U.S. patent application number 10/057505 was filed with the patent office on 2002-11-07 for tandem fluorescent protein constructs.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Cubitt, Andrew, Heim, Roger, Tsien, Roger Y..
Application Number | 20020164674 10/057505 |
Document ID | / |
Family ID | 24379467 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164674 |
Kind Code |
A1 |
Tsien, Roger Y. ; et
al. |
November 7, 2002 |
Tandem fluorescent protein constructs
Abstract
This invention provides tandem fluorescent protein construct
including a donor fluorescent protein moiety, an acceptor
fluorescent protein moiety and a linker moiety that couples the
donor and acceptor moieties. The donor and acceptor moieties
exhibit fluorescence resonance energy transfer which is eliminated
upon cleavage. The constructs are useful in enzymatic assays.
Inventors: |
Tsien, Roger Y.; (La Jolla,
CA) ; Heim, Roger; (Del Mar, CA) ; Cubitt,
Andrew; (San Diego, CA) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
|
Family ID: |
24379467 |
Appl. No.: |
10/057505 |
Filed: |
January 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10057505 |
Jan 25, 2002 |
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09396003 |
Sep 13, 1999 |
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09396003 |
Sep 13, 1999 |
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08792553 |
Jan 31, 1997 |
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5981200 |
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08792553 |
Jan 31, 1997 |
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08594575 |
Jan 31, 1996 |
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Current U.S.
Class: |
435/23 ;
530/350 |
Current CPC
Class: |
C07K 14/43595 20130101;
C12Q 1/485 20130101; C12Q 1/37 20130101; G01N 2333/43595 20130101;
G01N 33/542 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/23 ;
530/350 |
International
Class: |
C12Q 001/37; C07K
014/435 |
Claims
What is claimed is:
1. A tandem fluorescent protein construct comprising a donor
fluorescent protein moiety, an acceptor fluorescent protein moiety
and a linker moiety that couples the donor and acceptor moieties,
wherein the donor and acceptor moieties exhibit fluorescence
resonance energy transfer when the donor moiety is excited.
2. The construct of claim 1 wherein the donor moiety and acceptor
moiety are Aequorea-related fluorescent protein moieties.
3. The construct of claim 2 wherein the donor moiety is P4-3 or W7
and the acceptor moiety is S65C or S65T.
4. The construct of claim 1 wherein the linker moiety comprises a
cleavage recognition site for an enzyme.
5. The construct of claim 4 wherein the linker moiety is a peptide
moiety.
6. The construct of claim 5 comprising a fusion protein including
the donor moiety, the peptide moiety and the acceptor moiety in a
single polypeptide.
7. The construct of claim 6 wherein the linker moiety comprises
between about 5 amino acids and about 50 amino acids.
8. The construct of claim 7 wherein the linker moiety comprises
between about 10 amino acids and about 30 amino acids.
9. The construct of claim 8 wherein the donor moiety is P4-3 or W7
and the acceptor moiety is S65C or S65T.
10. The construct of claim 7 comprising a cleavage recognition site
for trypsin, enterokinase, HIV-1 protease, prohormone convertase,
interleukin-1b-converting enzyme, adenovirus endopeptidase,
cytomegalovirus assemblin, leishmanolysin, b-Secretase for APP,
thrombin, renin, angiotensin-converting enzyme, cathepsin D or a
kininogenase.
11. The construct of claim 6 wherein the donor moiety is positioned
at the amino terminus of the polypeptide relative to the acceptor
moiety.
12. The construct of claim 7 wherein the linker moiety comprises a
cleavage site having a randomized amino acid sequence.
13. The construct of claim 1 wherein the linker moiety has a length
between about 1 nm and about 10 nm.
14. The construct of claim 4 comprising a cleavage recognition site
for b-lactamase.
15. The construct of claim 1 wherein the linker moiety comprises a
cross-linker moiety.
16. A recombinant nucleic acid coding for expression of a tandem
fluorescent protein construct, the construct comprising a donor
fluorescent protein moiety, an acceptor fluorescent protein moiety
and a peptide linker moiety in a single polypeptide, wherein the
donor and acceptor moieties exhibit fluorescence resonance energy
transfer when the donor moiety is excited.
17. The recombinant nucleic acid of claim 16 wherein the peptide
linker moiety comprises a cleavage recognition site for a
protease.
18. The recombinant nucleic acid of claim 17 wherein the donor
moiety is selected from the group comprising W1B, Topaz, P4-3 and
W7 and the acceptor moiety is selected from the group comprising
Topaz, Emerald, S65C and S65T.
19. An expression vector comprising expression control sequences
operatively linked to a sequence coding for the expression of a
tandem fluorescent protein construct, the construct comprising a
donor fluorescent protein moiety, an acceptor fluorescent protein
moiety and a peptide linker moiety in a single peptide, wherein the
donor and acceptor moieties exhibit fluorescence resonance energy
transfer when the donor moiety is excited.
20. An expression vector of claim 19 adapted for function in a
prokaryotic cell.
21. An expression vector of claim 19 adapted for function in a
eukaryotic cell.
22. A host cell transfected with an expression vector comprising an
expression control sequence operatively linked to a sequence coding
for the expression of a tandem fluorescent protein construct, the
construct comprising a donor fluorescent protein moiety, an
acceptor fluorescent protein moiety and a peptide linker moiety in
a single polypeptide, wherein the donor and acceptor moieties
exhibit fluorescence resonance energy transfer when the donor
moiety is excited.
23. The cell of claim 22 further comprising a protease that is not
normally expressed by said cell.
24. The cell of claim 22 that is E. coli.
25. The cell of claim 22 that is a eukaryotic cell.
26. The cell of claim 22 that is a cultured mammalian cell.
27. A method for determining whether a sample contains an enzyme
comprising: contacting the sample with a tandem fluorescent protein
construct which comprises a donor fluorescent protein moiety, an
acceptor fluorescent protein moiety and a linker moiety that
couples the donor and acceptor moieties and that comprises a
cleavage recognition site specific for the enzyme, wherein the
donor and acceptor moieties exhibit fluorescence resonance energy
transfer when the donor moiety is excited; exciting the donor
moiety; and determining the degree of fluorescence resonance energy
transfer in the sample, whereby a degree of fluorescence resonance
energy transfer that is lower than an expected amount indicates the
presence of an enzyme.
28. The method of claim 27 for determining the amount of an enzyme
in a sample wherein determining the degree of fluorescence
resonance energy transfer in the sample comprises determining the
degree at a first and second time after contacting the sample with
a tandem fluorescent protein construct, and determining the
difference in the degree of fluorescence resonance energy transfer,
whereby the difference in the degree of fluorescence resonance
energy transfer reflects the amount of enzyme in the sample.
29. The method of claim 27 wherein the step of determining the
degree of fluorescence resonance energy transfer in the sample
comprises determining the amount of fluorescence from the donor
moiety.
30. The method of claim 27 wherein the step of determining the
degree of fluorescence resonance energy transfer in the sample
comprises determining the amount of fluorescence from the acceptor
donor moiety.
31. The method of claim 27 wherein the step of determining the
degree of fluorescence resonance energy transfer in the sample
comprises determining the ratio of the amount of fluorescence from
the donor moiety and the amount of fluorescence from the acceptor
moiety.
32. The method of claim 27 wherein the step of determining the
degree of fluorescence resonance energy transfer in the sample
comprises determining the excitation state lifetime of the donor
moiety.
33. The method of claim 27 wherein the enzyme is a protease and the
linker moiety is a peptide moiety having the cleavage recognition
site.
34. The method of claim 33 wherein the donor fluorescent protein
moiety is an Aequorea-related fluorescent protein.
35. The method of claim 34 wherein the donor moiety is P4-3 or W7
and the acceptor moiety is S6SC or S65T.
36. A method of determining the amount of activity of an enzyme in
a cell comprising the steps of: providing a cell that expresses a
tandem fluorescent protein construct, the construct comprising a
donor fluorescent protein moiety, an acceptor fluorescent protein
moiety and a peptide linker moiety, wherein the peptide linker
moiety comprises a cleavage recognition amino acid sequence
specific for the enzyme, and wherein the donor and acceptor
moieties exhibit fluorescence resonance energy transfer when the
donor moiety is excited; exciting the donor moiety; and determining
the degree of fluorescence resonance energy transfer in the cell,
whereby the degree of fluorescence resonance energy transfer
relates the amount of enzyme activity in the cell.
37. The method of claim 36 wherein the cell is transfected with an
expression vector comprising expression control sequences operably
linked to a nucleic acid sequence coding for the expression of the
enzyme.
38. The method of claim 37 wherein the donor fluorescent protein
moiety is an Aequorea-related fluorescent protein.
39. The method of claim 38 wherein the donor moiety is P4-3 or W7
and the acceptor moiety is S65C or S65T.
40. The method of claim 36 wherein the step of providing a cell
comprises inducing expression of the construct to produce a sudden
increase in the expression of the construct, and the step of
determining the degree of fluorescence resonance energy transfer
comprises determining the degree at a first and a second time after
expression of the construct and determining the difference between
the first and second time, whereby the difference reflects the
amount of enzyme.
41. A method of determining the amount of activity of an enzyme in
a sample from an organism comprising the steps of: providing a
sample from an organism having a cell that expresses a tandem
fluorescent protein construct, the construct comprising a donor
fluorescent protein moiety, an acceptor fluorescent protein moiety
and a peptide linker moiety, wherein the peptide linker moiety
comprises a cleavage recognition amino acid sequence specific for
the enzyme, and wherein the donor and acceptor moieties exhibit
fluorescence resonance energy transfer when the donor moiety is
excited; exciting the donor moiety; and determining the degree of
fluorescence resonance energy transfer in the sample, whereby the
degree of fluorescence resonance energy transfer reflects the
amount of enzyme activity in the cell.
42. A method for determining whether a compound alters the activity
of an enzyme comprising the steps of: contacting a sample
containing a known amount of the enzyme with the compound and with
a tandem fluorescent protein construct which comprises a donor
fluorescent protein moiety, an acceptor fluorescent protein moiety
and a linker moiety that couples the donor and acceptor moieties
and that comprises a cleavage recognition site specific for the
enzyme, wherein the donor and acceptor moieties exhibit
fluorescence resonance energy transfer when the donor moiety is
excited; exciting the donor moiety; and determining the amount of
enzyme activity in the sample as a function of the degree of
fluorescence resonance energy transfer in the sample.
43. The method of claim 42 wherein the enzyme is a protease and the
compound is at a predetermined concentration of at least 1
.mu.M.
44. The method of claim 42 further comprising the step of comparing
the amount of activity in the sample with a standard activity for
the same amount of the enzyme, whereby a difference between the
amount of enzyme activity in the sample and the standard activity
indicates that the compound alters the activity of the enzyme.
45. A method for determining whether a compound alters the activity
of an enzyme in a cell comprising the steps of: providing first and
second cells that express a tandem fluorescent protein construct,
the construct comprising a donor fluorescent protein moiety, an
acceptor fluorescent protein moiety and a peptide linker moiety,
wherein the peptide linker moiety comprises a cleavage recognition
amino acid sequence specific for the enzyme, and wherein the donor
and acceptor moieties exhibit fluorescence resonance energy
transfer when the donor moiety is excited; contacting the first
cell with an amount of the compound; contacting the second cell
with a different amount of the compound; exciting the donor moiety
in the first and second cell; determining the degree of
fluorescence resonance energy transfer in the first and second
cells; and comparing the degree of fluorescence resonance energy
transfer in the first and second cells, whereby a difference in the
degree of fluorescence resonance energy transfer indicates that the
compound alters the activity of the enzyme.
