP reduction at a 25 .mu.M
concentration. The IC.sub.50 value of
ROX5-C.sub.14H.sub.24O.sub.4--Cl toxicity and the "maximum
non-toxic concentration" of ROX5-C.sub.14H.sub.24O.sub.4--Cl
decreased in a time-dependent manner reaching 12.5 .mu.M and 6.25
.mu.M, respectively.
D. Detection of DhaA.D106C in CHO Cells Contacted with TAMRA- or
DiAc-FAM-containing Substrates and a Fixative
CHO cells (ATCC, passage 4) were seeded into 8-well chamber slides
(German coverglass system) at low density in DMEM:F12 media (Gibco)
containing 10% FBS and 1 mM glutamine (growth media) without
antibiotics. Two days later, cells were inspected using an inverted
phase microscope. Two visual criteria were confirmed before
applying the transfection reagents: 1) the level of cellular
confluence per chamber was approximately 60-80%, and 2) >90% of
the cells were adherent and showed a flattened morphology. The
media was replaced with 150 .mu.l of fresh pre-warmed growth media
and cells were incubated for approximately 1 hour.
Cells were transfected using the Transit TKO system (Miris). The
TKO lipid was diluted by adding 7 .mu.l of lipid per 100 .mu.l of
serum-free DMEM:F12 media, and then 1.2 .mu.g of transfection-grade
DhaA.D106C DNA was added per 100 .mu.l of lipid containing media.
The mixture was incubated at room temperature for 15 minutes, and
then 25 .mu.l aliquots were transferred into individual culture
chambers (0.3 .mu.g DNA). Cells were returned to the incubator for
5-6 hours, washed two times with growth media, 300 .mu.l of fresh
growth media was added, and then cells were incubated for an
additional 24 hours.
Transfected or non-transfected control cells were incubated with
12.5 .mu.M TAMRA-C.sub.14H.sub.24O.sub.4--Cl or 12.5 .mu.M
DiAc-FAM-C.sub.14H.sub.24O.sub.4--Cl in 10% FBS/DMEM for 30 minutes
at 37.degree. C. and 5% CO.sub.2. Cells were washed with warm
growth media three times, 300 .mu.l fresh growth media was added,
and then cells were incubated for 1 hour.
Growth media was replaced with warm PBS and live cells were
visualized using a Zeiss Axiovert 100 inverted microscope equipped
with a rhodamine filter set (Exciter filter=540, Emission
filter=560LP) and a fluorescein filter set (Exciter filter=490,
Emission filter=520), and a Spot CCD camera. Images were captured
with exposure times of 0.15-0.60 seconds at gain settings of 4 or
16.
Discreet and specifically labeled transfected cells were evident in
both TAMRA-C.sub.14H.sub.24O.sub.4--Cl and
DiAc-FAM-C.sub.14H.sub.24O.sub.4--Cl labeled cells. The majority of
cells were non-transfected cells and they did not retain the
label.
The PBS was removed and cells were fixed with 3.7%
paraformaldehyde/0.1% Triton in PBS for 15 minutes. The fixative
was removed, PBS was added, and a second set of images was captured
for both TAMRA-C.sub.14H.sub.24O.sub.4--Cl and
DiAc-FAM-C.sub.14H.sub.24O.sub.4--Cl labeled cells.
The PBS was replaced with 50% methanol in PBS and cells were
incubated for 15 minutes, followed by a 15 minute incubation in 95%
methanol. A third set of images was captured and then an equal
volume mixture of methanol and acetone was applied to the cells and
incubated for 15 minutes. The media was replaced with PBS and a
fourth set of images was collected.
Results suggested that the binding of the substrates to the DhaA.D
106C mutant was stable following fixation with paraformaldehyde and
subsequent processing of fixed cell samples in methanol and
acetone. Furthermore, the brightness of the TAMRA or FAM
fluorescence was unchanged under these conditions.
EXAMPLE VI
Mutant Beta-Lactamase (blaZ)-based Tethering
The serine-.beta.-lactamases, enzymes that confer bacterial
resistance to .beta.-lactam antibiotic, likely use the hydroxyl
group of a serine residue (Ser70 in the class A consensus numbering
scheme of Ambler et al. (1991)) to degrade a wide range of
.beta.-lactam compounds. The reaction begins with the formation of
a precovalent encounter complex (FIG. 20A), and moves through a
high-energy acylation tetrahedral intermediate (FIG. 20B) to form a
transiently stable acyl-enzyme intermediate, forming an ester
through the catalytic residue Ser70 (FIG. 20C). Subsequently, the
acyl-enzyme is attacked by hydrolytic water (FIG. 20D) to form a
high-energy deacylation intermediate (FIG. 20E) (Minasov et al.,
2002), which collapses to form the hydrolyzed product (FIG. 20F).
The product is then expelled, regenerating free enzyme. As in
serine proteases, this mechanism requires a catalytic base to
activate the serine nucleophile to attack the amide bond of the
substrate and, following formation of the acyl-enzyme intermediate,
to activate the hydrolytic water for attack on the ester center of
the adduct.
A. Mutant .beta.-Lactamase and Fusions Thereof
Materials and Methods
The plasmid pTS32 harboring Staphylococcus aureus PC1 blaZ gene
(Zawadzke et al., 1995) was kindly provided by Dr. O. Herzberg
(University of Maryland Biotechnology Institute). The blaZ gene has
the following sequence:
TABLE-US-00004 (SEQ ID NO: 36) AGCTTACTAT GCCATTATTA ATAACTTAGC
CATTTCAACA CCTTCTTTCA AATATTTATAATAAACTATT GACACCGATA TTACAATTGT
AATATTATTG ATTTATAAAA ATTACAACTGTAATATCGGA GGGTTTATTT TGAAAAAGTT
AATATTTTTA ATTGTAATTG CTTTAGTTTTAAGTGCATGT AATTCAAACA GTTCACATGC
CAAAGAGTTA AATGATTTAG AAAAAAAATATAATGCTCAT ATTGGTGTTT ATGCTTTAGA
TACTAAAAGT GGTAAGGAAG TAAAATTTAATTCAGATAAG AGATTTGCCT ATGCTTCAAC
TTCAAAAGCG ATAAATAGTG CTATTTTGTTAGAACAAGTA CCTTATAATA AGTTAAATAA
AAAAGTACAT ATTAACAAAG ATGATATAGTTGCTTATTCT CCTATTTTAG AAAAATATGT
AGGAAAAGAT ATCACTTTAA AAGCACTTATTGAGGCTTCA ATGACATATA GTGATAATAC
AGCAAACAAT AAAATTATAA AAGAAATCGGTGGAATCAAA AAAGTTAAAC AACGTCTAAA
AGAACTAGGA GATAAAGTAA CAAATCCAGTTAGATATGAG ATAGAATTAA ATTACTATTC
ACCAAAGAGC AAAAAAGATA CTTCAACACCTGCTGCCTTC GGTAAGACCC TTAATAAACT
TATCGCCAAT GGAAAATTAA GCAAAGAAAACAAAAAATTC TTACTTGATT TAATGTTAAA
TAATAAAAGC GGAGATACTT TAATTAAAGACGGTGTTCCA AAAGACTATA AGGTTGCTGA
TAAAAGTGGT CAAGCAATAA CATATGCTTCTAGAAATGAT GTTGCTTTTG TTTATCCTAA
GGGCCAATCT GAACCTATTG TTTTAGTCATTTTTACGAAT AAAGACAATA AAAGTGATAA
GCCAAATGAT AAGTTGATAA GTGAAACCGCCAAGAGTGTA ATGAAGGAAT TTTAATATTC
TAAATGCATA ATAAATACTG ATAACATCTTATATTTTGTA TTATATTTTG TATTATCGTT
GAC.
GST-blaZ (WT and E166D, N170Q, or E166D:N170Q mutants) fusion
cassettes were constructed by introducing point mutations into the
blaZ gene and cloning the blaZ coding regions into SalI/AgeI sites
of pGEX5.times.3 vector. The internal mutagenic primers were as
follows: E166D (5'-CCAGTTAGATATGACATAGAATTAAATTACTATTCACC-3', SEQ
ID NO:56; 5'-GGTGAATAGTAATTTAATTCTATGTCATATCTAACTGG-3', SEQ ID
NO:57); N170Q (5'-CCAGTTAGATATGAGATAGAATTACAGTACTATTCACC-3', SEQ ID
NO:58; and 5'-GGTGAATAGTACTGTAATTCTATCTCATATCTAACTGG-3', SEQ ID
NO:59); and E166D:N170Q
(5'CCAGTTAGATATGACATAGAATTACAGTACTATTCACC-3'; SEQ ID NO:60 and
5'-GGTGAATAGTACTGTAATTCTATGTCATATCTAACTGG-3; SEQ ID NO:61). Two
external primers (5'-CAACAGGTCGACGCCGCCATGAAAGAGTTAAATGATTTAG-3',
SEQ ID NO:62; and 5-GTAGTCACCGGTAAATTCCTTCATTACACTCTTGGC-3', SEQ ID
NO:63) were designed to add N-terminal SalI site and a Kozak
sequence to the 5' coding region, add an AgeI site to the 3' coding
regions of blaZ, and to amplify a 806 bp fragment from a blaZ.WT
template. The resulting fragment was inserted into the SalI/AgeI
site of the vector pGEX-5.times.-3 containing a glutathione
S-transferase (GST) gene, a sequence coding a Factor Xa cleavage
site, and multiple cloning sites (MCS) followed by a sequence
coding for Flag and stop codons. These gene fusion constructs were
confirmed by DNA sequencing.