46. A tandem fluorescent protein construct comprising a donor
moiety, an acceptor moiety and a linker moiety that couples a donor
and acceptor moiety, wherein one of the donor or acceptor moieties
is a fluorescent protein and one is a non-protein compound
fluorescent moiety, and wherein the donor and acceptor moieties
exhibit fluorescence resonance energy transfer when the donor
moiety is excited.
47. The construct of claim 46 wherein the fluorescent protein
moiety is an Aequorea-related florescent protein moiety.
48. A method of testing for cleavage enzyme activity comprising:
contacting a cleavage enzyme with a tandem fluorescent protein
construct which comprises a donor fluorescent protein moiety, an
acceptor fluorescent protein moiety and a linker moiety that
couples the donor and acceptor moieties and that comprises at least
one cleavage recognition site, wherein the donor and acceptor
moieties exhibit fluorescence resonance energy transfer when the
donor moiety is excited; exciting the donor moiety; and determining
the presence of fluorescence resonance energy transfer between the
donor and the acceptor moieties, wherein a decrease in fluorescence
resonance energy transfer indicates the presence of a cleavage
recognition site that is cleaved by the cleavage enzyme.
49. The method of claim 48 wherein the cleavage enzyme is a
protease.
50. The method of claim 49 wherein the cleavage enzyme is an orphan
protease.
51. The method of claim 50 wherein the cleavage recognition site
has a random amino acid sequence.
52. The method of claim 51 wherein the cleavage enzyme is contacted
with the tandem fluorescent protein construct by expressing the
cleavage enzyme and the tandem fluorescent-protein construct in a
cell.
52. The method of claim 51 wherein the cleavage enzyme is expressed
using an inducable promoter and optionally exposing the cell to an
inducer of the inducable promoter for less than two hours, wherein
the cleavage enzyme is transiently expressed.
53. The method of claim 52 wherein the cleavage enzyme and the
tandem fluorescent protein construct have a signal sequence
directing expression of protein into a vesicle.
54. The method of claim 51 wherein the cell is part of a library of
individual clones, wherein different said clones have been
transfected with tandem fluorescent protein constructs having
different cleavage recognition sites.
55. The method of claim 54 further comprising selecting clones from
said library that have cleavage recognition sites cleaved by
proteases.
56. The method of claim 55 wherein the selecting of the clones
comprises identifying the clones with a Fluorescent Activated Cell
Sorter (FACS) or luminescent assay based sorter.
Description
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/594575, filed Jan. 31, 1996.
BACKGROUND OF THE INVENTION
[0002] Proteases play essential roles in many disease processes
such as Alzheimer's, hypertension, inflammation, apoptosis, and
AIDS. Compounds that block or enhance their activity have potential
as therapeutic agents. Because the normal substrates of peptidases
are linear peptides and because established procedures exist for
making non-peptidic analogs, compounds that effect the activity of
proteases are natural subjects of combinatorial chemistry.
Screening compounds produced by combinatorial chemistry requires
convenient enzymatic assays.
[0003] The most convenient existing assays for proteases are based
on fluorescence resonance energy transfer from a donor fluorophore
to a quencher placed at opposite ends of a short peptide chain
containing the potential cleavage site. Knight CG, "Fluorimetric
assays of proteolytic enzymes," Methods in Enzymol. (1995)
248:18-34. Proteolysis separates the fluorophore and quencher,
resulting in increased intensity in the emission of the donor
fluorophore. Existing protease assays use short peptide substrates
incorporating unnatural chromophoric amino acids, assembled by
solid phase peptide synthesis. However, solid phase synthesis poses
certain problems of effort and expense.
[0004] It is useful to perform enzymatic assays in vivo, in order
to more closely mimic conditions in which intracellular proteases
act. Conventional artificial substrates prepared by solid-phase
synthesis would require microinjection into individual cells, which
is impractical as a high-throughput screen. Also, short unfolded
peptides are generally rapidly degraded by nonspecific mechanisms
inside cells.
[0005] The Edans fluorophore is the current mainstay of existing
fluorometric assays. Fluorophores with greater extinction
coefficients and quantum yields are desirable. The Edans
fluorophore often is coupled with a non-fluorescent quencher such
as Dabcyl. However, assays performed with such agents rely on the
absolute measurement of fluorescence from the donor. This amount is
contaminated by other factors including turbidity or background
absorbances of the sample, fluctuations in the excitation
intensity, and variations in the absolute amount of substrate.
SUMMARY OF THE INVENTION
[0006] This invention provides tandem fluorescent protein
constructs and methods for using them in enzymatic assays both in
vitro and in vivo. Tandem fluorescent protein constructs comprise a
donor fluorescent protein moiety, an acceptor fluorescent protein
moiety and a linker moiety that couples the donor and acceptor
moieties, wherein the donor and acceptor moieties exhibit
fluorescence resonance energy transfer when the donor moiety is
excited. The fluorescent protein moieties can be Aequorea-related
fluorescent protein moieties, such as green fluorescent protein and
blue fluorescent protein. In one aspect, the linker moiety
comprises a cleavage recognition site for an enzyme, and is,
preferably, a peptide of between 5 and 50 amino acids. In one
embodiment, the construct is a fusion protein in which the donor
moiety, the peptide moiety and the acceptor moiety are part of a
single polypeptide.
[0007] This invention also provides recombinant nucleic acids
coding for expression of tandem fluorescent protein constructs in
which a donor fluorescent protein moiety, an acceptor fluorescent
protein moiety and a peptide linker moiety are encoded in a single
polypeptide. The invention also provides expression vectors
comprising expression control sequences operatively linked to a
recombinant nucleic acid coding for the expression of a tandem
fluorescent protein construct, as well as host cells transfected
with those expression vectors.
[0008] The tandem constructs of this invention are useful in assays
for determining whether a sample contains an enzyme. The methods
involve contacting the sample with a tandem fluorescent protein
construct. The donor moiety is excited. Then the degree of
fluorescence resonance energy transfer in the sample is determined.
A degree of fluorescence resonance energy transfer that is lower
than an expected amount indicates the presence of an enzyme. The
degree of fluorescence resonance energy transfer in the sample can
be determined as a function of the amount of fluorescence from the
donor moiety, the amount of fluorescence from the acceptor donor
moiety, the ratio of the amount of fluorescence from the donor
moiety to the amount of fluorescence from the acceptor moiety or
the excitation state lifetime of the donor moiety.
[0009] The assay also is useful for determining the amount of
enzyme in a sample by determining the degree of fluorescence
resonance energy transfer at a first and second time after contact
between the enzyme and the tandem construct, and determining the
difference in the degree of fluorescence resonance energy transfer.
The difference in the degree of fluorescence resonance energy
transfer reflects the amount of enzyme in the sample.
[0010] The invention also provides methods for determining the
amount of activity of an enzyme in a cell. The methods involve
providing a cell that expresses a tandem fluorescent protein
construct, for example by transfecting the cell with an appropriate
expression vector. The cell is exposed to light in order to excite
the donor moiety. Then the degree of fluorescence resonance energy
transfer in the cell is determined. The degree of fluorescence
resonance energy transfer reflects to the amount of enzyme activity
in the cell.
[0011] Similarly, the invention provides methods of determining the
amount of activity of an enzyme in a sample from an organism. The
methods involve providing a sample from an organism having a cell
that expresses a tandem fluorescent protein construct. The donor
moiety in the sample is excited. Then the degree of fluorescence
resonance energy transfer in the sample is determined. The degree
of fluorescence resonance energy transfer reflects the amount of
enzyme activity in the cell.
[0012] The assay methods also can be used to determine whether a
compound alters the activity of an enzyme, i.e., screening assays.
The methods involve contacting a sample containing an amount of the
enzyme with the compound and with a tandem fluorescent protein
construct; exciting the donor moiety; determining the amount of
enzyme activity in the sample as a function of the degree of
fluorescence resonance energy transfer in the sample; and comparing
the amount of activity in the sample with a standard activity for
the same amount of the enzyme. A difference between the amount of
enzyme activity in the sample and the standard activity indicates
that the compound alters the activity of the enzyme.
[0013] Similar methods, are useful for determining whether a
compound alters the activity of an enzyme in a cell. The methods
involve providing first and second cells that express a tandem
fluorescent protein construct; contacting the first cell with an
amount of the compound; contacting the second cell with a different
amount of the compound; exciting the donor moiety in the first and
second cell; determining the degree of fluorescence resonance
energy transfer in the first and second cells; and comparing the
degree of fluorescence resonance energy transfer in the first and
second cells. A difference in the degree of fluorescence resonance
energy transfer indicates that the compound alters the activity of
the enzyme.
[0014] Assays of the invention are also useful for determining and
characterizing substrate cleavage sequences of proteases or for
identifying proteases, such as orphan proteases. In one embodiment
the method involves the replacement of a defined linker moiety
amino acid sequence with one that contains a randomized selection
of amino acids. A library of fluorescent protein moieties each
linked by a randomized linker moiety can be generated using
recombinant engineering techniques or synthetic chemistry
techniques. Screening the members of the library can be
accomplished by measuring a signal related to cleavage, such as
fluorescence energy transfer, after contacting the cleavage enzyme
with each of the library members of the tandem fluorescent protein
construct. A degree of fluorescence resonance energy transfer that
is lower than an expected amount indicates the presence of a linker
sequence that can be cleaved by the enzyme. The degree of
fluorescence resonance energy transfer in the sample can be
determined as a function of the amount of fluorescence from the
donor moiety, the amount of fluorescence from the acceptor donor
moiety, or the ratio of the amount of fluorescence from the donor
moiety to the amount of fluorescence from the acceptor moiety or
the excitation state lifetime of the donor moiety.
[0015] Libraries of fluorescent proteins can be expressed in cells
and used to characterize the recognition motif of proteases
expressed within cells, where the enzyme is in its native context.
This method provides the additional advantage of assessing the
specificity of any given linker sequence to cleavage by other
enzymes other than the target enzyme. The methods consist of the
generation of a library of recombinant host cells, each of which
expresses a tandem fluorescent protein construct linked through a
randomized candidate linker substrate. Each cell is expanded into a
clonal population that is genetically homogeneous and the degree of
energy transfer is measured from each clonal population.
Optionally, FRETS can be measured before and at least one specified
time after a known change in intracellular protease activity. A
change in the degree of fluorescence resonance energy transfer
demonstrates that the cell contains a tandem construct and linker
sequence that can be cleaved by the enzyme activity in the cell.
Such methods are particular suited to Fluorescent Activated Cell
Sorter (FACS) clonal selection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 depicts the nucleotide sequence and deduced amino
acid sequence of a wild-type Aequorea green fluorescent
protein.
[0017] FIG. 2 depicts a tandem construct of the invention involved
in FRET.
[0018] FIG. 3 depicts fluorescence emission spectra of a
composition containing a tandem S65C--linker--P4-3 fluorescent
protein construct excited at 368 nm after exposure to trypsin for
0, 2, 5, 10 and 47 minutes.
[0019] FIG. 4 depicts fluorescence emission spectra intensity of a
composition containing a tandem S65C--linker--P4-3 fluorescent
protein construct excited at 368 nm after exposure to calpain for
0, 2, 6 and 15 minutes.
[0020] FIG. 5 depicts fluorescence emission spectra of a
composition-containing a tandem S65C--linker--P4 fluorescent
protein construct excited at 368 nm after exposure to enterokinase
for 0, 2, 20 and 144 minutes.
[0021] FIG. 6 depicts fluorescence emission spectra of a
composition containing a tandem S65T--linker--W7 fluorescent
protein construct excited at 432 nm before and after exposure to
trypsin.
[0022] FIG. 7 depicts fluorescence emission spectra of a
composition containing a tandem P4-3--linker--W7 fluorescent
protein construct excited at 368 nm before and after exposure to
trypsin.
[0023] FIG. 8 depicts fluorescence emission spectra of a
composition containing a tandem W1B--linker--10c fluorescent
protein construct excited at 433 nm before and after exposure to
trypsin.
[0024] FIG. 9 depicts the time course of fluorescent ratio changes
upon cleavage of a composition containing the tandem WlB
--linker--10c fluorescent protein construct measured at different
protein concentrations after exposure to trypsin measured in a
fluorescent 96 well plate reader.
[0025] FIG. 10 depicts a method of generating fluorescent tandem
constructs separated by a randomized linker region for use in
identifying cleavage specificities or orphan proteases.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Definitions
[0027] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used herein and the laboratory
procedures in cell culture, molecular genetics, and nucleic acid
chemistry and hybridization described below are those well known
and commonly employed in the art. Standard techniques Are used for
recombinant nucleic acid methods, polynucleotide synthesis, and
microbial culture and transformation (e.g., electroporation,
lipofection). Generally, enzymatic reactions and purification steps
are performed according to the manufacturer's specifications. 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., which is incorporated herein by reference) which are
provided throughout this document. The nomenclature used herein and
the laboratory procedures in analytical chemistry, organic
synthetic chemistry, and pharmaceutical formulation described below
are those well known and commonly employed in the art. Standard
techniques are used for chemical syntheses, chemical analyses,
pharmaceutical formulation and delivery, and treatment of patients.
As employed throughout the disclosure, the following terms, unless
otherwise indicated, shall be understood to have the following
meanings:
[0028] "Moiety" refers to the radical of a molecule that is
attached to another moiety. Thus, a "fluorescent protein moiety" is
the radical of a fluorescent protein coupled to the linker moiety.
By the same token, the term "linker moiety" refers to the radical
of a molecular linker that is coupled to both the donor and
acceptor protein moieties.
[0029] "Fluorescent protein" refers to any protein capable of
fluorescence when excited with appropriate electromagnetic
radiation. This includes fluorescent proteins whose amino acid
sequences are either natural or engineered.
[0030] "Peptide" refers to a polymer in which the monomers are
amino acids and are joined together through amide bonds,
alternatively referred to as a polypeptide. When the amino acids
are a-amino acids, either the L-optical isomer or the D-optical
isomer may be used. Additionally, unnatural amino acids, for
example, b-alanine, phenylglycine and homoarginin are also meant to
be included. Commonly encountered amino acids which are not
gene-encoded may also be used in the present invention. All of the
amino acids used in the present invention may be either the D- or
L-isomer. The L-isomers are preferred. In addition, other
peptidomimetics are also useful in the linker moieties of the
present invention. For a general review see Spatola, A. F., in
Chemistry and Biochemistry of Amino Acids, Peptides and Proteins,
B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).
[0031] "Naturally-occurring" as used herein, as applied to an
object, refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0032] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0033] "Control sequence" refers to polynucleotide sequences which
are necessary to effect the expression of coding and non-coding
sequences to which they are ligated. The nature of such control
sequences differs depending upon the host organism; in prokaryotes,
such control sequences generally include promoter, ribosomal
binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include, at a minimum, components whose presence can
influence expression, and can also include additional components
whose presence is advantageous, for examples leader sequences and
fusion partner sequences.
[0034] "Polynucleotide" refers to a polymeric form of nucleotides
of at least 10 bases in length, either ribonucleotides or
deoxynucleotides or a modified form of either type of nucleotide.
The term includes single and double stranded forms of DNA.
[0035] "Modulation " refers to the capacity to either enhance or
inhibit a functional property of biological activity or process
(e.g., enzyme activity or receptor binding); such enhancement or
inhibition may be contingent on the occurrence of a specific event,
such as activation of a signal transduction pathway, and/or may be
manifest only in particular cell types.
[0036] The term "modulator" refers to a chemical compound
(naturally occurring or non-naturally occurring), such as a
biological macromolecule (e.g. nucleic acid, protein, non-peptide,
or organic molecule), or an extract made from biological materials
such as bacteria, plants, fungi, or animal (particularly mammalian)
cells or tissues. Modulators are evaluated for potential activity
as inhibitors or activators (directly or indirectly) of a
biological process or processes (e.g., agonist, partial antagonist,
partial agonist, antagonist, antineoplastic agents, cytotoxic
agents, inhibitors of neoplastic transformation or cell
proliferation, cell proliferation-promoting agents, and the like)
by inclusion in screening assays described herein. The activities
(or activity) of a modulator may be known, unknown or partial
known. Such modulators can be screened using the methods described
herein.
[0037] The term "test compound" refers to a compound to be tested
by one or more screening method(s) of the invention as a putative
modulator. Usually, various predetermined concentrations are used
for screening such as 0.01 uM, 0.1 uM, 1.0 uM, and 10.0 uM. Test
compound controls can include the measurement of a signal in the
absence of the test compound or comparison to a compound known to
modulate the target.
[0038] Introduction
[0039] It has been discovered that fluorescent proteins having the
proper emission and excitation spectra that are brought into
physically close proximity with one another can exhibit
fluorescence resonance energy transfer ("FRET"). This invention
takes advantage of that discovery to provide tandem fluorescent
protein constructs in which two fluorescent protein moieties
capable of exhibiting FRET are coupled through a linker to form a
tandem construct. The protein moieties are chosen such that the
excitation spectrum of one of the moieties (the acceptor moiety)
overlaps with the emission spectrum of the excited protein moiety
(the donor moiety). The donor moiety is excited by light of
appropriate intensity within the donor's excitation spectrum. The
donor then emits the absorbed energy as fluorescent light. The
fluorescent energy it produces is quenched by the acceptor
fluorescent protein moiety. FRET can be manifested as a reduction
in the intensity of the fluorescent signal from the donor,
reduction in the lifetime of its excited state, and re-emission of
fluorescent light at the longer wavelengths (lower energies)
characteristic of the acceptor. When the linker that connects the
donor and acceptor moieties is cleaved, the fluorescent proteins
physically separate, and FRET is diminished or eliminated. This has
also been described in U.S. patent application 08/594,575, filed
Jan. 31, 1996, which is herein incorporated by reference.
[0040] One can take advantage of the FRET exhibited by the tandem
fluorescent protein constructs of the invention in performing
enzymatic assays. An embodiment of this process is depicted in FIG.
2. A recombinant nucleic acid encodes a single polypeptide
including a poly-histidinyl tag, a blue fluorescent protein donor
moiety, a peptide linker moiety comprising a protease recognition
site and a green fluorescent protein acceptor moiety. The nucleic
acid can be expressed into a tandem fluorescent protein construct
of the invention. In this example, a tandem construct contains a
blue fluorescent protein (such as P4-3, TABLE I) as the donor
moiety and a green fluorescent protein (such as S65C, TABLE I) as
the acceptor moiety.
[0041] The construct is exposed to light at, for example, 368 nm, a
wavelength that is near the excitation maximum of P4-3. This
wavelength excites S65C only minimally. Upon excitation, some
portion of the energy absorbed by the blue fluorescent protein
moiety is transferred to the acceptor moiety through FRET. As a
result of this quenching, the blue fluorescent light emitted by the
blue fluorescent protein is less bright than would be expected if
the blue fluorescent protein existed in isolation. The acceptor
moiety (S65C) may re-emit the energy at longer wavelength, in this
case, green fluorescent light.
[0042] After cleavage of the linker moiety by an enzyme, the blue
and green fluorescent proteins physically separate and FRET is
lost. Over time, as increasing amounts of the tandem construct are
cleaved, the intensity of visible blue fluorescent light emitted by
the blue fluorescent protein increases, while the intensity of
visible green light emitted by the green fluorescent protein as a
result of FRET, decreases.
[0043] The tandem fluorescent protein constructs of this invention
are useful as substrates to study agents or conditions that cleave
the linker. In particular, this invention contemplates tandem
constructs in which the linker is a peptide moiety containing an
amino acid sequence that is a cleavage site for a protease of
interest. The amount of the protease in a sample is determined by
contacting the sample with a tandem fluorescent protein construct
and measuring changes in fluorescence of the donor moiety, the
acceptor moiety or the relative fluorescence of both. In one
embodiment, the tandem construct is a recombinant fusion protein
produced by expression of a nucleic acid that encodes a single
polypeptide containing the donor moiety, the peptide linker moiety
and the acceptor moiety. Fusion proteins can be used for, among
other things, monitoring the activity of a protease inside the cell
that expresses the recombinant tandem construct. The distance
between fluorescent proteins in the construct can be regulated
based on the length of the linking moiety. Therefore, tandem
constructs of this invention whose linker moieties do not include
cleavage sites also are useful as agents for studying FRET between
fluorescent proteins.
[0044] Advantages of tandem fluorescent protein constructs include
the greater extinction coefficient and quantum yield of many of
these proteins compared with those of the Edans fluorophore. Also,
the acceptor in a tandem construct is, itself, a fluorophore rather
than a non-fluorescent quencher like Dabcyl. Thus, the enzyme's
substrate (i.e., the tandem construct) and products (i.e., the
moieties after cleavage) are both fluorescent but with different
fluorescent characteristics.
[0045] In particular, the substrate and cleavage products exhibit
different ratios between the amount of light emitted by the donor
and acceptor moieties. Therefore, the ratio between the two
fluorescences measures the degree of conversion of substrate to
products, independent of the absolute amount of either, the optical
thickness of the sample, the brightness of the excitation lamp, the
sensitivity of the detector, etc. Furthermore, the Aequorea-related
fluorescent protein moieties tend to be protease resistant.
Therefore, they are likely to survive as fluorescent moieties even
after the linker moiety is cleaved.
[0046] II. Tandem Fluorescent Protein Constructs
[0047] The tandem fluorescent protein constructs of the invention
usually comprise three elements: a donor fluorescent protein
moiety, an acceptor fluorescent protein moiety and a linker moiety
that couples the donor and acceptor moieties. The donor fluorescent
protein moiety is capable of absorbing a photon and transferring
energy to another fluorescent moiety. The acceptor fluorescent
protein moiety is capable of absorbing energy and emitting a
photon. The linker moiety connects the donor fluorescent protein
moiety to the acceptor fluorescent protein moiety. In many
instances the linker moiety will covalently connect the donor
fluorescent protein moiety and the acceptor fluorescent protein
moiety. It is desirable, as described in greater detail herein, to
select a donor fluorescent protein moiety with an emission spectrum
that overlaps with the excitation spectrum of an acceptor
fluorescent protein moiety. In some embodiments of the invention
the overlap in emission and excitation spectra will facilitate
FRET, Such an overlap is not necessary, however, if intrinsic
fluorescence is measured instead of FRET. Any fluorescent protein
may be used in the invention, including proteins that have
fluoresce due intramolecular rearrangements or the addition of
cofactors that promote fluorescence.
[0048] For example, green fluorescent proteins ("GFPs") of
cnidarians, which act as their energy-transfer acceptors in
bioluminescence, can be used in the invention. A green fluorescent
protein, as used herein, is a protein that fluoresces green light,
and a blue fluorescent protein is a protein that fluoresces blue
light. GFPs have been isolated from the Pacific Northwest
jellyfish, Aequorea Victoria, the sea pansy, Renilla reniformis,
and Phialidium gregarium. W. W. Ward et al., Photochem. Photobiol.,
35:803-808 (1982); L. D. Levine et al., Comp. Biochem. Physiol.,
72B:77-85 (1982).
[0049] A variety of Aequorea-related GFPs having useful excitation
and emission spectra have been engineered by modifying the amino
acid sequence of a naturally occurring GFP from Aequorea victoria.