The GST-b/aZ (WT or mutants) fusion proteins were overexpressed in
competent E. coli BL21 (.lamda. DE3) cells and purified essentially
as described for DhaA and GST-DhaA fusion proteins (except the
potassium phosphate buffer (0.1 M. pH 6.8) was used instead of
Buffer A). Homogeneity of the proteins was verified by
SDS-PAGE.
The chromogenic substrate 6-.beta.-[(Furylacryloyl)amido]
penicillanic acid triethylamine salt (FAP) was purchased from
Calbiochem (La Jolla, Calif.). Hydrolysis of FAP was monitored by
loss of adsorbance at 344 nm (deltaE=1330 M.sup.-1 cm.sup.-1) on a
Beckman Du640 spectrophotometer (Beckman Coulter, Fullerton,
Calif.). All assays were performed at 25.degree. C. in 0.1 M
potassium phosphate buffer at pH 6.8.
In CCF2, the cephalosporin core links a 7-hydroxycoumarin to a
fluorescein. In the intact molecule, excitation of the coumarin
(E.sub.ex-409 nm) results in FRET to the fluorescein, which emits
green light (E.sub.em-520 nm). Cleavage of CCF2 by .beta.-lactamase
results in spatial separation of the two dyes, disrupting FRET such
that excitation of coumarin now gives rise to blue fluorescence
(E.sub.ex-447 nm). CCF2 was purchased from Aurora Biosciences
Corporation (San Diego, Calif.). Reduction of the FRET signal and
an increase in blue fluorescence were measured on Fluorescence
Multiwell Plate Reader CytoFluorII (PerSeptive Biosystems,
Framingham, Mass., USA).
Results
All .beta.-lactamases, including .beta.-lactamase from
Staphylococcus aureus PC1, hydrolyze .beta.-lactams of different
chemical structure. The efficiency of hydrolysis depends on the
type of the enzyme and chemical structure of the substrate.
Penicillin is considered to be a preferred substrate for
.beta.-lactamase from Staphylococcus aureus PC1.
The effect of point mutation(s) on the ability of .beta.-lactamase
to hydrolyze penicillins was studied as described in Zawadzke et
al. (1995). As shown in FIG. 20, a GST-.beta.-lactamase PC1 fusion
protein efficiently hydrolyzed FAP. Hydrolysis of FAP by
blaZ.E166D, blaZ.N170Q or blaZ.E166D:N170Q blaZ mutants could not
be detected even after 60 minutes of co-incubation. Therefore,
these mutations lead to significant inactivation of blaz.
To show that blaZ.E166D, blaZ.N170Q, or blaZ.E166D: N170Q mutants
bind .beta.-lactams, and therefore different functional groups
could be tethered to these proteins via .beta.-lactams, GST fusions
of these mutants were incubated with BOCELLIN.TM. FL, a fluorescent
penicillin (Molecular Probes Inc., Eugene, Oreg.). Proteins were
resolved on SDS-PAGE and analyzed on fluoroimager (Hitachi, Japan)
at an E.sub.ex/E.sub.em appropriate for the particular fluorophore.
The data in FIG. 22 show that all blaZ mutants bind bocellin.
Moreover, the bond between blaZ mutants and fluorescent substrates
was very strong, and probably covalent, since boiling with SDS
followed by SDS-PAGE did not disrupt the bond. Also, the binding
efficiency of double mutant blaZ.E166D:N170Q (judged by the
strength of the fluorescent signal of protein-bound fluorophore)
was much higher than binding efficiency of either of the single
mutants, and the binding efficiency of blaZ.N170Q was higher than
binding efficiency of blaZ.E166D. These data, in combination with
current understanding of the role of the individual amino acids in
hydrolysis of beta-lactams, show that additional mutations (e.g., a
mutation of an auxiliary amino acid) can improve efficiency of
tethering of functional groups to a mutated protein.
The effect of point mutation(s) on the ability of .beta.-lactamase
to hydrolyze cephalosporins was also studied using CCF2, a
FRET-based substrate described by Zlokarnik et al. (1998). As shown
in FIG. 23, the GST-.beta.-lactamase PC1 fusion protein efficiently
hydrolyzed CCF2 (lane 2). Single point mutations (i.e., E166D or
N170Q) reduced the ability of the fusion proteins to hydrolyze CCF2
(lanes 3 and 4). The replacement of two amino acids
(blaZ.E166D:N170Q mutants, lane 5) had an even more pronounced
effect on the CCF2 hydrolysis. However, all blaz mutants were
capable of hydrolyzing CCF2.
Thus, an amino acid substitution at position 166 or 170, e.g.,
Glu166Asp or Asn170Gly enables the mutant beta-lactamase to trap a
substrate and therefore tether the functional group of the
substrate to the mutant beta-lactamase via a stable, e.g.,
covalent, bond. Moreover, mutation of an amino acid that has an
auxiliary effect on H.sub.2O activation increased the efficiency of
tethering.
EXAMPLE VII
Targeting of DhaA.H272F to the Nucleus and Cytosol of Living
Cells
Materials and Methods
A GFP-connector-DhaA.H272F-NLS3 fusion cassette was constructed by
inserting a sequence encoding NLS3 (three tandem repeats of the
Nuclear Localization Sequence (NLS) from simian virus large
T-antigen) into the AgeI/BamHI sites of a
pClneo.GFP-connector-DhaA.H272F-Flag vector. Two complementary
oligonucleotides
(5'-CCGGTGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAA
AGGTAGATCCAAAAAAGAAGAGAAAGGTATGAG-3', sense, SEQ ID NO:37, and
5'-GATCCTCATACCTTTCTCTTCTTTTTTGGATCTACCTTTCTCTTCTTTTTTG
GATCTAACCTTTCTCTTCTTTTTTGGATCA-3', antisense, SEQ ID NO:38) coding
for the NLS3 peptide, were annealed. The annealed DNA had an AgeI
site at 5' end and a BamHI site at the 3' end. The annealed DNA was
subcloned into the GFP-connector-DhaA.H272F-Flag construct at the
AgeI/BamHI sites. The sequence of the gene fusion construct was
confirmed by DNA sequencing.
A DhaA.H272F-.beta.-arresting fusion cassette was constructed by
replacing the pGFP.sup.2 coding region in Packard's vector encoding
GFP.sup.2-.beta.-arrestin2 (Packard #6310176-1F1) with the
DhaA.H272F-Flag coding region. Two primers
(5'-ATTATGCTGAGTGATATCCC-3'; SEQ ID NO:39, and
5'-CTCGGTACCAAGCTCCTTGTAGTCA-3'; SEQ ID NO:40) were designed to add
a KpnI site to the 3' coding region of DhaA, and to amplify a 930
bp fragment from a pGEX5.times.-3. DhaA.H272F-Flag template. The
pGFP.sup.2 coding region was excised with NheI and KpnI restriction
enzymes, then the 930 bp fragment containing encoding DhaA.H272F
was inserted into the NheI and KpnI sites of the
GFP.sup.2-.beta.-arrestin2 coding vector. The sequence of the
fusion construct was confirmed by DNA sequencing.
CHO-K1 or 3T3 cells transiently expressing
GFP-connector-DhaA.H272F-NLS3, GFP.sup.2-.beta.-arrestin2 or
DhaA.H272F-.beta.-arrestin2 fusion proteins were plated in LT-II
chambers (Nunc) at a density of 30,000 cells/cm.sup.2. The next
day, media was replaced with fresh media containing 25 .mu.M of
TAMRA-C.sub.14H.sub.24O.sub.4--Cl and the cells were placed back
into a CO.sub.2 incubator for 60 minutes. At the end of the
incubation, substrate media was removed, cells were quickly washed
with PBS (pH 7.4; four consecutive washes: 1.0 ml/cm 2; 5 seconds
each), and new media was added to the cells. The cells were placed
back into a CO.sub.2 incubator and after 60 minutes the cells were
quickly washed with PBS (pH 7.4; 1.0 ml/cm.sup.2). Fluorescent
images of the cells were taken on confocal microscope Pascal-5
(Carl Zeiss) with filter sets appropriate for the detection of GFP
and TAMRA.
Results
As shown by the images in FIG. 24, GFP and TAMRA were co-localized
in the cell nucleus of cells expression
GFP-connector-DhaA.H272F-NLS3 and contacted with
TAMRA-C.sub.14H.sub.24O.sub.4--Cl.
As shown by the images in FIG. 25, GFP-.beta.-arrestin2 expressing
cells have a typical .beta.-arrestin2 cytosolic localization. A
fluoroscan of the SDS-PAGE gel of DhaA.H272F-.beta.-arrestin2
showed strong binding of a TAMRA containing DhaA substrate to cells
expressing DhaA.H272F-.beta.-arrestin2.
EXAMPLE VIII
Site-Directed Mutagenesis of DhaA Catalytic Residue 130
Haloalkane dehalogenases use a three-step mechanism for cleavage of
the carbon-halogen bond. This reaction is catalyzed by a triad of
amino acid residues composed of a nucleophile, base and acid which,
for the haloalkane dehalogenase from Xanthobacter autotrophicus
(DhlA), are residues Asp124, His289 and Asp260, respectively
(Franken et al., 1991), and in Rhodococcus dehalogenase enzyme
(DhaA), Asp106, His272 and Glu130 (Newman et al., 1999).