(D. C. Prasher et al., Gene, 111:229-233 (1992); R. Heim et al.,
Proc. Natl. Acad. Sci., USA, 91:12501-04 (1994); U.S patent
application 08/337,915, filed Nov. 10 1994; International
application PCT/US95/14692, filed Nov. 10, 1995; U.S. patent
application 08/706,408, filed Aug. 30, 1996.) As used herein, a
fluorescent protein is an Aequorea-related fluorescent protein if
any contiguous sequence of 150 amino acids of the fluorescent
protein has at least 85% sequence identity with an amino acid
sequence, either contiguous or non-contiguous, from the wild type
Aequorea green fluorescent protein of SEQ ID NO: 2. More
preferably, a fluorescent protein is an Aequorea-related
fluorescent protein if any contiguous sequence of 200 amino acids
of the fluorescent protein has at least 95% sequence identity with
an amino acid sequence, either contiguous or non-contiguous, from
the wild type Aequorea green fluorescent protein of SEQ ID NO: 2.
Similarly, the fluorescent protein may be related to Renilla or
Phialidium wild-type fluorescent proteins using the same
standards.
[0050] Aequorea-related fluorescent proteins include, for example,
wild-type (native) Aequorea victoria GFP, whose nucleotide (SEQ ID
NO: 1) and deduced amino acid (SEQ ID NO: 2) sequences are
presented in FIG. 1; and those Aequorea-related engineered versions
described in TABLE I. Several of these, i.e., P4, P4-3, W7 and W2
fluoresce at a distinctly shorter wavelength than wild type.
1TABLE I Emission Extinct. Coefficient Clone Mutation(s) Excitation
max (nm) max (nm) (M.sup.1cm.sup.-1) Quantum yield Wild type none
395 (475) 508 21,000 (7,150) 0.77 P4 Y66H 383 447 13,500 0.21 P4-3
Y66H; YI45F 381 445 14,000 0.38 W7 Y66W; N146I 433 (453) 475 (501)
18,000 (17,100) 0.67 M153T V163A N212K W2 Y66W; I123V 432 (453) 480
10,000 (9,600) 0.72 Y145H HI48R M153T V163A N212K S65T S65T 489 511
39,200 0.68 P4-1 S65T; M153A 504 (396) 514 14,500 (8,600) 0.53
K238E S65A S65A 471 504 S65C S65C 479 507 S65L S65L 484 510 Y66F
Y66F 360 442 Y66W Y66W 458 480 10c S65G; V68L 513 527 S72A; T203Y
W1B F64L; S65T 432 (453) 476 (503) Y66W; N146I M153T V163A N212K
Emerald S65T; S72A 487 508 N149K M153T I167T Sapphire S72A; Y145F
395 511 T203I
[0051] This invention contemplates the use of other fluorescent
proteins in tandem constructs. The cloning and expression of yellow
fluorescent protein from Vibrio fischeri strain Y-1 has been
described by T. O. Baldwin et al., Biochemistry (1990) 29:5509-15.
This protein requires flavins as fluorescent co-factors. The
cloning of Peridinin-chlorophyll a binding protein from the
dinoflagellate Symbiodinium sp. was described by B. J. Morris et
al., Plant Molecular Biology, (1994) 24:673:77. One useful aspect
of this protein is that it fluoresces in red. The cloning of
phycobiliproteins from marine cyanobacteria such as Synechococcus,
e.g., phycoerythrin and phycocyanin, is described in S. M. Wilbanks
et al., J. Biol. Chem. (1993) 268:1226-35. These proteins require
phycobilins as fluorescent co-factors, whose insertion into the
proteins involves auxiliary enzymes. The proteins fluoresce at
yellow to red wavelengths.
[0052] For FRET, the donor fluorescent protein moiety and the
acceptor fluorescent protein moiety are selected so that the donor
and acceptor moieties exhibit fluorescence resonance energy
transfer when the donor moiety is excited. One factor to be
considered in choosing the fluorescent protein moiety pair is the
efficiency of fluorescence resonance energy transfer between them.
Preferably, the efficiency of FRET between the donor and acceptor
moieties is at least 10%, more preferably at least 50% and even
more preferably at least 80%. The efficiency of FRET can easily be
empirically tested using the methods described herein and known in
the art, particularly, using the conditions set forth in the
Examples.
[0053] The efficiency of FRET is dependent on the separation
distance and the orientation of the donor and acceptor moieties, as
described by the Forster equation, the fluorescent quantum yield of
the donor moiety and the energetic overlap with the acceptor
moiety. Forster derived the relationship:
E=(F.sup.0-F)/F.sup.0=R.sub.0.sup.6/(R.sup.6+R.sub.0.sup.6)
[0054] where E is the efficiency of FRET, F and F.sup.0 are the
fluorescence intensities of the donor in the presence and absence
of the acceptor, respectively, and R is the distance between the
donor and the acceptor. R.sub.0, the distance at which the energy
transfer efficiency is 50%, is given (in .ANG.) by
R.sub.0=9.79.times.10.sup.3(K.sup.2QJn.sup.-4).sup.1/6
[0055] where K.sup.2 is an orientation factor having an average
value close to 0.67 for freely mobile donors and acceptors, Q is
the quantum yield of the unquenched fluorescent donor, n is the
refractive index of the intervening medium, and J is the overlap
integral, which expresses in quantitative terms the degree of
spectral overlap,
J=.intg..circle-solid..sub.0.epsilon..sub..lambda.F.sub..lambda..lambda..s-
up.4d.lambda./.intg..circle-solid..sub.0F.sub..lambda.d.lambda.
[0056] where .epsilon..sub..lambda. is the molar absorptivity of
the acceptor in M.sup.-1 cm.sup.-1 and F.sub..lambda. is the donor
fluorescence at wavelength 1 measured in cm. Forster, T. (1948)
Ann.Physik 2:55-75. Tables of spectral overlap integrals are
readily available to those working in the field (for example,
Berlman, I. B. Energy transfer parameters of aromatic compounds,
Academic Press, New York and London (1973)).
[0057] The characteristic distance R.sub.0 at which FRET is 50%
efficient depends on the quantum yield of the donor i.e., the
shorter-wavelength fluorophore, the extinction coefficient of the
acceptor, i.e., the longer-wavelength fluorophore, and the overlap
between the donor's emission spectrum and the acceptor's excitation
spectrum. Calculated values of R.sub.0 for P4-3 to S65T and S65C
are both 4.03 nm because the slightly higher extinction coefficient
of S65T compensates for its slightly longer emission wavelength. R.
Heim et al., "Improved green fluorescence," Nature (1995)
373:663-664.
[0058] The efficiency of FRET between the two fluorescent proteins
can also be adjusted by changing ability of the two fluorescent
proteins to dimerize or closely associate. If the two fluorescent
proteins are known or determined to closely associate, an increase
or decrease in dimerization can be promoted by adjusting the length
of the linker moiety between the two fluorescent proteins. Such
dimerization can change .LAMBDA..sup.2, R, J, and Q and
dimerization changes directly affect the fluorescence spectra
compared to undimerized protein. Consequently, for FRET aspects of
the invention, the change in intrinsic fluorescence can be used to
adjust the amount of FRET between the donor and the acceptor, as
well as dimerization induced changes in FRET distances. Such
dimerization induced changes in FRET distance can be optimized for
maximal changes in FRET upon cleavage of a linker moiety by
empirically determining the length of the linker moiety that
produces the best FRET. Usually, such linkers will be comparable to
a length of 14 to 30 amino acids.
[0059] The ability of two fluorescent proteins to dimerize could be
increased by selecting amino acid positions that interact in the
dimer and making changes of the amino acids at such positions that
increase the hydrophobic or ionic interactions, or decrease the
steric repulsions. Conversely, ability of two fluorescent-proteins
to dimerize could be decreased by selecting amino acid positions
that interact in the dimer and making changes in the amino acids at
such positions that decrease the hydrophobic or ionic interaction,
or increase the steric repulsions. Thus, intramolecular
interactions responsible for the association of fluorescent protein
moieties in a tandem fluorescent protein or intermolecular
interactions between two fluorescent proteins in free solution can
be enhanced or attenuated.
[0060] For example, Aequorea derived fluorescent proteins and
related proteins, especially at high concentrations of free
protein, exist as dimers. The dimerization domain can be identified
in the wild type protein using the crystal structure. Yang,F., et
al The Molecular structure of Green Fluorescent Protein. Nature.
Biotech. (1996) 14 1246-1251. In the case of wildtype GFP, the
hydrophobic amino acids, Ala 206, Leu 221, and Phe 223 interact
during dimerization. The tendency of a tandem GFP (or two GFPs in
free solution)to non-covalently associate at these positions could
be increased by increasing the hydrophobicity of amino acids at
positions 206 or 221, thereby increasing the strength of
hydrophobic interactions between the two fluorescent proteins.
[0061] For example, replacement of Ala 206, or Leu 221 by any of
the amino acids, Val, Ile or Phe would increase their
hydrophobicity, and potentially strengthen the hydrophobic
interaction between two GFPs. Alternatively, the amino acids could
be changed to positively charged amino acids in one fluorescent
protein (for example lys or Arg) and to negatively charged amino
acids in the second fluorescent protein of the construct (for
example Glu or Asp) thereby creating additional electrostatic
interactions between two GFPs. Similarly the amino acids Tyr 39,
Glu 142, Asn 144, Asn 146, Ser 147, Asn 149, Tyr 151, Arg 168, Asn
170, Glu 172, Tyr 200, Ser 202, Gln 204 and Ser 208 could be
changed according to the methods described herein to enhance
intramolecular interactions between tandem-. fluorescent proteins
or intermolecular interactions between to GFPs in free
solution.
[0062] The length of the linker moiety is chosen to optimize both
FRET and the kinetics and specificity of enzymatic cleavage. The
average distance between the donor and acceptor moieties should be
between about 1 nm and about 10 nm, preferably between about 1 nm
and about 6 nm, and more preferably between about 1 nm and about 4
nm. If the linker is too short, the protein moieties may sterically
interfere with each other's folding or with the ability of the
cleavage enzyme to attack the linker. In embodiments of the
invention where dimerization is desired the linker length will
typically be a length comparable the length of at least 12 amino
acids, preferably at least 18 amino acids and more preferably at
least 24 amino acids. Only in rare instances will the linker length
be longer than the length of about 40 to 50 amino acids. However,
embodiments of the invention comprise linker moieties having 150 to
200 amino acids.
[0063] The effect of linker length on the ability of tandemly
linked fluorescent proteins to become fluorescent was determined
for a modified GFP tandem protein, as shown in TABLE II. The
modified GFP tandem protein was expressed in bacteria and grown at
37(C.
2TABLE II Fluorescence of 1.sup.st Fluorescence of 2.sup.nd Linker
Length Fluorescent protein Fluorescent protein in amino acids
(Sapphire) (10C) 12 6.8 .times. 10.sup.4 6.2 .times. 10.sup.4 14
8.9 .times. 10.sup.4 8.4 .times. 10.sup.4 16 1.1 .times. 10.sup.5
1.0 .times. 10.sup.5 18 1.3 .times. 10.sup.5 1.2 .times. 10.sup.5
20 1.5 .times. 10.sup.5 1.4 .times. 10.sup.5 22 2.9 .times.
10.sup.5 1.6 .times. 10.sup.5 24 1.1 .times. 10.sup.6 7.8 .times.
10.sup.4 25 2.0 .times. 10.sup.6 1.2 .times. 10.sup.6
[0064] Tandem-fluorescent proteins of the invention comprising the
general form Sapphire--linker--10C (10C is also known as Topaz)
were expressed in the bacterial cells JM109 (DE3). The linker
moiety was constructed with variable numbers of amino acids to
evaluate the influence of linker size on fluorescence development.
The linker sequences of TABLE II are described as SEQ ID NO.: 26 to
31, respectively. The composition of the 25 amino acid linker is
identical to that used in the tandem fluorescent protein constructs
in the Examples. After overnight growth at 37(C the bacterial
colonies were examined to determine their relative fluorescence by
resuspension in PBS after normalization for the number of bacteria
present by measuring the optical density at 600 nm.