Unlike the haloalkane dehalogenase nucleophile and base residues,
the role of the third member of the catalytic triad is not yet
fully understood. The catalytic acid is hydrogen bonded to the
catalytic His residue and may assist the His residue in its
function by increasing the basicity of nitrogen in the imidazole
ring. Krooshof et al. (1997), using site-directed mutagenesis to
study the role of the DhlA catalytic acid Asp260, demonstrated that
a D260N mutant was catalytically inactive. Furthermore, this
residue apparently had an important structural role since the
mutant protein accumulated mainly in inclusion bodies. The
haloalkane dehalogenase from Sphinogomonas paucimobilis (LinB) is
the enzyme involved in .gamma.-hexachlorocyclohexane degradation
(Nagata et al., 1997). Hynkova et al., (1999) replaced the putative
catalytic residue (Glu-132) of the LinB with glutamine (Q) residue.
However, no activity was observed for the E132Q mutant even at very
high substrate concentrations.
To examine the role of the DhaA catalytic triad acid Glu130 in
protein production and on the ability of the mutant protein to form
covalent alkyl-enzyme intermediates with a fluorescent-labeled
haloalkane substrate, site-directed mutagenesis was employed to
replace the DhaA glutamate (E) residue at position 130 with
glutamine, leucine and alanine.
Materials and Methods
Strains and plasmids. Ultracompetent E. coli XL10 Gold (Stratagene;
Tet.sup.r .DELTA.(mcrA)183 .DELTA.(mcrCB-hsdSMR-mrr)173 endA1
supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F' proAB
lacI.sup.qZ.DELTA.M15 Tn10 (Tet.sup.r) Amy Cam.sup.r]) was used to
as a host in transformation of site-directed mutagenesis reactions.
E. coli strain JM109 (e14-(McrA-) recA1 endA1 gyrA96 thi-1
hsdR17(rK-mK+) supE44 relA1 .DELTA.(lac-proAB) [F' traD36proAB
lac.sup.qZ.DELTA.M15]) was used as the host for gene expression and
whole cell enzyme labeling studies. A GST-DhaA-FLAG gene fusion
cloned into plasmid pGEX5.times.3, designated
pGEX5.times.3DhaAWT.FLAG, was used as the starting template for
E130 mutagenesis. A mutant plasmid containing a H272F mutation in
DhaA, designated pGEX5.times.3DhaAH272F-FLAG, was used as a
positive control in labeling studies and the cloning vector
pGEX5.times.3 was used as a negative control.
Site-directed mutagenesis of the DhaA E130 residue. The sequence of
the oligonucleotides used for mutagenesis is shown below. The
underlined nucleotides indicate the position of the altered codons.
The oligonucleotides were synthesized by Integrated DNA
Technologies (Coralville, Iowa) at the 100 nmole scale and modified
by phosphorylation at the 5' end.
TABLE-US-00005 Dhaa E130Q (SEQ ID NO: 41) 5'
CAAAGGTATTGCATGTATGGCGTTCATCCGGCCTATCCCG 3' DhaA E130L (SEQ ID NO:
42) 5' GTCAAAGGTATTGCATGTATGCTGTTCATCCGGCCTATCCCGAC 3' DhaA E130A
(SEQ ID NO: 43) 5' AGGTATTGCATGTATGGCGTTCATCCGGCCTATCCC 3'
Site-directed mutagenesis was performed using the QuikChange Multi
kit according to the manufacturer's instructions (Stratagene, La
Jolla, Calif.). The mutagenesis reactions were introduced into
competent E. coli XL10 Gold cells and transformants were selected
on LB agar plates containing ampicillin (100 .mu.g/mL). Plasmid DNA
isolated from individual transformants was initially screened for
the loss of an EcoRI site due to replacement of the glutamate codon
(GAAttc). Clones suspected of containing the desired codon change
from each reaction were selected and subjected to DNA sequence
analysis (SeqWright, Houston, Tex.). The primer used to confirm the
sequence of the mutants in the pGEX5.times.3 vector was as follows:
5' GGGCTGGCAAGCCACGTTTGGTG 3' (SEQ ID NO:44).
DhaA mutant analysis. The three DhaA E130 substitution mutants were
compared to the following constructs: Wild-type DhaA, DhaA.H272F,
and a DhaA negative control (pGEX5.times.3 vector only). Overnight
cultures of each clone were grown in 2 mL of LB containing
ampicillin (100 .mu.g/mL) by shaking at 30.degree. C. The overnight
cultures were diluted 1:50 into a sterile flask containing 50 mL
fresh LB medium and ampicillin (100 .mu.g/mL). The cultures were
incubated with shaking at 25.degree. C. to minimize the production
of insoluble protein species. When the cultures reached mid-log
phase (OD.sub.600=0.6), IPTG (0.1 mM) was added and the cultures
were incubated with shaking at 25.degree. C. for an additional 22
hours. For labeling of whole cells with a tetramethylrhodamine
(TAMRA) haloalkane conjugated substrate, the cell density of each
culture was adjusted to OD.sub.600=1 prior to adding substrate to a
concentration of 15 .mu.M. The cells were incubated with gentle
agitation at 4.degree. C. for approximately 18 hours. Following
incubation, 20 .mu.l of cells from each labeling reaction was added
to 6 .mu.l of 4.times.SDS loading dye and the samples were boiled
for about 3 minutes prior to being loaded onto a 4-20% acrylamide
gel (Tris glycine). For in vitro labeling studies, crude lysates of
IPTG induced cultures were prepared by collecting 3 mL of cells
(OD.sub.600=1) and resuspending the resulting pellet in 75 .mu.L
PBS. Following a freeze/thaw step, 225 .mu.L of 1.times. Cell
Culture Lysis Reagent (Promega Corp., Madison, Wis.) containing
1.25 mg/mL lysozyme was added to facilitate lysis of the cells. A
20 .mu.L sample of each lysate was combined with 25 .mu.L of
1.times. PBS. The TAMRA labeled haloalkane substrate was added to a
final concentration of 25 .mu.M. The labeling reactions were
incubated at room temperature for 2 hours. A 25 .mu.l sample of
each labeling reaction was added to 6 .mu.l 4.times.SDS loading dye
and the samples were boiled for about 3 minutes prior to being
loaded onto a 4-20% acrylamide gel (Tris glycine). The gels were
imaged using a FluorImager SI instrument (Amersham Biosciences,
Piscataway, N.J.) set to detect emission at 570 nm.
Cell-free lysates were generated by centrifugation of crude lysates
for 15 minutes at 14,000 RPM. Protein production was monitored by
SDS-PAGE and Western blot analysis. Proteins transferred to a PVDF
membrane were incubated with an anti-FLAG.sup.R antibody conjugated
with alkaline phosphatase (AP) (Sigma, St. Louis, Mo.). The blot
was developed with the Western Blue stabilized substrate for
alkaline phosphatase (Promega Corp., Madison, Wis.).
Results
The role of the DhaA catalytic acid in the hydrolysis of the
alkyl-enzyme intermediate was probed by site-directed mutagenesis.
The DhaA codon E130 was replaced with a codon for glutamine (Q),
leucine (L) or alanine (A), as these substitutions would likely be
least disruptive to the structure of the enzyme. Following
mutagenesis, restriction endonuclease screening and DNA sequence
analysis was used to verify the desired codon changes. Sequence
verified DhaA.E130Q, DhaA.E130L and DhaA.E130A clones, designated
C1, A5 and A12, respectively, were chosen for further analysis. The
E130 mutants were analyzed for protein expression and for their
ability to form a covalent alkyl-enzyme intermediate with a TAMRA
labeled haloalkane substrate. The three E130 gene variants were
over-expressed in E. coli JM109 cells following induction with
IPTG. SDS-PAGE analysis of crude cell lysates showed that cultures
expressing the wild-type and mutant dhaA genes accumulated protein
to approximately the same level (FIG. 26; lanes 2, 4, 6, 8, 10, and
12). Furthermore, the DhaA protein that was produced by the
wild-type and H272F constructs was for the most part soluble since
the amount of protein did not change appreciably after
centrifugation (FIG. 26; lanes 3 and 5). The abundant 22 kDa
protein bands present in the vector only lanes (FIG. 26; lanes 6
and 7) represented the GST protein. These results, however, are in
stark contrast to the DhaA.E130Q, DhaA.E130L and DhaA.E130A mutants
that appeared to accumulate predominantly insoluble DhaA protein.
This conclusion is based on the observation that after
centrifugation, there was a significant loss in the amount of DhaA
protein present in cell-free lysates (FIG. 26; lanes 9, 11, and
13). Nevertheless, a protein band that comigrates with DhaA was
clearly observed in each DhaA.E130 mutant lanes after
centrifugation (+s) suggesting the presence of soluble enzyme.
Western analysis was, therefore, used to determine if the protein
bands observed in the DhaA.E130 mutants following centrifugation
represented soluble DhaA material. The immunoblot shown in FIG. 27
confirmed the presence of soluble DhaA protein in each of the
DhaA.E130 mutant cell-free lysates (lanes 9, 11, and 13).
The DhaA.E130 mutants were also examined for their ability to
generate an alkyl-enzyme covalent intermediate. Crude lysates
prepared from IPTG induced cultures of the various constructs were
incubated in the presence of the TAMRA labeled substrate. FIG. 28
showed that the DhaA.H272F mutant (lane 3) was very efficient at
producing this intermediate. No such product could be detected with
either the WT DhaA or negative control lysates. Upon initial
examination, the DhaA.E130 mutants did not appear to produce
detectable levels of the covalent product. However, upon closer
inspection of the fluoroimage extremely faint bands were observed
that could potentially represent minute amounts of the covalent
intermediate (FIG. 28; lanes 5-7). Based on these results, the
ability of whole cells to generate a covalent, fluorescent
alkyl-enzyme intermediate was investigated.
FIG. 29 shows the results of an in vivo labeling experiment
comparing each of the DhaA.E 130 mutants with positive (DhaA.H272F
mutant) and negative (DhaA-) controls. As expected, the DhaA.H272F
mutant was capable of generating a covalent alkyl-enzyme
intermediate as evidenced by the single fluorescent band near the
molecular weight predicted for the GST-DhaA-Flag fusion (FIG. 29,
lane 3). As previously observed with the in vitro labeling results,
no such product could be detected with either the wild-type or
negative control cultures (FIG. 29, lanes 2 and 3) but very faint
fluorescent bands migrating at the correct position were again
detected with all three DhaA.E130 substituted mutants (FIG. 29,
lanes 5-7). These results point to the possibility that the
DhaA.E130Q, L and A mutants have the ability to trap covalent
alkyl-enzyme intermediates. The efficiency of this reaction,
however, appears to proceed at a dramatically reduced rate compared
to the DhaA.H272F mutant enzyme.
The results of this mutagenesis study suggest that the DhaA
catalytic acid residue DhaA.E130 plays an important structural role
in the correct folding of the enzyme. The DhaA protein was clearly
sensitive to substitutions at this amino acid position as evidenced
by the presence of largely insoluble protein complexes in the
DhaA.E130Q, DhaA.E130L and DhaA.E130A crude lysates. Nevertheless,
based on SDS-PAGE and immunoblot analyses, a significant quantity
of soluble DhaA protein was detected in the cell-free lysates of
all three DhaA.E130 mutants.
EXAMPLE IX
Capturing of DhaA.H272F-Flag and DhaA.H272F-Flag Renilla Luciferase
Fusion Proteins Expressed in Living Mammalian Cells
Materials and Methods
CHO-K1 cells were plated in 24 well plates (Labsystems) at a
density of 30,000 cells/cm.sup.2 and transfected with a
pCIneo.DhaA.WT-Flag or pCIneo.hRLuc-connector-DhaA.H272F-Flag
vector. Twenty-four hours later, media was replaced with fresh
media containing 25 .mu.M biotin-C.sub.18H.sub.32O.sub.4--Cl and
0.1% DMSO, or 0.1% DMSO alone, and the cells were placed in a
CO.sub.2 incubator for 60 minutes. At the end of the incubation,
the media was removed, cells were quickly washed with PBS (pH 7.4;
four consecutive washes; 1.0 ml/cm.sup.2; 5 seconds each) and new
media was added to the cells. In some experiments, the media was
not changed. The cells were placed back in a CO.sub.2
incubator.
After 60 minutes, media was removed, and the cells were collected
in PBS (pH=7.4, 200 .mu.l/well, RT) containing protease inhibitors
(Sigma #P8340). The cells were lysed by trituriation through a
needle (IM1 23GTW). Then, cell lysates were incubated with
MagnaBind Streptavidin coated beads (Pierce #21344) according to
the manufacturer's protocol. Briefly, cell lysates were incubated
with heads for 60 minutes at room temperature (RT) using a rotating
disk. Unbound material was collected; beads were washed with PBS
(3.times.500 .mu.l, pH=7.4, RT) and resuspended in SDS-sample
buffer (for SDS-PAGE analysis) or PBS (pH=7.4, for determination of
R.Luc activity). Proteins were resolved on SDS-PAGE, transferred to
a nitrocellulose membrane, analyzed with anti-Flag-Ab or
anti-R.Luc-Ab, and bound antibody detected by an enhanced
chemiluminescence (ECL) system (Pharmacia-Amersham). Activity of
hR.Luc bound to beads was determined using Promega's "Renilla
Luciferase Assay System" according to the manufacturer's
protocol.
Results
Capturing of proteins expressed in living cells allows for analysis
of those proteins with a variety of analytic methods/techniques. A
number of capturing tools are available although most of those
tools require generation of a highly specific antibody or
genetically fusing a protein of interest with specific tag
peptides/proteins (Jarvik and Telmer, 1998; Ragaut et al., 1999).
However, those tags have only limited use for live cell imaging. To
capture DhaA.H272F and functional proteins fused to DhaA.H272F,
SA-coated beads were used (Savage et al., 1992).
Biotin-C.sub.18H.sub.32O.sub.4--Cl was efficiently hydrolyzed by
wild-type DhaA, and covalently bound to DhaA.H272F and DhaA.H272F
fusion proteins in vitro and in vivo. Moreover, binding was
observed both in E. coli and in mammalian cells. Control
experiments indicated that about 80% of the DhaA.H272F-Flag protein
expressed in CHO-K1 cells was labeled after a 60 minute
treatment.
CHO-K1 cells transiently expressing DhaA.H272F-Flag were treated
with biotin-C.sub.18H.sub.32O.sub.4--Cl.
Biotin-C.sub.18H.sub.32O.sub.4--Cl treated cells were lysed and
cell lysates were incubated with SA-coated beads. Binding of
DhaA.H272F to beads was analyzed by Western blot using
anti-Flag.sup.R antibody. As shown in FIG. 30D, DhaA.H272F-Flag
capturing was not detected in the absence of
biotin-C.sub.18H.sub.32O.sub.4--Cl treatment. At the same time,
more than 50% of the DhaA.H272F-Flag expressed in cells was
captured on SA-coated beads if the cells were treated with
biotin-C.sub.18H.sub.32O.sub.4--Cl.
To show the capturing of functionally active proteins fused to
DhaA.H272F-Flag, cells were transfected with a vector encoding
hR.Luc-connector-DhaA.H272F-Flag, and the luciferase activity
captured on the beads measured. As shown in FIG. 30C, significant
luciferase activity was detected on beads incubated with a lysate
of biotin-C.sub.18H.sub.32O.sub.4--Cl treated cells. At the same
time, no luciferase activity was detected on beads incubated with a
lysate from cells that were not treated with
biotin-C.sub.18H.sub.32O.sub.4--Cl. Moreover, no hR.Luc activity
was detected on beads incubated with lysate from the cells treated
with biotin-C.sub.18H.sub.32O.sub.4--Cl when free
biotin-C.sub.18H.sub.32O.sub.4--Cl was not washed out.
Taken together, these data show that functionally active protein
(hR.Luc) fused to the DhaA.H272F can be efficiently captured using
biotin-C.sub.18H.sub.32O.sub.4--Cl and SA-coated beads. The capture
is biotin-dependent, and can be competed-off by excess of
biotin-C.sub.18H.sub.32O.sub.4--Cl. As a significant inhibitory
effect of the beads on the hR.Luc activity was observed (data not
shown), SDS-PAGE and Western blot analysis with anti-R.Luc antibody
were used to estimate the efficiency of capture of
hR.Luc-connector-DhaA.H272F-Flag fusion protein. As shown in FIG.
30D, more than 50% of hR.Luc-connector-DhaA.H272F-Flag fusion
protein can be captured in biotin-dependent manner. This is in good
agreement with the capturing efficiency of DhaA.H272F-Flag (see
FIG. 30A).
EXAMPLE X cl Optimized DhaA Gene
DhaA General Sequence Design
A synthetic DhaA.H272F gene was prepared which had a human codon
bias, low CG content, selected restriction enzyme recognition sites
and a reduced number of transcription regulatory sites. Relative to
the amino sequence encoded by a wild-type DhaA gene which lacks a
signal sequence (SEQ ID NO:51), and/or to DhaA.H272F, the amino
acid sequence of a codon-optimized DhaA gene and flanking sequences
included: 1) a Gly inserted at position 2, due to introduction of
an improved Kozak sequence (GCCACCATGG; SEQ ID NO:45) and a BamHI
site (thus the H272F active site mutation in DhaA mutants with the
Gly insertion is at position 273); 2) a A292G substitution due to
introduction of a SmaI/XmaI/AvaI site which, in the DhaA mutant
with the Gly insertion, is at position 293; 3) the addition of
Ala-Gly at the C-terminus due to introduction of a NaeI (NgoMIV)
site; 4) the addition of NheI, PvuII, EcoRV and NcoI sites in the
5' flanking sequence; 5) the addition of NNNN in the 5' flanking
sequence to eliminate search algorithm errors at the end and to
maintain the ORFI (i.e.,
NNN-NGC-TAG-CCA-GCT-GGC-GAT-ATC-GCC-ACC-ATG-GGA; SEQ ID NO:46); 6)
at the 3' end a NotI site, the addition of NNNN to eliminate search
algorithm errors at the end, a Pad site with ORF Leu-Ile-Lys, and
two stop codons, at least one of which is a TAA (i.e.,
TAATAGTTAATTAAGTAAGCGGCCGCNNNN; SEQ ID NO:47). SEQ ID NO:51 has the
following sequence:
TABLE-US-00006 atgtcagaaatcggtacaggcttccccttcgacccccattatgtggaagt
cctgggcgagcgtatgcactacgtcgatgttggaccgcgggatggcacgc
ctgtgctgttcctgcacggtaacccgacctcgtcctacctgtggcgcaac
atcatcccgcatgtagcaccgagtcatcggtgcattgctccagacctgat
cgggatgggaaaatcggacaaaccagacctcgattatttcttcgacgacc
acgtccgctacctcgatgccttcatcgaagccttgggtttggaagaggtc
gtcctggtcatccacgactggggctcagctctcggattccactgggccaa
gcgcaatccggaacgggtcaaaggtattgcatgtatggaattcatccggc
ctatcccgacgtgggacgaatggccggaattcgcccgtgagaccttccag
gccttccggaccgccgacgtcggccgagagttgatcatcgatcagaacgc
tttcatcgagggtgcgctcccgaaatgcgtcgtccgtccgcttacggagg
tcgagatggaccactatcgcgagcccttcctcaagcctgttgaccgagag
ccactgtggcgattccccaacgagctgcccatcgccggtgagcccgcgaa
catcgtcgcgctcgtcgaggcatacatgaactggctgcaccagtcacctg
tcccgaagttgttgttctggggcacacccggcgtactgatccccccggcc
gaagccgcgagacttgccgaaagcctccccaactgcaagacagtggacat
cggcccgggattgcactacctccaggaagacaacccggaccttatcggca
gtgagatcgcgcgctggctccccgcactctag
Codon Selection
Codon usage data was obtained from the Codon Usage Database
(http://www.kazusa.or.jip/codon/), which is based on: GenBank
Release 131.0 of 15 Aug. 2002 (See, Nakamura et al., 2000). Codon
usage tables were downloaded for: HS: Homo sapiens [gbpri] 50,031
CDS's (21,930,294 codons); MM: Mus musculus [gbrod] 23,113 CDS's
(10,345,401 codons): EC: Escherichia coli [gbbct] 11,985 CDS's
(3,688,954 codons); and EC K12: Escherichia coli K12 [gbbct] 4,291
CDS's (1,363,716 codons). HS and MM were compared and found to he
closely similar, thus the HS table was used. EC and EC K12 were
compared and found to be closely similar, therefore the EC K12
table was employed.