[0065] When the intramolecular dimerization of a tandem fluorescent
protein construct is preferred, the three dimensional structure and
flexability of the linker shouldpermit the fluorescent protein
moieties to associate. When the linker moiety contains a cleavage
site, the length of the linker can be between about 5 and about 50
amino acids and more preferably between about 12 and 30 amino
acids. Longer linkers may create too many sites which are
vulnerable to attack by enzymes other than the one being
assayed.
[0066] To optimize the efficiency and detectability of FRET within
the tandem fluorescent protein construct, several factors need to
be balanced. The emission spectrum of the donor moiety should
overlap as much as possible with the excitation spectrum of the
acceptor moiety to maximize the overlap integral J. Also, the
quantum yield of the donor moiety and the extinction coefficient of
the acceptor should likewise be as high as possible to maximize
R.sub.0. However, the excitation spectra of the donor and acceptor
moieties should overlap as little as possible so that a wavelength
region can be found at which the donor can be excited efficiently
without directly exciting the acceptor. Fluorescence arising from
direct excitation of the acceptor is difficult to distinguish from
fluorescence arising from FRETS Similarly, the emission spectra of
the donor and acceptor moieties should overlap as little as
possible so that the two emissions can be clearly distinguished.
High fluorescence quantum yield of the acceptor moiety is desirable
if the emission from the acceptor is to be measured either as the
sole readout or as part of an emission ratio. In a preferred
embodiment, the donor moiety is excited by ultraviolet (<400 nm)
and emits blue light (<500 nm), whereas the acceptor is
efficiently excited by blue but not by ultraviolet light and emits
green light (>500 nm), for example, P4-3 and S65C.
[0067] In the tandem constructs of the invention, the donor and
acceptor moieties are connected through a linker moiety. The linker
moiety is, preferably, a peptide moiety, but can be another organic
molecular moiety, as well. In a preferred embodiment, the linker
moiety includes a cleavage recognition site specific for an enzyme
or other cleavage agent of interest. A cleavage site in the linker
moiety is useful because when a tandem construct is mixed with the
cleavage agent, the linker is a substrate for cleavage by the
cleavage agent. Rupture of the linker moiety results in separation
of the fluorescent protein moieties that is measurable as a change
in FRET.
[0068] When the cleavage agent of interest is a protease, the
linker can comprise a peptide containing a cleavage recognition
sequence for the protease. A cleavage recognition sequence for a
protease is a specific amino acid sequence recognized by the
protease during proteolytic cleavage. In particular, the linker can
contain any of the amino acid sequences in TABLE III. The sites are
recognized by the enzymes as indicated and the site of cleavage is
marked by a hyphen. Other protease cleavage sites also are known in
the art and can be included in the linker moiety.
3TABLE III Protease Sequence HIV-1 protease SQNY-PIVQ (SEQ ID NO:
3) KARVL-AEAMS (SEQ ID NO: 4) Prohormone convertase PSPREGKR-SY
(SEQ ID NO: 5) Interleukin-1b-converting YVAD-G (SEQ ID NO: 6)
enzyme Adenovirus endopeptidase MFGG-AKKR (SEQ ID NO: 7)
Cytomegalovirus assemblin GVVNA-SSRLA (SEQ ID NO: 8) Leishmanolysin
LIAY-LKKAT (SEQ ID NO: 9) b-Secretase for amyloid VKM-DAEF (SEQ ID
NO: 10) precursor protein Thrombin FLAEGGGVR-GPRVVERH (SEQ ID NO:
11) Rerun and DRVYIHPF-HL-VIH (SEQ ID angiotensin-converting NO:
12) enzyme Cathepsin D KPALF-FRL (SEQ ID NO: 13) Kininogenases
including QPLGQTSLMK-RPPGFSPFR-SVQVM- KT kallikrein QEGS (SEQ ID
NO: 14)
[0069] See, e.g., Matayoshi et al. (1990) Science 247:954, Dunn et
al. (1994) Meth. Enzymol. 241:254, Seidah & Chretien (1994)
Meth. Enzymol. 244:175, Thornberry (1994) Meth. Enzymol. 244:615,
Weber & Tihanyi (1994) Meth. Enzymol. 244:595, Smith et al.
(1994) Meth. Enzymol. 244:412, Bouvier et al. (1995) Meth. Enzymol.
248:614, Hardy et al. (1994) in Amyloid Protein Precursor in
Development, Aginq, and Alzheimer's Disease, ed. C. L. Masters et
al. pp. 190-198.
[0070] In the case of a known protease with cleavage activity of
unknown or partially defined specificity, a library of randomized
linker sequences can be used in place of a predetermined linker
sequence in the tandem fluorescent protein construct in order to
determine the sequences cleaved by a protease. The method can be
used with a recombinant protease constructed with a novel cleavage
specificity. This method can also be used to determine the
specificity of cleavage of an orphan protein that reveals sequence
homology to a known protease structure or group of proteases.
[0071] In one embodiment, a genetically engineered library of
tandem fluorescent protein constructs having randomized linkers can
be used to define the function of an orphan protease. Optionally,
the orphan protease, especially to if is thought to be expressed
relatively inactive precursor, can be coexpressed with the tandem
fluorescent protein construct. The protease may also be coexpressed
with the tandem construct and under the control of an inducable
promoter.
[0072] As used herein, a "library" refers to a collection
containing at least 5 different members, preferably at least 100
different members and more preferably at least 200 different
members. Each member of a tandem fluorescent substrate library
comprises 2 tandemly linked fluorescent protein moieties separated
by a peptide linker moiety of variable amino acid composition The
amino acid sequences for the peptide linked moiety may be
completely random or biased towards a particular sequence based on
the homology between other proteases and the protease being tested.
The library can be chemically synthesized, which is particularly
desirable if d-amino acids are to be included. In most instances,
however, the library will be expressed in bacteria or a mammalian
cell.
[0073] For example, the library can contain linkers with a diverse
collection of amino acids in which most or all of the amino acid
positions are randomized. Alternatively, the library can contain
variable peptide moieties in which only a few, e.g., one to ten,
amino acid positions are varied, but in which the probability of
substitution is very high.
[0074] Preferably, libraries of tandem fluorescent protein
candidate substrates are created by expressing protein from
libraries of recombinant nucleic acid molecules having expression
control sequences operatively linked to nucleic acid sequences that
code for the expression of different fluorescent protein candidate
substrates. Methods of making nucleic acid molecules encoding a
diverse collection of peptides are described in, for example, U.S.
Pat. No. 5,432,018 (Dower et al.), U.S. Pat. No. 5,223,409 (Ladner
et al.) and International patent publication WO 92/06176 (Huse et
al.).
[0075] For expression of tandem fluorescent protein candidate
substrates, recombinant nucleic acid molecules are used to
transfect cells, such that a cell contains a member of the library.
This produces, in turn, a library of host cells capable of
expressing a library of different fluorescent protein candidate
substrates. The library of host cells is useful in the screening
methods of this invention.
[0076] In one method of creating such a library, a diverse
collection of oligonucleotides having random codon sequences are
combined to create polynucleotides encoding peptides having a
desired number of amino acids for the linker moiety. The
oligonuclectides preferably are prepared by chemical synthesis. The
polynucleotides encoding peptide linker moiety of variable
composition can then be ligated to the 5' or 3' end of a nucleic
acid encoding one of the tandem fluorescent protein moieties, using
methods known in the art. This creates a recombinant nucleic acid
molecule coding for the expression of a fluorescent protein
candidate substrate having a variable linker peptide moiety fused
to the amino or carboxy-terminus of one of the tandem fluorescent
proteins. This recombinant nucleic acid molecule is then inserted
into an expression vector in which the second fluorescent has
already been inserted to create a recombinant nucleic acid molecule
comprising expression control sequences operatively linked to the
sequences encoding the tandemly repeated fluorescent proteins
separated by the linker moieties (FIG. 10).
[0077] To generate the collection of oligonucleotides which forms a
series of codons encoding a random collection of amino acids that
is ultimately cloned into the vector, a codon motif is used, such
as (NNK).sub.x, where N may be A, C, G, or T (nominally equimolar),
K is G or T (nominally equimolar), and x is the desired number of
amino acids in the peptide moiety, e.g., 15 to produce a library of
15-mer peptides. The third position may also be G or C, designated
"S". Thus, NNK or NNS (i) code for all the amino acids, (ii) code
for only one stop codon, and (iii) reduce the range of codon bias
from 6:1 to 3:1. The expression of peptides from randomly generated
mixtures of oligonucleotides in appropriate recombinant vectors is
discussed in Oliphant et al., Gene 44:177-183 (1986), incorporated
herein by reference.
[0078] An exemplified codon motif (NNK).sub.6 produces 32 codons,
one for each of 12 amino acids, two for each of five amino acids,
three for each of three amino acids and one (amber) stop codon.
Although this motif produces a codon distribution as equitable as
available with standard methods of oligonucleotide synthesis, it
results an a bias for amino acids encoded by two or threes
alternative codons.
[0079] An alternative approach to minimize the bias against
one-codon residues involves the synthesis of 20 activated
tri-nucleotides, each representing the codon for one of the 20
genetically encoded amino acids. These are synthesized by
conventional means, removed from the support but maintaining the
base and 5-HO-protecting groups, and activating by the addition of
3'O-phosphoramidite (and phosphate protection with beta-cyanoethyl
groups) by the method used for the activation of mononucleosides,
as generally described in McBride and Caruthers, Tetrahedron
Letters 22:245 (1983). Degenerate "oligocodons" are prepared using
these trimers as building blocks. The trimers are mixed at the
desired molar ratios and installed in the synthesizer. The ratios
will usually be approximately equimolar, but may be a controlled
unequal ratio to obtain the over- to under-representation of
certain amino acids coded for by the degenerate oligonucleotide
collection. The condensation of the trimers to form the oligocodons
is done essentially as described for conventional synthesis
employing activated mononucleosides as building blocks. See
generally, Atkinson and Smith, Oligonucleotide Synthesis, M. J.
Gait, ed. p35-82 (1984). Thus, this procedure generates a
population of oligonucleotides for cloning that is capable of
encoding an equal distribution (or a controlled unequal
distribution) of the possible peptide sequences.
[0080] Because protease cleavage recognition sequences generally
are only a few amino acids in length, the linker moiety can include
the recognition sequence within flexible spacer amino acid
sequences, such as GGGGS (SEQ ID NO: 15). For example, a linker
moiety including a cleavage recognition sequence for Adenovirus
endopeptidase could have the sequence GGGGGGSMFG GAKKRSGGGG GG (SEQ
ID NO: 16).
[0081] Alternatively, the linker moiety can be an organic molecular
moiety that can contain a cleavage site for an enzyme that is not a
protease. The molecular structure is selected so that the distance
between the fluorescent moieties allows FRET (i.e., less than about
10 nm). For example, the linker moiety can contain a structure that
is recognized by b-lactamase, rendering the tandem complex a
substrate for this enzyme. One structure for such a linker moiety
is: 1
[0082] in which one of X and Y is the donor moiety and the other is
the acceptor moiety. R' can be, for example, H. lower alkyl or
lower alkoxy of up to 15 carbon. R" can be H,
physiologically-acceptable metal and ammonium cations, alkyl,
alkoxy or aromatic groups of up to 15 carbon atoms. (See, e.g.,
Bundgaard, H., Design of prodrugs, Elsevier Science publishers
(1985); Bioreversible Carriers in Drug Design, New York:Pergamon
Press (1987); Ferres, H. (1980) Chem. Ind. June:435-440.) Z' and Z"
are parts of the linker moiety having fewer than about 20 carbon
atoms. Z" includes a heteroatom, such as oxygen or, preferably,
sulfur, attached to the cephalosporin side chain to act as a
nucleofuge. Such linker moieties also are described in U.S. patent
application Ser. No. 08/407,547, filed Mar. 20, 1995.