The overall strategy for selecting codons was to adapt codon usage
for optimal expression in mammalian cells while avoiding low-usage
E. coli codons. One "best" codon was selected for each amino acid
and used to back-translate the desired protein sequence to yield a
starting gene sequence. Another selection criteria was to avoid
high usage frequency HS codons which contain CG dinucleotides, as
methylation of CG has been implicated in transcriptional gene
regulation and can cause down-regulation of gene expression in
stable cell lines. Thus, all codons containing CG (8 human codons)
and TA (4 human codons, except for Tyr codons) were excluded.
Codons ending in C were also avoided as they might form a CG with a
downstream codon. Of the remaining codons, those with highest usage
in HS were selected, unless a codon with a slightly lower usage had
substantially higher usage in E. coli.
DhaA Gene Sequences
To generate a starting DhaA sequence, codon usage tables in Vector
NTI 8.0 (Informax) were employed. The DhaA.v2.1 protein sequence
(SEQ ID NO:48) was back translated to create a starting gene
sequence, hDhaA.v2.1-0, and flanking regions were then added, as
described above, to create hDhaA.v2. 1-OF (SEQ ID NO:49).
TABLE-US-00007 DhaA.v2.1: (SEQ ID NO: 48)
MGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYL
WRNIIPHVAPSHRCIAPDLIGMGKSDKPDLDYFFDDHVRYLDAFIEAL
GLEEVVLVIHDWGSALGFHWAKRNPERVKGIACMEFIRPIPTWDEWPE
FARETFQAFRTADVGRELIIDQNAFIEGALPKCVVRPLTEVEMDHYRE
PFLKPVDREPLWRFPNELPIAGEPANIVALVEAYMNWLHQSPVPKLLF
WGTPGVLIPPAEAARLAESLPNCKTVDIGPGLFYLQEDNPDLIGSEIA RWLPGLAG
hDhaA.v2.1-0F: (SEQ ID NO: 49)
NNNNGCTAGCCAGCTGGCGATATCGCCACCATGGGATCCGAGATTGGG
ACAGGGTTTCCTTTTGATCCTCATTATGTGGAGGTGCTGGGGGAGAGA
ATGCATTATGTGGATGTGGGGCCTAGAGATGGGACACCTGTGCTGTTT
CTGCATGGGAATCCTACATCTTCTTATCTGTGGAGAAATATTATTCCT
CATGTGGCTCCTTCTCATAGATGTATTGCTCCTGATCTGATTGGGATG
GGGAAGTCTGATAAGCCTGATCTGGATTATTTTTTTGATGATCATGTG
AGATATCTGGATGCTTTTATTGAGGCTCTGGGGCTGGAGGAGGTGGTG
CTGGTGATTCATGATTGGGGGTCTGCTCTGGGGTTTCATTGGGCTAAG
AGAAATCCTGAGAGAGTGAAGGGGATTGCTTGTATGGAGTTTATTAGA
CCTATTCCTACATGGGATGAGTGGCCTGAGTTTGCTAGAGAGACATTT
CAGGCTTTTAGAACAGCTGATGTGGGGAGAGAGCTGATTATTGATCAG
AATGCTTTTATTGAGGGGGCTCTGCCTAAGTGTGTGGTGAGACCTCTG
ACAGAGGTGGAGATGGATCATTATAGAGAGCCTTTTCTGAAGCCTGTG
GATAGAGAGCCTCTGTGGAGATTTCCTAATGAGCTGCCTATTGCTGGG
GAGCCTGCTAATATTGTGGCTCTGGTGGAGGCTTATATGAATTGGCTG
CATCAGTCTCCTGTGCCTAAGCTGCTGTTTTGGGGGACACCTGGGGTG
CTGATTCCTCCTGCTGAGGCTGCTAGACTGGCTGAGTCTCTGCCTAAT
TGTAAGACAGTGGATATTGGGCCTGGGCTGTTTTATCTGCAGGAGGAT
AATCCTGATCTGATTGGGTCTGAGATTGCTAGATGGCTGCCCGGGCTG
GCCGGCTAATAGTTAATTAAGTAAGCGGCCGCNNNN
Further Optimization
Programs and databases used for identification and removal of
sequence motifs were from Genomatix Software GmbH (Munich, Germany,
http://www.genomatix.de): GEMS Launcher Release 3.5.1 (April 2003),
MatInspector professional Release 6.1 (January 2003), Matrix Family
Library Ver 3.1.1 (April 2003, including 318 vertebrate matrices in
128 families), ModelInspector professional Release 4.8 (October
2002), Model Library Ver 3.1 (March 2003, 226 modules),
SequenceShaper tool, and User Defined Matrices. The sequence motifs
to be removed from starting gene sequences in order of priority
were restriction enzyme recognition sequences listed below;
transcription factor binding sequences including promoter modules
(i.e., 2 transcription factor binding sites with defined
orientation) with a default score or greater, and vertebrate
transcription factor binding sequences with a minimum score of
=0.75/matrix=optimized; eukaryotic transcription regulatory sites
including a Kozak sequence, splice donor/acceptor sequences, polyA
addition sequences; and prokaryotic transcription regulatory
sequences including E. coli promoters and E. coli RBS if less than
20 bp upstream of a Met codon.
User-defined Matrices
Subset DhaA
Format: Matrix name (core similarity threshold/matrix similarity
threshold): U$AatII (0.75/1.00), U$BamHI (0.75/1.00), U$BglI
(0.75/1.00), USBglII (0.75/1.00), U$BsaI (0.75/1.00), U$BsmAI
(0.75/1.00), USBsmBI (0.75/1.00), U$BstEII (0.75/1.00), U$BstXI
(0.75/1.00), U$Csp45I (0.75/1.00), U$CspI (0.75/1.00), U$DraI
(0.75/1.00), U$EC-P-10 (1.00/Optimized), U$EC-P-35
(1.00/Optimized), U$EC-Prom (1.00/Optimized), U$EC-RBS (0.75/1.00),
U$EcoRI (0.75/1.00), U$EcoRV (0.75/1.00), U$HindIII (0.75/1.00),
U$Kozak (0.75/Optimized), U$KpnI (0.75/1.00), U$MluI (0.75/1.00),
U$NaeI (0.75/1.00), U$NcoI (0.75/1.00), U$NdeI (0.75/1.00), U$NheI
(0.75/1.00), U$NotI (0.75/1.00), U$NsiI (0.75/1.00), U$PacI
(0.75/1.00), U$pflMI (0.75/1.00), U$PmeI (0.75/1.00), U$PolyAsig
(0.75/1.00), USPstI (0.75/1.00), USPvuII (0.75/1.00), U$SacI
(0.75/1.00), U$SacII (0.75/1.00), U$SalI (0.75/1.00), U$SfiI
(0.75/1.00), U$SgfI (0.75/1.00), U$SmaI (0.75/1.00), USSnaBI
(0.75/1.00), USSpeI (0.75/1.00), U$Splice-A (0.75/Optimized),
U$Splice-D (0.75/Optimized), U$XbaI (0.75/1.00), U$XcmI
(0.75/1.00), USXhoI (0.75/1.00), and ALL vertebrates.lib.
Subset DhaA-EC
Without E. coli specific sequences: U$AatII (0.75/1.00), U$BamHI
(0.75/1.00), U$BglI (0.75/1.00), U$BglII (0.75/1.00), U$BsaI
(0.75/1.00), U$BsmAI (0.75/1.00), U$BsmBI (0.75/1.00), U$BstEII
(0.75/1.00), U$BstXI (0.75/1.00), U$Csp451 (0.75/1.00), U$CspI
(0.75/1.00), U$DraI (0.75/1.00), USEcoRI (0.75/1.00), U$EcoRV
(0.75/1.00), U$HindIII (0.75/1.00), U$Kozak (0.75/Optimized),
U$KpnI (0.75/1.00), U$MluI (0.75/1.00), U$NaeI (0.75/1.00), U$NcoI
(0.75/1.00), U$NdeI (0.75/1.00), U$NheI (0.75/1.00), U$NotI
(0.75/1.00), U$NsiI (0.75/1.00), U$PacI (0.75/1.00), U$PflMI
(0.75/1.00), U$PmeI (0.75/1.00), U$PolyAsig (0.75/1.00), USPstI
(0.75/1.00), USPvuII (0.75/1.00), U$SacI (0.75/1.00), U$SacII
(0.75/1.00), U$SalI (0.75/1.00), U$SfiI (0.75/1.00), U$SgfI
(0.75/1.00), U$SmaI (0.75/1.00), USSnaBI (0.75/1.00), USSpeI
(0.75/1.00), U$Splice-A (0.75/Optimized), U$Splice-D
(0.75/Optimized), U$XbaI (0.75/1.00), U$XcmI (0.75/1.00), USXhoI
(0.75/1.00), and ALL vertebrates.lib.