[0083] This invention contemplates tandem fluorescent protein
constructs produced in the form of a fusion protein by recombinant
DNA technology as well as constructs produced by chemically
coupling fluorescent proteins to a linker. In either case, the
fluorescent proteins for use as donor or acceptor moieties in a
tandem construct of the invention preferably are produced
recombinantly.
[0084] Recombinant production of fluorescent proteins involves
expressing nucleic acids having sequences that encode the proteins.
Nucleic acids encoding fluorescent proteins can be obtained by
methods known in the art. For example, a nucleic acid encoding the
protein can be isolated by polymerase chain reaction of cDNA from
A. victoria using primers based on the DNA sequence of A. victoria
green fluorescent protein, as presented in FIG. 1. PCR methods are
described in, for example, U.S. Pat. No. 4,683,195; Mullis et al.
(1987) Cold Spring Harbor Symp. Quant. Biol. 51:263; and Erlich,
ed., PCR Technology, (Stockton Press, NY, 1989). Mutant versions of
fluorescent proteins can be made by site-specific mutagenesis of
other nucleic acids encoding fluorescent proteins, or by random
mutagenesis caused by increasing the error rate of PCR of the
original polynucleotide with 0.1 mM MnCl.sub.2 and unbalanced
nucleotide concentrations. See, e.g., U.S. patent application
08/337,915, filed Nov. 10, 1994 or International application
PCT/US95/14692, filed Nov. 10, 1995.
[0085] The construction of expression vectors and the expression of
genes in transfected cells involves the use of molecular cloning
techniques also well known in the art. Sambrook et al., Molecular
Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., (1989) and Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., (Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (most recent Supplement).
[0086] Nucleic acids used to transfect cells with sequences coding
for expression of the polypeptide of interest generally will be in
the form-of an expression vector including expression control
sequences operatively linked to a nucleotide sequence coding for
expression of the polypeptide. As used, the term "nucleotide
sequence coding for expression of" a polypeptide refers to a
sequence that, upon transcription and translation of mRNA, produces
the polypeptide. This can include sequences containing, e.g.,
introns. As used herein, the term "expression control sequences"
refers to nucleic acid sequences that regulate the expression of a
nucleic acid sequence to which it is operatively linked. Expression
control sequences are "operatively linked" to a nucleic acid
sequence when the expression control sequences control and regulate
the transcription and, as appropriate, translation of the nucleic
acid sequence. Thus, expression control sequences can include
appropriate promoters, enhancers, transcription terminators, a
start codon (i.e., ATG) in front of a protein-encoding gene,
splicing signals for introns, maintenance of the correct reading
frame of that gene to permit proper translation of the mRNA, and
stop codons.
[0087] Recombinant fluorescent protein can be produced by
expression of nucleic acid encoding the protein in E. coli. The
fluorophore of Aequorea-related fluorescent proteins results from
cyclization and oxidation of residues 65-67. Aequorea-related
fluorescent proteins are best expressed by cells cultured between
about 20.degree. C. and 30.degree. C. After synthesis, these
enzymes are stable at higher temperatures (e.g., 37.degree. C.) and
can be used in assays at those temperatures.
[0088] The construct can also contain a tag to simplify isolation
of the tandem construct. For example, a polyhistidine tag of, e.g.,
six histidine residues, can be incorporated at the amino terminal
end of the fluorescent protein. The polyhistidine tag allows
convenient isolation of the protein in a single step by
nickel-chelate chromatography.
[0089] A. Recombinant Nucleic Acids Encoding Tandem Construct
Fusion Proteins
[0090] In a preferred embodiment, the tandem construct is a fusion
protein produced by recombinant DNA technology in which a single
polypeptide includes a donor moiety, a peptide linker moiety and an
acceptor moiety. The donor moiety can be positioned at the
amino-terminus relative to the acceptor moiety in the polypeptide.
Such a fusion protein has the generalized structure: (amino
terminus) donor fluorescent protein moiety--peptide linker
moiety--acceptor fluorescent protein moiety (carboxy terminus).
Alternatively, the donor moiety can be positioned at the
carboxy-terminus relative to the acceptor moiety within the fusion
protein. Such a fusion protein has the generalized structure:
(amino terminus) acceptor fluorescent protein moiety--peptide
linker moiety--donor fluorescent protein moiety (carboxy terminus).
The invention also envisions fusion proteins that contain extra
amino acid sequences at the amino and/or carboxy termini, for
example, polyhistidine tags.
[0091] Thus, tandem constructs encoded by a recombinant nucleic
acid include sequences coding for expression of a donor fluorescent
protein moiety, an acceptor fluorescent protein moiety and a
peptide linker moiety. The elements are selected so that upon
expression into a fusion protein, the donor and acceptor moieties
exhibit FRET when the donor moiety is excited.
[0092] The recombinant nucleic acid can be incorporated into an
expression vector comprising expression control sequences
operatively linked to the recombinant nucleic acid. The expression
vector can be adapted for function in prokaryotes or eukaryotes by
inclusion of appropriate promoters, replication sequences, markers,
etc.
[0093] The expression vector can be transfected into a host cell
for expression of the recombinant nucleic acid. Host cells can be
selected for high levels of expression in order to purify the
tandem construct fusion protein. E. coli is useful for this
purpose. Alternatively, the host cell can be a prokaryotic or
eukaryotic cell selected to study the activity of an enzyme
produced by the cell. In this case, the linker peptide is selected
to include an amino acid sequence recognized by the protease. The
cell can be, e.g., a cultured cell or a cell in vivo.
[0094] A primary advantage of tandem construct fusion proteins is
that they are prepared by normal protein biosynthesis, thus
completely avoiding organic synthesis and the requirement for
customized unnatural amino acid analogs. The constructs can be
expressed in E. coli in large scale for in vitro assays.
Purification from bacteria is simplified when the sequences include
polyhistidine tags for one-step purification by nickel-chelate
chromatography. Alternatively, the substrates can be expressed
directly in a desired host cell for assays in situ, which is
particularly advantageous if the proteases of interest are
membrane-bound or regulated in a complex fashion or not yet
abundant as purified stable enzymes. No other generalizable method
for continuous nondestructive assay of protease activity in living
cells or organisms presently exists.
[0095] B. Non-Recombinant Coupling Methods
[0096] Fluorescent proteins can be attached through non-recombinant
means. In one embodiment, the moieties are attached to a linker by
chemical means. This is preferred if the linker moiety is not a
peptide. In this case, the linker moiety can comprise a
cross-linker moiety. A number of cross-linkers are well known in
the art, including homo- or hetero-bifunctional cross-linkers, such
as BMH, SPDP, etc. In general, the linker should have a length so
as to separate the moieties by about 10 .ANG. to about 100 .ANG..
This is more critical than the particular chemical composition of
the linker. Chemical methods for specifically linking molecules to
the amino- or carboxy-terminus of a protein are reviewed by R. E.
Offord, "Chemical Approaches to Protein Engineering," in Protein
Engineering--A Practical Approach, (1992) A. R. Rees, M. Sternberg
and R. Wetzel, eds., Oxford University Press.
[0097] When the protein moieties are to be chemically coupled,
fluorescent proteins can be isolated from natural sources by means
known in the art. One method involves purifying the proteins to
electrophoretic homogeneity. Also, J. R. Deschamps et al. describe
a method of purifying recombinant Aequorea GFP in Protein
Expression and Purification, (1995) 6:555-558.
[0098] In another embodiment, the moieties are coupled by attaching
each to a nucleic acid molecule. The nucleic acids have sequences
of sufficient length and areas of sufficient complementarity to
allow hybridization between them, thereby linking the moieties
through hydrogen bonds. When the linker contains the sequence of a
restriction site, this embodiment allows one to assay for the
presence of restriction enzymes by monitoring FRET after the
nucleic acid is cleaved and the moieties physically separate.
[0099] In another embodiment, the moieties are coupled by attaching
each to a polypeptide pair capable of bonding through dimerization.
For example, the peptide can include sequences that form a leucine
zipper, shown to enable dimerization of a protein to which it was
attached. See A. Blondel et al., "Engineering the quaternary
structure of an exported protein with a leucine zipper," Protein
Engineering (1991) 4:457-461. The linker containing the leucine
zipper in the Blondel et al. article had the sequence: IQRMKQLED
KVEELLSKNY HLENEVARLK KLVGER (SEQ ID NO: 17). In another
embodiment, a peptide linker moiety can comprise the sequence
SKVILF (SEQ OID NO: 18), which also is capable of dimerization. See
WO 94/28173.
[0100] C. Alternative Fluorescent Protein Constructs
[0101] This invention also contemplates tandem constructs
possessing a single fluorescent protein moiety that functions as
donor or acceptor and a non-protein compound fluorescent moiety
that functions as donor or quencher. In one embodiment, the
construct comprises a donor fluorescent protein moiety, a
non-protein compound acceptor fluorescent moiety and a linker
moiety that couples the donor and acceptor moieties. Alternatively,
a tandem construct can comprise a non-protein compound donor
fluorescent moiety, an acceptor fluorescent protein moiety and a
linker moiety that couples the donor and acceptor moieties.
Non-protein compound fluorescent donor moieties of particular
interest include coumarins and fluoresceins; particular quenchers
of interest include fluoresceins, rhodols, rhodamines and azo dyes.
Acceptable fluorescent dyes are described, for example, in U.S.
application Ser. No. 08/407,544, filed Mar. 20, 1995. The donor and
acceptor moieties of these constructs are chosen with many of the
same considerations for FRET as for tandem fluorescent protein
constructs having two fluorescent protein moieties.
[0102] III. Enzymatic Assays Using Tandem Fluorescent Protein
Constructs
[0103] Tandem fluorescent protein constructs are useful in
enzymatic assays. These assays take advantage of the fact that
cleavage of the linker moiety and separation of the fluorescent
moieties results in a measurable change in FRET. Methods for
determining whether a sample has activity of an enzyme involve
contacting the sample with a tandem fluorescent protein construct
in which the linker moiety that couples the donor and acceptor
moieties contains a cleavage recognition site specific for the
enzyme. Then the donor moiety is excited with light in its
excitation spectrum. If the linker moiety is cleaved, the donor and
acceptor are free to drift apart, increasing the distance between
the donor and acceptor and preventing FRET. Then, the degree of
FRET in the sample is determined. A degree of FRET that is lower
than the amount expected in a sample in which the tandem construct
is not cleaved indicates that the enzyme is present.
[0104] The amount of activity of an enzyme in a sample can be
determined by determining the degree of FRET in the sample at a
first and second time after contact between the sample and the
tandem construct, determining the difference in the degree of FRET.
The amount of enzyme in the sample can be calculated as a function
of the difference in the degree of FRET using appropriate
standards. The faster or larger the loss of FRET, the more enzyme
activity must have been present in the sample.
[0105] The degree of FRET can be determined by any spectral or
fluorescence lifetime characteristic of the excited construct, for
example, by determining the intensity of the fluorescent signal
from the donor, the intensity of fluorescent signal from the
acceptor, the ratio of the fluorescence amplitudes near the
acceptor's emission maxima to the fluorescence amplitudes near the
donor's emission maximum, or the excited state lifetime of the
donor.
[0106] For example, cleavage of the linker increases the intensity
of fluorescence from the donor, decreases the intensity of
fluorescence from the acceptor, decreases the ratio of fluorescence
amplitudes from the acceptor to that from the donor, and increases
the excited state lifetime of the donor.
[0107] Preferably, changes in the degree of FRET are determined as
a function of the change in the ratio of the amount of fluorescence
from the donor and acceptor moieties, a process referred to as
"ratioing." Changes in the absolute amount of substrate, excitation
intensity, and turbidity or other background absorbances in the
sample at the excitation wavelength affect the intensities of
fluorescence from both the donor and acceptor approximately in
parallel. Therefore the ratio of the two emission intensities is a
more robust and preferred measure of cleavage than either intensity
alone.
[0108] The excitation state lifetime of the donor moiety is,
likewise, independent of the absolute amount of substrate,
excitation intensity, or turbidity or other background absorbances.
Its measurement requires equipment with nanosecond time
resolution.
[0109] Fluorescence in a sample is measured using a fluorimeter. In
general, excitation radiation, from an excitation source having a
first wavelength, passes through excitation optics. The excitation
optics cause the excitation radiation to excite the sample. In
response, fluorescent proteins in the sample emit radiation which
has a wavelength that is different from the excitation wavelength.
Collection optics then collect the emission from the sample. The
device can include a temperature controller to maintain the sample
at a specific temperature while it is being scanned. According to
one embodiment, a multi-axis translation stage moves a microtiter
plate holding a plurality of samples in order to position different
wells to be exposed. The multi-axis translation stage, temperature
controller, auto-focusing feature, and electronics associated with
imaging and data collection can be managed by an appropriately
programmed digital computer. The computer also can transform the
data collected during the assay into another format for
presentation.
[0110] Methods of performing assays on fluorescent materials are
well known in the art and are described in, e.g., Lakowicz, J. R.,
Principles of Fluorescence Spectroscopy, New York:Plenum Press
(1983); Herman, B., Resonance energy transfer microscopy, in:
Fluorescence Microscopy of Living Cells in Culture, Part B, Methods
in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y. -L., San
Diego: Academic Press (1989), pp. 219-243; Turro, N. J., Modern
Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing
Col, Inc. (1978), pp. 296-361.
[0111] Enzymatic assays also can be performed on living cells in
vivo, or from samples derived from organisms transfected to express
the tandem construct. Because tandem construct fusion proteins can
be expressed recombinantly inside a cell, the amount of enzyme
activity in the cell or organism of which it is a part can be
determined by determining changes in fluorescence of cells or
samples from the organism.
[0112] In one embodiment, a cell is transiently or stably
transfected with an expression vector encoding a tandem fluorescent
protein construct containing a linker moiety that is specifically
cleaved by the enzyme to be assayed. This expression vector
optionally includes controlling nucleotide sequences such as
promotor or enhancing elements. The enzyme to be assayed may either
be intrinsic to the cell or may be introduced by stable
transfection or transient co-transfection with another expression
vector encoding the enzyme and optionally including controlling
nucleotide sequences such as promoter or enhancer elements. The
fluorescent protein construct and the enzyme preferably are
expressed in the same cellular compartment so that they have more
opportunity to come into contact.
[0113] If the cell does not possess enzyme activity, the efficiency
of FRET in the cell is high, and the fluorescence characteristics
of the cell reflect this efficiency. If the cell possesses a high
degree of enzyme activity, most of the tandem construct expressed
by the cell will be cleaved. In this case, the efficiency of FRET
is low, reflecting a large amount or high efficiency of the
cleavage enzyme relative to the rate of synthesis of the tandem
fluorescent protein construct. If the level of enzyme activity in
the cell is such that an equilibrium is reached between expression
and cleavage of the tandem construct, the fluorescence
characteristics will reflect this equilibrium level. In one aspect,
this method can be used to compare mutant cells to identify which
ones possess greater or less enzymatic activity. Such cells can be
sorted by a fluorescent cell sorter based on fluorescence.
[0114] A contemplated variation of the above assay is to use the
controlling nucleotide sequences to produce a sudden increase
in-the expression of either the tandem fluorescent protein
construct or the enzyme being assayed, e.g., by inducing expression
of the construct. The efficiency of FRET is monitored at one or
more time intervals after the onset of increased expression. A low
efficiency or rapid decline of FRET reflects a large amount or high
efficiency of the cleavage enzyme. This kinetic determination has
the advantage of minimizing any dependency of the assay on the
rates of degradation or loss of the fluorescent protein
moieties.
[0115] Libraries of host cells expressing tandem fluorescent
protein candidate substrates are useful in identifying linker
sequences that can be cleaved by a target protease. In general, one
begins with a library of recombinant host cells, each of which
expresses a different fluorescent protein candidate substrate. Each
cell is expanded into a clonal population that is genetically
homogeneous. The method consists of measuring FRET from each clonal
population before and at least one specified time after a known
change in intracellular protease activity. This could be achieved
using a fluorimeter, a 96 well plate reader, or by FACS
(fluorescence Activated Cell Sorting) anlysis and sorting. This
change in protease activity could be produced by transfection with
a gene encoding the protease, or infection of a cell by a virus, or
induction of protease gene expression using expression control
elements, or by any condition that post-translationally modulates
the activity of a protease that has already been expressed. An
example of the latter is the activation of Calpain 1 by increases
in intracellular calcium. The nucleic acids from cells exhibiting a
change in FRET can be isolated for example by PCR amplification,
and the linker sequences that could be cleaved by the protease
identified by sequencing. The results from these studies could used
as the basis for the generation of more targeted libraries to
identify optimal cleavage motifs through repeated rounds of
analysis and selection of clones exhibiting the largest and most
rapid changes in FRET in the presence, but not the absence ok the
protease.
[0116] In another embodiment, the vector may be incorporated into
an entire organism by standard transgenic or gene replacement
techniques. An expression vector capable of expressing the enzyme
optionally may be incorporated into the entire organism by standard
transgenic or gene replacement techniques. Then, a sample from the
organism containing the tandem construct or the cleaved moieties is
tested. For example, cell or tissue homogenates, individual cells,
or samples of body fluids, such as blood, can be tested.
[0117] The enzymatic assays of the invention can be used in drug
screening assays to identify compounds that alter the activity of
an enzyme. In one embodiment, the assay is performed on a sample in
vitro containing the enzyme. A sample containing a known amount of
enzyme is mixed with a tandem construct of the invention and with a
test compound. The amount of the enzyme activity in the sample is
then determined as above, e.g., by determining the degree of
fluorescence at a first and second time after contact between the
sample, the tandem construct and the compound. Then the amount of
activity per mole of enzyme in the presence of the test compound is
compared with the activity per mole of enzyme in the absence of the
test compound. A difference indicates that the test compound alters
the activity of the enzyme.
[0118] In another embodiment, the ability of a compound to alter
enzyme activity in vivo is determined. In an in vivo assay, cells
transfected with a expression vector encoding a tandem construct of
the invention are exposed to different amounts of the test
compound, and the effect on fluorescence in each cell can be
determined. Typically, the difference is calibrated against
standard measurements to yield an absolute amount of enzyme
activity. A test compound that inhibits or blocks the expression of
the enzyme can be detected by increased FRET in treated cells
compared to untreated controls.
[0119] The following examples are offered by way of illustration,
not by way of limitation.
EXAMPLES
Example 1
[0120] Construction of Tandem Fluorescent Protein Constructs
[0121] Mutant Green Fluorescent Proteins were created as follows.
Random mutagenesis of the Aequorea green fluorescent protein (FIG.
1) was performed by increasing the error rate of the PCR with 0.1
mM MnCl.sub.2 and unbalanced nucleotide concentrations. The
templates used for PCR encoded the GFP mutants S65T, Y66H and Y66W.
They had been cloned into the BamH1 site of the expression vector
pRSETB (Invitrogen), which includes a T7 promoter and a
polyhistidine tag. The GFP coding region (shown in bold) was
flanked by the following 5' and 3' sequences: 5'-G GAT CCC CCC GCT
GAA TTC ATG (SEQ ID NO: 19) . . . AAA TAA TAA GGA TCC (SEQ ID NO:
20) -3'. The 5' primer for the mutagenic PCR was the T7 primer
matching the vector sequence; the 3' primer was 5'-GGT AAG CTT TTA
TTT GTA TAG TTC ATC CAT GCC-3' (SEQ ID NO: 21), specific for the 3'
end of GFP, creating a HindIII restriction site next to the stop
codon.
[0122] Amplification was over 25 cycles (1 min at 94.degree. C., 1
min 52.degree. C., 1 min 72.degree. C.) using the AmpliTaq
polymerase from Perkin Elmer). Four separate reactions were run in
which the concentration of a different nucleotide was lowered from
200 .mu.M to 50 .mu.M. The PCR products were combined, digested
with BamHI and HindIII and ligated to the pRSETB cut with BamHI and
HindIII. The ligation mixture was dialyzed against water, dried and
subsequently transformed into the bacterial strain BL21(DE3) by
electroporation (50 .mu.l electrocompetent cells in 0.1 cm
cuvettes, 1900 V, 200 ohm, 25 .mu.F). Colonies on agar were
visually screened for brightness as previously described. R. Heim
et al., "Wavelength mutations and post-translational autooxidation
of green fluorescent protein," Proc Natl Acad Sci USA 1994,
91:12501-12504. On the order of 7000 colonies were examined in each
successful round of mutagenesis, which is not claimed to be
exhaustive. The selected clones were sequenced with the Sequenase
version 2.0 kit from United States Biochemical.
[0123] A nucleic acid sequence encoding a tandem GFP-BFP construct
fusion protein was produced as follows. The DNA of the GFP mutant
S65C (Heim R, Cubitt A B, Tsien R Y, "Improved green fluorescence,"
Nature 1995, 373:663-664) was amplified by PCR (1 cycle 3 min
94.degree. C., 2 min 33.degree. C., 2 min 72.degree. C.; 20 cycles
1 min 94.degree. C., 1 min 44.degree. C., 1 min 72.degree. C.) with
Pfu polymerase (Stratagene) using the primers 5'-AGA AAG GCT AGC
AAA GGA GAA GAA C-3' (SEQ ID NO: 22) and 5'-T CAG TCT AGA TTT GTA
TAG TTC ATC-3' (SEQ ID NO: 23) to create a NheI site and a (NheI
compatible) XbaI site and to eliminate the GFP stop codon. The
restricted product was cloned in-frame into the NheI site of the
construct pRSETB-Y66H/Y145F, between a polyhistidine tag and an
enterokinase cleavage site. When translated this fusion gives the
following sequence: MRGSHHHHHH GMA (SEQ ID NO: 24)--(S2 . . .
GFP:S65C . . . K238 "S65C")--SSMTGGQQMG RDLYDDDDKD PPAEF (SEQ ID
NO: 25)--(GFP:Y66H/Y145F "P4-3"). The linker moiety includes
cleavage recognition sites for many proteases, including trypsin,
enterokinase and calpain:
4 calpain / SSMTGGQQMG RDLYDDDDKD PPAEF (SEQ ID NO:25) / / trypsin
trypsin, enterokinase
[0124] Several other constructs were constructed and tested using
the same linker moiety. One of these has the structure
S65C--linker--P4. Another had the structure S65C--linker--W7. A
third construct had the structure S65T--linker--W7. A fourth
construct had the structure P4-3--linker--W7.
[0125] Cultures with freshly transformed E. coli cells were grown
at 37.degree. C. to an optical density of 0.8 at 600 nm, then
induced with 0.4 mM isopropylthiogalactoside overnight at room
temperature. Expression levels were roughly equivalent between
mutants and are typical for the T7 expression system used. Cells
were washed in PBS pH 7.4, resuspended in 50 mM Tris pH 8.0, 300 mM
NaCl and lysed in a French press. The polyhistidine-tagged GFP
proteins were purified from cleared lysates on nickel-chelate
columns (Qiagen) using 100 mM imidazole in the above buffer to
elute the protein. Samples used for proteolytic experiments were
further purified by MonoQ FPLC to remove monomeric GFP. Protein
concentrations were estimated with bicinchoninic acid (BCA kit from
Pierce) using bovine serum albumin as a standard.