Strategy for Removal of Sequence Motifs
The undesired sequence motifs specified above were removed from the
starting gene sequence by selecting alternate codons that allowed
retention of the specified protein and flanking sequences.
Alternate codons were selected in a way to conform to the overall
codon selection strategy as much as possible.
A. General Steps
Identify undesired sequence matches with MatInspector using matrix
family subset "DhaA" or "DhaA-EC" and with ModelInspector using
default settings. Identify possible replacement codons to remove
undesired sequence matches with SequenceShaper (keep ORF).
Incorporate all changes into a new version of the synthetic gene
sequence and re-analyze with MatInspector and ModelInspector. B.
Specific Steps Remove undesired sequence matches using subset
"DhaA-EC" and SequenceShaper default remaining thresholds
(0.70/Opt-0.20). For sequence matches that cannot be removed with
this approach use lower SequenceShaper remaining thresholds (e.g.,
0.70/Opt-0.05). For sequence matches that still cannot be removed,
try different combinations of manually chosen replacement codons
(especially if more than 3 base changes might be needed). If that
introduces new sequence matches, try to remove those using the
steps above (a different starting sequence sometimes allows a
different removal solution). Use subset "DhaA" to check whether
problematic E. coli sequences motifs were introduced, and if so try
to remove them using an analogous approach to that described above
for non E. coli sequences.
Use an analogous strategy for the flanking (non-open reading frame)
sequences.
C. Identification and Removal of Putative CpG Islands
Software used: EMBOSS CpGPlot/CpGReport
http://www.ebi.ac.uk/emboss/cpgplot/index.html) (see,
Gardiner-Garden et al., 1987).
Parameters: default (modified): Window: 100; Step: 1; Obs/Exp: 0.6;
MinPC: 50; Length: 100; Reverse: no; Complement: no. After the
removal of undesired sequence motifs, the gene sequence was checked
for putative CpG islands of at least 100 bases using the software
described above. If CpG islands were identified, they were removed
by selecting, at some of the CG di-nucleotide positions, alternate
codons that allowed retention of the specified protein and flanking
sequences, but did not introduce new undesired sequence motifs.
D. Restriction Sites
A unique MunI/MfeI (C'AATTG) site was introduced to allow removal
of the C-terminal 34 amino acids, including a putative
myristylation site (GSEIAR) near the C-terminus. Another unique
site, a NruI site, was introduced to allow removal of the
C-terminal 80-100 amino acids.
Results
Sequence Comparisons
An optimized DhaA gene has the following sequence: hDhaA.v2.1-6F
(FINAL, with flanking sequences)
TABLE-US-00008 (SEQ ID NO: 50)
NNNNGCTAGCCAGCTGGCgcgGATATCGCCACCATGGGATCCGAGATT
GGGACAGGGTTcCCTTTTGATCCTCAcTATGTtGAaGTGCTGGGgGAa
AGAATGCAcTAcGTGGATGTGGGGCCTAGAGATGGGACcCCaGTGCTG
TTcCTcCAcGGGAAcCCTACATCTagcTAcCTGTGGAGaAAtATTATa
CCTCATGTtGCTCCTagtCATAGgTGcATTGCTCCTGATCTGATcGGG
ATGGGGAAGTCTGATAAGCCTGActtaGAcTAcTTTTTTGATGAtCAT
GTtcGATActTGGATGCTTTcATTGAGGCTCTGGGGCTGGAGGAGGTG
GTGCTGGTGATaCAcGAcTGGGGGTCTGCTCTGGGGTTTCAcTGGGCT
AAaAGgAATCCgGAGAGAGTGAAGGGGATTGCTTGcATGGAgTTTATT
cGACCTATTCCTACtTGGGAtGAaTGGCCaGAGTTTGCcAGAGAGACA
TTTCAaGCcTTTAGAACtGCcGATGTGGGcAGgGAGCTGATTATaGAc
CAGAATGCTTTcATcGAGGGGGCTCTGCCTAAaTGTGTaGTcAGACCT
CTcACtGAAGTaGAGATGGAcCATTATAGAGAGCCcTTTCTGAAGCCT
GTGGATcGcGAGCCTCTGTGGAGgTTtCCaAATGAGCTGCCTATTGCT
GGGGAGCCTGCTAATATTGTGGCTCTGGTGGAaGCcTATATGAAcTGG
CTGCATCAGagTCCaGTGCCcAAGCTaCTcTTTTGGGGGACtCCgGGa
GTtCTGATTCCTCCTGCcGAGGCTGCTAGACTGGCTGAaTCcCTGCCc
AAtTGTAAGACcGTGGAcATcGGcCCtGGgCTGTTTTAcCTcCAaGAG
GAcAAcCCTGATCTcATcGGGTCTGAGATcGCacGgTGGCTGCCCGGG
CTGGCCGGCTAATAGTTAATTAAGTAgGCGGCCGCNNNN
A comparison of the nucleic acid sequence identity of different
DhaA genes (without flanking sequences) is shown in Table III.
TABLE-US-00009 TABLE III DhaA DhaA.v2.1 hDhaA.v.2.1-0 hDhaA.v2.1-6
DhaA 100 98 72 75 DhaA.v2.1.sup.a 100 74 76 hDhaA.v.2.1-0.sup.b 100
88 hDhaA.v2.1-6 100 .sup.aGly added at position 2, 11272F, A292G,
Ala-Gly added to C-terminus .sup.bcodon optimized
The GC content of different DhaA genes (without flanking sequences)
is provided in Table IV.
TABLE-US-00010 TABLE IV GC content CG di-nucleotides H. sapiens 53%
DhaA 60% 85 DhaA.v2.1 60% 87 hDhaA.v.2.1-0 49% 3 hDhsA.v2.1-6 52%
21
Vertebrate transcription factor binding sequence families (core
similarity: 0.75/matrix similarity: opt) and promoter modules
(default parameters: optimized threshold or 80% of maximum score)
found in different DhaA genes are shown in Table V.
TABLE-US-00011 TABLE V TF binding sequences Promoter modules Gene
name 5' F/ORF/3' F 5' F/ORF/3' F DhaA --/82/-- --/5/-- DhaA.v2.1-F
3/82/12 0/5/0 hDhaA.v.2.1 -OF 3/87/12 0/0/0 hDhaA.v2.1-6F 1/3/8
0/0/0
Note: 3 bp insertion before EcoRV in hDhaA.v.2.1-OF and in
hDhaA.v2.1-6F to remove 5' binding sequence matches in 3' flanking
region.
The remaining transcription factor binding sequence matches in
hDhaA.v2.1-6F included in the 5' flanking region: Family: V$NEUR
(NeuroD, Beta2, HLH domain), best match: DNA binding site for
NEUROD1 (BETA-2 /E47 dimer) (MEDLINE 9108015); in the open reading
frame: Family: V$GATA (GATA binding factors), best match:
GATA-binding factor 1 (MEDLINE 94085373), Family: V$PCAT (Promoter
CCAAT binding factors), best match: cellular and viral CCAAT box,
(MEDLINE 90230299), Family: V$RXRF (RXR heterodimer binding sites),
best match: Famesoid X-activated receptor (RXR/FXR dimer) (MEDLINE
11792716); and in the 3' flanking region: Family: V$HNF1 (Hepatic
Nuclear Factor 1), best match: Hepatic nuclear factor 1 (MEDLINE
95194383), Family: V$BRNF (Bm POU domain factors), best match: POU
transcription factor Bm-3 (MEDLINE 9111308), Family: V$RBIT
(Regulator of B-Cell IgH transcription), best match: Bright, B cell
regulator of IgH transcription (MEDLINE 96127903) Family: V$CREB
(Camp-Responsive Element Binding proteins), best match: E4BP4, bZIP
domain, transcriptional repressor (MEDLINE 92318924), Family:
V$HOMS (Homeodomain subfamily S8), best match: Binding site for S8
type homeodomains (MEDLINE 94051593), Family: V$NKXH
(NKX/DLX--Homeodomain sites), best match: DLX-1, -2, and -5 binding
sites (MEDLINE 11798166) Family: V$TBPF (Tata-Binding Protein
Factor), best match: Avian C-type LTR TATA box (MEDLINE 6322120),
and Family: V$NKXH (NKX/XDLX-Homeodomain sites), best match:
Prostate-specific homeodomain protein NKX3.1 (MEDLINE
10871372).
The other sequence motifs remaining in hDhaA.v2.1-6F in the open
reading frame were for an E. coli RBS (AAGG) 11 b upstream of a Met
codon which was not removed due to retain the protein sequence
(Lys-Gly: AA(A/G)-GGN), and a BsmAI restriction site (GTCTC) which
was not removed due to introduction of transcription factor binding
site sequences.
The putative CpG islands in the coding sequence for each of the
DhaA genes was analyzed as in EMBOSS CpGPlot/CpGReport with default
parameters, and the results are shown in Table VI.
TABLE-US-00012 TABLE VI Gene name CpG Islands > 100 bp Length bp
(location in ORF) DhaA 1 775 by (49 . . . 823) DhaA.v2.1 1 784 by
(49 . . . 832) hDhaA.v.2.1-0 0 -- hDhaA.v2.1-6 0 --
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All publications, patents and patent applications are incorporated
herein by reference. While in the foregoing specification this
invention has been described in relation to certain preferred
embodiments thereof, and many details have been set forth for
purposes of illustration, it will be apparent to those skilled in
the art that the invention is susceptible to additional embodiments
and that certain of the details described herein may be varied
considerably without departing from the basic principles of the
invention.
SEQUENCE LISTINGS
1
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 64 <210>
SEQ ID NO 1 <211> LENGTH: 31 <212> TYPE: DNA
<213> ORGANISM: Rhodococcus rhodochrous <400> SEQUENCE:
1 gcttcacttg tcgtcatcgt ccttgtagtc a 31 <210> SEQ ID NO 2
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
Rhodococcus rhodochrous <400> SEQUENCE: 2 gcttcacttg
tcgtcatcgt ccttgtagtc a 31 <210> SEQ ID NO 3 <211>
LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: A
synthetic primer <400> SEQUENCE: 3 ccgggattgt tctacctcca
ggaagac 27 <210> SEQ ID NO 4 <211> LENGTH: 27
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
primer <400> SEQUENCE: 4 ccgggattgg cctacctcca ggaagac 27
<210> SEQ ID NO 5 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 5 ccgggattgc agtacctcca ggaagac 27 <210> SEQ ID NO
6 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 6
ccgggattgg gctacctcca ggaagac 27 <210> SEQ ID NO 7
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic primer <400> SEQUENCE: 7 acgcgtcgac
gccgccatgt cagaaatcgg tacaggc 37 <210> SEQ ID NO 8
<211> LENGTH: 55 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic primer <400> SEQUENCE: 8 ataagaatgc
ggccgctcaa gcgcttcaac cggtgagtgc ggggagccag cgcgc 55 <210>
SEQ ID NO 9 <211> LENGTH: 35 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic oligonucleotide
<400> SEQUENCE: 9 ccggtgacta caaggacgat gacgacaagt gaagc 35
<210> SEQ ID NO 10 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic oligonucleotide
<400> SEQUENCE: 10 gcttcacttg tcgtcatcgt ccttgtagtc a 31
<210> SEQ ID NO 11 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 11 gcttcacttg tcgtcatcgt ccttgtagtc a 31 <210> SEQ
ID NO 12 <211> LENGTH: 31 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 12
gcttcacttg tcgtcatcgt ccttgtagtc a 31 <210> SEQ ID NO 13
<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic primer <400> SEQUENCE: 13 cttgggtttg
gaagaggtcg tcctggtcat ccactgctgg ggc 43 <210> SEQ ID NO 14
<211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic primer <400> SEQUENCE: 14 tgagccccag
cagtggatga ccaggacgac ctcttccaaa cc 42 <210> SEQ ID NO 15
<211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic primer <400> SEQUENCE: 15 ggaatgggcc
ctctagagcg acgatgtca 29 <210> SEQ ID NO 16 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: A
synthetic primer <400> SEQUENCE: 16 cagtcagtca cgatggatcc
gctcaa 26 <210> SEQ ID NO 17 <400> SEQUENCE: 17 000
<210> SEQ ID NO 18 <400> SEQUENCE: 18 000 <210>
SEQ ID NO 19 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic affinity molecule
<400> SEQUENCE: 19 His His His His His 1 5 <210> SEQ ID
NO 20 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: A synthetic affinity molecule <400>
SEQUENCE: 20 His His His His His His 1 5 <210> SEQ ID NO 21
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic affinity molecule <400> SEQUENCE: 21
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> SEQ ID
NO 22 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic affinity molecule
<400> SEQUENCE: 22 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5
<210> SEQ ID NO 23 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic affinity molecule
<400> SEQUENCE: 23 Trp Ser His Pro Gln Phe Glu Lys 1 5
<210> SEQ ID NO 24 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic affinity molecule
<400> SEQUENCE: 24 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5
<210> SEQ ID NO 25 <400> SEQUENCE: 25 000 <210>
SEQ ID NO 26 <211> LENGTH: 45 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic oligonucleotide
<400> SEQUENCE: 26 atcgaaggtc gtgggatccc caggaattcc
cgggtcgacg ccgcc 45 <210> SEQ ID NO 27 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
peptide <400> SEQUENCE: 27 Ile Glu Gly Arg Gly Ile Pro Arg
Asn Ser Arg Val Asp Ala Ala 1 5 10 15 <210> SEQ ID NO 28
<211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic oligonucleotide <400> SEQUENCE: 28
tccggatcaa gcttgggcga cgaggtggac ggcgggccct ctagagccac c 51
<210> SEQ ID NO 29 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic peptide <400>
SEQUENCE: 29 Ser Gly Ser Ser Leu Gly Asp Glu Val Asp Gly Gly Pro
Ser Arg Ala 1 5 10 15 Thr <210> SEQ ID NO 30 <211>
LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: A
synthetic oligonucleotide <400> SEQUENCE: 30 accggttccg
gatcaagctt gcggtaccgc gggccctcta gagcc 45 <210> SEQ ID NO 31
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic peptide <400> SEQUENCE: 31 Thr Gly
Ser Gly Ser Ser Leu Arg Tyr Arg Gly Pro Ser Arg Ala 1 5 10 15
<210> SEQ ID NO 32 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic oligonucleotide
<400> SEQUENCE: 32 tccggatcaa gcttgcggta ccgcgggccc
tctagagccg tcgacgccgc c 51 <210> SEQ ID NO 33 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: A
synthetic peptide <400> SEQUENCE: 33 Ser Gly Ser Ser Leu Arg
Tyr Arg Gly Pro Ser Arg Ala Val Asp Ala 1 5 10 15 Ala <210>
SEQ ID NO 34 <211> LENGTH: 43 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic oligonucleotide
<400> SEQUENCE: 34 cttgggtttg gaagaggtcg tcctggtcat
ccaccagtgg ggc 43 <210> SEQ ID NO 35 <211> LENGTH: 42
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
oligonucleotide <400> SEQUENCE: 35 tgagccccac tggtggatga
ccaggacgac ctcttccaaa cc 42 <210> SEQ ID NO 36 <211>
LENGTH: 1053 <212> TYPE: DNA <213> ORGANISM:
Staphylococcus aureus <400> SEQUENCE: 36 agcttactat
gccattatta ataacttagc catttcaaca ccttctttca aatatttata 60
ataaactatt gacaccgata ttacaattgt aatattattg atttataaaa attacaactg
120 taatatcgga gggtttattt tgaaaaagtt aatattttta attgtaattg
ctttagtttt 180 aagtgcatgt aattcaaaca gttcacatgc caaagagtta
aatgatttag aaaaaaaata 240 taatgctcat attggtgttt atgctttaga
tactaaaagt ggtaaggaag taaaatttaa 300 ttcagataag agatttgcct
atgcttcaac ttcaaaagcg ataaatagtg ctattttgtt 360 agaacaagta
ccttataata agttaaataa aaaagtacat attaacaaag atgatatagt 420
tgcttattct cctattttag aaaaatatgt aggaaaagat atcactttaa aagcacttat
480 tgaggcttca atgacatata gtgataatac agcaaacaat aaaattataa
aagaaatcgg 540 tggaatcaaa aaagttaaac aacgtctaaa agaactagga
gataaagtaa caaatccagt 600 tagatatgag atagaattaa attactattc
accaaagagc aaaaaagata cttcaacacc 660 tgctgccttc ggtaagaccc
ttaataaact tatcgccaat ggaaaattaa gcaaagaaaa 720 caaaaaattc
ttacttgatt taatgttaaa taataaaagc ggagatactt taattaaaga 780
cggtgttcca aaagactata aggttgctga taaaagtggt caagcaataa catatgcttc
840 tagaaatgat gttgcttttg tttatcctaa gggccaatct gaacctattg
ttttagtcat 900 ttttacgaat aaagacaata aaagtgataa gccaaatgat
aagttgataa gtgaaaccgc 960 caagagtgta atgaaggaat tttaatattc
taaatgcata ataaatactg ataacatctt 1020 atattttgta ttatattttg
tattatcgtt gac 1053 <210> SEQ ID NO 37 <211> LENGTH: 81
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
oligonucleotide <400> SEQUENCE: 37 ccggtgatcc aaaaaagaag
agaaaggtag atccaaaaaa gaagagaaag gtagatccaa 60 aaaagaagag
aaaggtatga g 81 <210> SEQ ID NO 38 <211> LENGTH: 81
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
oligonucleotide <400> SEQUENCE: 38 gatcctcata cctttctctt
cttttttgga tctacctttc tcttcttttt tggatctacc 60 tttctcttct
tttttggatc a 81 <210> SEQ ID NO 39 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
oligonucleotide <400> SEQUENCE: 39
attatgctga gtgatatccc 20 <210> SEQ ID NO 40 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: A
synthetic oligonucleotide <400> SEQUENCE: 40 ctcggtacca
agctccttgt agtca 25 <210> SEQ ID NO 41 <211> LENGTH: 40
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
oligonucleotide <400> SEQUENCE: 41 caaaggtatt gcatgtatgc
agttcatccg gcctatcccg 40 <210> SEQ ID NO 42 <211>
LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: A
synthetic oligonucleotide <400> SEQUENCE: 42 gtcaaaggta
ttgcatgtat gctgttcatc cggcctatcc cgac 44 <210> SEQ ID NO 43
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic oligonucleotide <400> SEQUENCE: 43