[0126] Example 2
[0127] Cleavage Measurements
[0128] Proteolytic cleavage of 10 .mu.g of the various GFP-BFP
fusion proteins were performed in 500 .mu.l PBS pH 7.4 with 0.1
.mu.g trypsin (Sigma, grade III) and emission spectra were recorded
at different time intervals. Analogous cleavage experiments were
done also with enterokinase (Sigma) and calpain.
[0129] Excitation spectra were obtained by collecting emission at
the respective peak wavelengths and were corrected by a Rhodamine B
quantum counter. Emission spectra were likewise measured at the
respective excitation peaks and were corrected using factors from
the fluorometer manufacturer (Spex Industries, Edison, N.J.). In
cleavage experiments emission spectra were recorded at excitation
368 nm or at 432 nm. For measuring molar extinction coefficients,
20 to 30 .mu.g of protein were used in 1 ml of PBS pH 7.4. The
extinction coefficients in TABLE I necessarily assume that the
protein is homogeneous and properly folded; if this assumption is
incorrect, the real extinction coefficients could be yet higher.
Quantum yields of wild-type GFP, S65T, and P4-1 mutants were
estimated by comparison with fluorescein in 0.1 N NaOH as a
standard of quantum yield 0.91. J. N. Miller, ed., Standards in
Fluorescence Spectrometry, New York: Chapman and Hall (1981).
Mutants P4 and P4-3 were likewise compared to 9-aminoacridine in
water (quantum yield 0.98). W2 and W7 were compared to both
standards, which gave concordant results.
[0130] Excited at 368 nm, the uncleaved S65C--linker--P4-3
construct emitted bright green light that gradually dimmed upon
cleavage of the linker to separate the protein domains. As the
cleavage by trypsin progressed (0, 2, 5, 10, and 47 min), more blue
light was emitted. There was no further change after 47
minutes.
[0131] The emission spectrum of the intact fusion protein (FIG. 3)
shows that FRET is fairly efficient, because UV excitation causes
substantial green emission from the acceptor S65C. After
proteolytic cleavage of the spacer, which permits the two domains
to diffuse apart, the green emission almost completely disappears,
whereas the blue emission from the Y66H/Y145F is enhanced because
its excited state is no longer being quenched by the acceptor.
Control experiments with the same proteolytic conditions applied to
either GFP mutant alone showed no effect, arguing that the GFP
domains per se are resistant to proteolysis, as is known to be the
case for the native protein. W. W. Ward et al., "Spectral
perturbations of the Aequorea green-fluorescent protein,"
Photochem. Photobiol. (1982) 35:803-808.
[0132] Similar result were obtained when the S65C--linker--P4-3
fusion construct was cleaved with calpain and excited at 368 nm.
(See FIG. 4.)
[0133] The tandem construct S65C--linker--P4 was exposed to
enterokinase and excited at 368 nm. FRET diminished over time,
demonstrating that one could detect cleavage of the linker by
enterokinase. (See FIG. 5.)
[0134] The tandem construct S65T--linker--W7 was exposed to
enterokinase and excited at 432 nm. Cleavage of the linker and
separation of the moieties was detectable as a decrease in FRET
over time. (See FIG. 6.)
[0135] The tandem construct P4-3--linker--W7 was exposed to trypsin
and excited at 368 nm. FIG. 7. demonstrates the change in FRET
resulting from cleavage.
[0136] The tandem construct W1B--linker--10c was exposed to trypsin
and excited at 433 nm. FIG. 8. demonstrates the change in FRET
resulting from cleavage.
[0137] FIG. 9 depicts fluorescent ratio changes upon cleavage of a
composition containing the tandem construct W1B--linker--10 c
fluorescent construct at different protein concentrations after
exposure to trypsin measured in a fluorescent 96 well microtitre
plate reader (a CytoFluor II Series 4000 Perseptive Biosystems.
Microtitre wells were excited with light at 395+/-25 nm, and the
emitted light measured at 460+/-20 nm and 530+/-15 nm using
appriopriate excitation and emission filter sets.
[0138] These different tandem fluorescent protein constructs
demonstrate that fluorescence resonance energy transfer can monitor
the distance between fluorescent protein domains. Disruption of
FRET between man-made chromophores in a short synthetic peptide has
been used before to assay proteases (G. A. Krafft et al.,
"Synthetic approaches to continuous assays of retroviral
proteases," Methods Enzymol. (1994) 241:70-86; C. G. Knight,
"Fluorimetric assays of proteolytic enzymes," Methods Enzymol.
(1995) 248:18-34), but use of fluorescent proteins as the
fluorophores gives the unique possibility of replacing organic
synthesis by molecular biology and monitoring proteases in situ in
living cells and organisms. FRET is also one of the few methods for
imaging dynamic non-covalent protein-protein associations in
situ.
[0139] The present invention provides novel tandem fluorescent
protein constructs and methods for their use. While specific
examples have been provided, the above description is illustrative
and not restrictive. Many variations of the invention will become
apparent to those skilled in the art upon review of this
specification. The scope of the invention should, therefore, be
determined not with reference to the above description, but instead
should be determined with reference to the appended claims along
with their full scope of equivalents.
[0140] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted.
Sequence CWU 1
1
31 1 716 DNA Aequorea victoria CDS (1)..(714) 1 atg agt aaa gga gaa
gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 Met Ser Lys Gly Glu
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 gaa tta gat
ggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag 96 Glu Leu Asp
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt
gaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt att tgc 144 Gly
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40
45 act act gga aaa cta cct gtt cca tgg cca aca ctt gtc act act ttc
192 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe
50 55 60 tct tat ggt gtt caa tgc ttt tca aga tac cca gat cat atg
aaa cgg 240 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
Lys Arg 65 70 75 80 cat gac ttt ttc aag agt gcc atg ccc gaa ggt tat
gta cag gaa aga 288 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
Val Gln Glu Arg 85 90 95 act ata ttt ttc aaa gat gac ggg aac tac
aag aca cgt gct gaa gtc 336 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggt gat acc ctt gtt
aat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly Asp Thr Leu Val
Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaa gat gga
aac att ctt gga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu Asp Gly
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tca cac
aat gta tac atc atg gca gac aaa caa aag aat gga 480 Tyr Asn Ser His
Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atc
aaa gtt aac ttc aaa att aga cac aac att gaa gat gga agc gtt 528 Ile
Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170
175 caa cta gca gac cat tat caa caa aat act cca att ggc gat ggc cct
576 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
180 185 190 gtc ctt tta cca gac aac cat tac ctg tcc aca caa tct gcc
ctt tcg 624 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
Leu Ser 195 200 205 aaa gat ccc aac gaa aag aga gac cac atg gtc ctt
ctt gag ttt gta 672 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
Leu Glu Phe Val 210 215 220 aca gct gct ggg att aca cat ggc atg gat
gaa cta tac aaa ta 716 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu
Leu Tyr Lys 225 230 235 2 238 PRT Aequorea victoria 2 Met Ser Lys
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu
Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25
30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
Thr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp
His Met Lys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu
Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly
Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn
Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155
160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val
165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln
Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met
Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly
Met Asp Glu Leu Tyr Lys 225 230 235 3 8 PRT Artificial sequence
Linker moiety 3 Ser Gln Asn Tyr Pro Ile Val Gly 1 5 4 10 PRT
Artificial sequence Linker moiety 4 Lys Ala Arg Val Leu Ala Glu Ala
Met Ser 1 5 10 5 10 PRT Artificial sequence Linker moiety 5 Pro Ser
Pro Arg Glu Gly Lys Arg Ser Tyr 1 5 10 6 5 PRT Artificial sequence
Linker moiety 6 Tyr Val Ala Asp Gly 1 5 7 8 PRT Artificial sequence
Linker moiety 7 Met Phe Gly Gly Ala Lys Lys Arg 1 5 8 10 PRT
Artificial sequence Linker moiety 8 Gly Val Val Asn Ala Ser Ser Arg
Leu Ala 1 5 10 9 9 PRT Artificial sequence Linker moiety 9 Leu Ile
Ala Tyr Leu Lys Lys Ala Thr 1 5 10 7 PRT Artificial sequence Linker
moiety 10 Val Lys Met Asp Ala Glu Phe 1 5 11 17 PRT Artificial
sequence Linker moiety 11 Phe Leu Ala Glu Gly Gly Gly Val Arg Gly
Pro Arg Val Val Glu Arg 1 5 10 15 His 12 13 PRT Artificial sequence
Linker moiety 12 Asp Arg Val Tyr Ile His Pro Phe His Leu Val Ile
His 1 5 10 13 8 PRT Artificial sequence Linker moiety 13 Lys Pro
Ala Leu Phe Phe Arg Leu 1 5 14 30 PRT Artificial sequence Linker
moiety 14 Gln Pro Leu Gly Gln Thr Ser Leu Met Lys Arg Pro Pro Gly
Phe Ser 1 5 10 15 Pro Phe Arg Ser Val Gln Val Met Lys Thr Gln Glu
Gly Ser 20 25 30 15 5 PRT Artificial sequence Cleavage recognition
sequence 15 Gly Gly Gly Gly Ser 1 5 16 22 PRT Artificial sequence
Linker moiety 16 Gly Gly Gly Gly Gly Gly Ser Met Phe Gly Gly Ala
Lys Lys Arg Ser 1 5 10 15 Gly Gly Gly Gly Gly Gly 20 17 35 PRT
Artificial sequence Linker moiety 17 Ile Gln Arg Met Lys Gln Leu
Glu Asp Lys Val Glu Glu Leu Leu Ser 1 5 10 15 Lys Asn Tyr His Leu
Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val 20 25 30 Gly Glu Arg 35
18 6 PRT Artificial sequence Linker moiety 18 Ser Lys Val Ile Leu
Phe 1 5 19 22 DNA Artificial sequence Primer sequence 19 ggatcccccc
gctgaattca tg 22 20 15 DNA Artificial sequence Primer sequence 20
aaataataag gatcc 15 21 33 DNA Artificial sequence PCR primer 21
ggtaagcttt tatttgtata gttcatccat gcc 33 22 24 DNA Artificial
sequence Primer sequence 22 agaaaggcta gcaaaggaga agaa 24 23 25 DNA
Artificial sequence Primer sequence 23 tcagtctaga tttgtatagt tcatc
25 24 10 PRT Artificial sequence Fusion sequence 24 Met Arg Gly Ser
His His His His His His 1 5 10 25 25 PRT Artificial sequence Linker
moiety 25 Ser Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr
Asp Asp 1 5 10 15 Asp Asp Lys Asp Pro Pro Ala Glu Phe 20 25 26 12
PRT Artificial sequence Linker sequence 26 Ala Asn Pro Leu Tyr Lys
Asp Ala Thr Asp Phe Thr 1 5 10 27 14 PRT Artificial sequence Linker
sequence 27 Thr Ala Asn Pro Leu Tyr Lys Asp Ala Thr Ser Asp Phe Thr
1 5 10 28 16 PRT Artificial sequence Linker sequence 28 Gly Thr Ala
Asn Pro Leu Tyr Lys Asp Ala Thr Ser Gly Asp Phe Thr 1 5 10 15 29 18
PRT Artificial sequence Linker sequence 29 Gly Thr Ala Asn Pro Leu
Tyr Lys Asp Ala Thr Ser Gly Ser Thr Asp 1 5 10 15 Phe Thr 30 20 PRT
Artificial sequence Linker sequence 30 Gly Thr Ala Asn Pro Leu Tyr
Lys Asp Ala Thr Ser Gly Ser Thr Gly 1 5 10 15 Ser Asp Phe Thr 20 31
22 PRT Artificial sequence Linker sequence 31 Gly Thr Ala Asn Pro
Leu Tyr Lys Asp Ala Thr Ser Gly Ser Thr Gly 1 5 10 15 Ser Gly Ser
Asp Phe Thr 20
* * * * *