aggtattgca tgtatggcgt tcatccggcc tatccc 36 <210> SEQ ID NO 44
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic primer <400> SEQUENCE: 44 gggctggcaa
gccacgtttg gtg 23 <210> SEQ ID NO 45 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
improved Kozak sequence <400> SEQUENCE: 45 gccaccatgg 10
<210> SEQ ID NO 46 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: 1-36 <223> OTHER INFORMATION: n = A, T, G, or C
<400> SEQUENCE: 46 nnnngctagc cagctggcga tatcgccacc atggga 36
<210> SEQ ID NO 47 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: 1-34 <223> OTHER INFORMATION: n = A, T, G, or C
<400> SEQUENCE: 47 taatagttaa ttaagtaagc ggccgcnnnn 30
<210> SEQ ID NO 48 <211> LENGTH: 296 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic peptide <400>
SEQUENCE: 48 Met Gly Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro
His Tyr Val 1 5 10 15 Glu Val Leu Gly Glu Arg Met His Tyr Val Asp
Val Gly Pro Arg Asp 20 25 30 Gly Thr Pro Val Leu Phe Leu His Gly
Asn Pro Thr Ser Ser Tyr Leu 35 40 45 Trp Arg Asn Ile Ile Pro His
Val Ala Pro Ser His Arg Cys Ile Ala 50 55 60 Pro Asp Leu Ile Gly
Met Gly Lys Ser Asp Lys Pro Asp Leu Asp Tyr 65 70 75 80 Phe Phe Asp
Asp His Val Arg Tyr Leu Asp Ala Phe Ile Glu Ala Leu 85 90 95 Gly
Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu 100 105
110 Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala
115 120 125 Cys Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp
Pro Glu 130 135 140 Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Ala
Asp Val Gly Arg 145 150 155 160 Glu Leu Ile Ile Asp Gln Asn Ala Phe
Ile Glu Gly Ala Leu Pro Lys 165 170 175 Cys Val Val Arg Pro Leu Thr
Glu Val Glu Met Asp His Tyr Arg Glu 180 185 190 Pro Phe Leu Lys Pro
Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn 195 200 205 Glu Leu Pro
Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu 210 215 220 Ala
Tyr Met Asn Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe 225 230
235 240 Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg
Leu 245 250 255 Ala Glu Ser Leu Pro Asn Cys Lys Thr Val Asp Ile Gly
Pro Gly Leu 260 265 270 Phe Tyr Leu Gln Glu Asp Asn Pro Asp Leu Ile
Gly Ser Glu Ile Ala 275 280 285 Arg Trp Leu Pro Gly Leu Ala Gly 290
295 <210> SEQ ID NO 49 <211> LENGTH: 948 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: A synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: 1-948 <223> OTHER INFORMATION: n = A, C, T, or G
<400> SEQUENCE: 49 nnnngctagc cagctggcga tatcgccacc
atgggatccg agattgggac agggtttcct 60 tttgatcctc attatgtgga
ggtgctgggg gagagaatgc attatgtgga tgtggggcct 120 agagatggga
cacctgtgct gtttctgcat gggaatccta catcttctta tctgtggaga 180
aatattattc ctcatgtggc tccttctcat agatgtattg ctcctgatct gattgggatg
240 gggaagtctg ataagcctga tctggattat ttttttgatg atcatgtgag
atatctggat 300 gcttttattg aggctctggg gctggaggag gtggtgctgg
tgattcatga ttgggggtct 360 gctctggggt ttcattgggc taagagaaat
cctgagagag tgaaggggat tgcttgtatg 420 gagtttatta gacctattcc
tacatgggat gagtggcctg agtttgctag agagacattt 480 caggctttta
gaacagctga tgtggggaga gagctgatta ttgatcagaa tgcttttatt 540
gagggggctc tgcctaagtg tgtggtgaga cctctgacag aggtggagat ggatcattat
600 agagagcctt ttctgaagcc tgtggataga gagcctctgt ggagatttcc
taatgagctg 660 cctattgctg gggagcctgc taatattgtg gctctggtgg
aggcttatat gaattggctg 720 catcagtctc ctgtgcctaa gctgctgttt
tgggggacac ctggggtgct gattcctcct 780 gctgaggctg ctagactggc
tgagtctctg cctaattgta agacagtgga tattgggcct 840 gggctgtttt
atctgcagga ggataatcct gatctgattg ggtctgagat tgctagatgg 900
ctgcccgggc tggccggcta atagttaatt aagtaagcgg ccgcnnnn 948
<210> SEQ ID NO 50 <211> LENGTH: 951 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: 1-951 <223> OTHER INFORMATION: n = A, T, G, or C
<400> SEQUENCE: 50 nnnngctagc cagctggcgc ggatatcgcc
accatgggat ccgagattgg gacagggttc 60 ccttttgatc ctcactatgt
tgaagtgctg ggggaaagaa tgcactacgt ggatgtgggg 120 cctagagatg
ggaccccagt gctgttcctc cacgggaacc ctacatctag ctacctgtgg 180
agaaatatta tacctcatgt tgctcctagt cataggtgca ttgctcctga tctgatcggg
240 atggggaagt ctgataagcc tgacttagac tacttttttg atgatcatgt
tcgatacttg 300 gatgctttca ttgaggctct ggggctggag gaggtggtgc
tggtgataca cgactggggg 360 tctgctctgg ggtttcactg ggctaaaagg
aatccggaga gagtgaaggg gattgcttgc 420 atggagttta ttcgacctat
tcctacttgg gatgaatggc cagagtttgc cagagagaca 480 tttcaagcct
ttagaactgc cgatgtgggc agggagctga ttatagacca gaatgctttc 540
atcgaggggg ctctgcctaa atgtgtagtc agacctctca ctgaagtaga gatggaccat
600 tatagagagc cctttctgaa gcctgtggat cgcgagcctc tgtggaggtt
tccaaatgag 660 ctgcctattg ctggggagcc tgctaatatt gtggctctgg
tggaagccta tatgaactgg 720 ctgcatcaga gtccagtgcc caagctactc
ttttggggga ctccgggagt tctgattcct 780 cctgccgagg ctgctagact
ggctgaatcc ctgcccaatt gtaagaccgt ggacatcggc 840 cctgggctgt
tttacctcca agaggacaac cctgatctca tcgggtctga gatcgcacgg 900
tggctgcccg ggctggccgg ctaatagtta attaagtagg cggccgcnnn n 951
<210> SEQ ID NO 51 <211> LENGTH: 882 <212> TYPE:
DNA <213> ORGANISM: Rhodococcus rhodochrous <400>
SEQUENCE: 51 atgtcagaaa tcggtacagg cttccccttc gacccccatt atgtggaagt
cctgggcgag 60 cgtatgcact acgtcgatgt tggaccgcgg gatggcacgc
ctgtgctgtt cctgcacggt 120 aacccgacct cgtcctacct gtggcgcaac
atcatcccgc atgtagcacc gagtcatcgg 180 tgcattgctc cagacctgat
cgggatggga aaatcggaca aaccagacct cgattatttc 240 ttcgacgacc
acgtccgcta cctcgatgcc ttcatcgaag ccttgggttt ggaagaggtc 300
gtcctggtca tccacgactg gggctcagct ctcggattcc actgggccaa gcgcaatccg
360 gaacgggtca aaggtattgc atgtatggaa ttcatccggc ctatcccgac
gtgggacgaa 420 tggccggaat tcgcccgtga gaccttccag gccttccgga
ccgccgacgt cggccgagag 480 ttgatcatcg atcagaacgc tttcatcgag
ggtgcgctcc cgaaatgcgt cgtccgtccg 540 cttacggagg tcgagatgga
ccactatcgc gagcccttcc tcaagcctgt tgaccgagag 600 ccactgtggc
gattccccaa cgagctgccc atcgccggtg agcccgcgaa catcgtcgcg 660
ctcgtcgagg catacatgaa ctggctgcac cagtcacctg tcccgaagtt gttgttctgg
720 ggcacacccg gcgtactgat ccccccggcc gaagccgcga gacttgccga
aagcctcccc 780 aactgcaaga cagtggacat cggcccggga ttgcactacc
tccaggaaga caacccggac 840 cttatcggca gtgagatcgc gcgctggctc
cccgcactct ag 882 <210> SEQ ID NO 52 <211> LENGTH: 43
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
oligonucleotide <400> SEQUENCE: 52 cttgggtttg gaagaggtcg
tcctggtcat ccacgaatgg ggc 43 <210> SEQ ID NO 53 <211>
LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: A
synthetic oligonucleotide <400> SEQUENCE: 53 tgagccccat
tcgtggatga ccaggacgac ctcttccaaa cc 42 <210> SEQ ID NO 54
<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic oligonucleotide <400> SEQUENCE: 54
cttgggtttg gaagaggtcg tcctggtcat ccactactgg ggc 43 <210> SEQ
ID NO 55 <211> LENGTH: 42 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: A synthetic oligonucleotide <400>
SEQUENCE: 55 tgagccccag tagtggatga ccaggacgac ctcttccaaa cc 42
<210> SEQ ID NO 56 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 56 ccagttagat atgacataga attaaattac tattcacc 38
<210> SEQ ID NO 57 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 57 ggtgaatagt aatttaattc tatgtcatat ctaactgg 38
<210> SEQ ID NO 58 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 58 ccagttagat atgagataga attacagtac tattcacc 38
<210> SEQ ID NO 59 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 59 ggtgaatagt actgtaattc tatctcatat ctaactgg 38
<210> SEQ ID NO 60 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 60 ccagttagat atgacataga attacagtac tattcacc 38
<210> SEQ ID NO 61 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 61 ggtgaatagt actgtaattc tatgtcatat ctaactgg 38
<210> SEQ ID NO 62 <211> LENGTH: 40 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 62 caacaggtcg acgccgccat gaaagagtta aatgatttag 40
<210> SEQ ID NO 63 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 63 gtagtcaccg gtaaattcct tcattacact cttggc 36 <210>
SEQ ID NO 64 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic peptide <400>
SEQUENCE: 64 Asp Glu Val Asp 1
* * * * *
References