U.S. patent application number 10/264127 was filed with the patent office on 2003-10-09 for sequencing by mass spectrometry.
This patent application is currently assigned to The Trustees of Boston University. Invention is credited to Gite, Sadanand, Olejnik, Jerzy, Rothschild, Kenneth J..
Application Number | 20030190643 10/264127 |
Document ID | / |
Family ID | 23510197 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030190643 |
Kind Code |
A1 |
Rothschild, Kenneth J. ; et
al. |
October 9, 2003 |
Sequencing by mass spectrometry
Abstract
This invention relates to non-radioactive markers that
facilitate the detection and analysis of nascent proteins
translated within cellular or cell-free translation systems.
Nascent proteins containing these markers can be rapidly and
efficiently detected, isolated and analyzed without the handling
and disposal problems associated with radioactive reagents.
Preferred markers are dipyrrometheneboron difluoride
(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)dyes.
Inventors: |
Rothschild, Kenneth J.;
(Newton, MA) ; Gite, Sadanand; (Cambridge, MA)
; Olejnik, Jerzy; (Brookline, MA) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
Suite 350
101 Howard Street
San Francisco
CA
94105
US
|
Assignee: |
The Trustees of Boston
University
|
Family ID: |
23510197 |
Appl. No.: |
10/264127 |
Filed: |
October 3, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10264127 |
Oct 3, 2002 |
|
|
|
09813197 |
Mar 20, 2001 |
|
|
|
09813197 |
Mar 20, 2001 |
|
|
|
09382736 |
Aug 25, 1999 |
|
|
|
6306628 |
|
|
|
|
Current U.S.
Class: |
435/6.13 ;
435/455; 435/69.1; 435/7.5 |
Current CPC
Class: |
C12N 15/10 20130101;
C12N 15/67 20130101; C12P 21/02 20130101; G01N 33/6803 20130101;
C12P 21/00 20130101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/455; 435/7.5 |
International
Class: |
C12Q 001/68; G01N
033/53; C12P 019/34; C12N 015/85 |
Claims
1. A method, comprising: a) providing a tRNA molecule and a mass
marker; b) aminoacylating said tRNA molecule with said mass marker
to create a misaminoacylated tRNA; c) introducing said
misaminoacylated tRNA into a translation system under conditions
such that said marker is incorporated into a nascent protein; and
d) detecting said mass marker by mass spectrometry.
2. The method of claim 1, further comprising after step c)
proteolyzing said nascent protein.
3. The method of claim 1, wherein detection comprises identifying a
spectrometric peak which correspond to mass of said nascent protein
plus the mass of the marker.
4. The method of claim 1, wherein the translation system comprises
a cellular or cell-free translation system.
5. The method of claim 1, wherein said mass marker is selected from
the group consisting of photocleavable markers, coupling agents and
combinations thereof.
6. The method of claim 1, wherein said mass marker comprises a
cleavable marker attached to an amino acid of said nascent protein
such that cleavage results in an unmodified amino acid.
7. The method of claim 1, wherein said mass marker comprises a
cleavable marker attached to an amino acid of said nascent protein
such that cleavage results in a modified amino acid.
8. The method of claim 1, wherein said mass marker comprises an
amino acid-photocleavable biotin conjugate.
9. The method of claim 1, wherein the nascent protein is selected
from recombinant gene products, gene fusion products, enzymes,
cytokines, carbohydrate and lipid binding proteins, nucleic acid
binding proteins, hormones, immunogenic proteins, human proteins,
viral proteins, bacterial proteins, parasitic proteins and
fragments and combinations thereof.
10. A method, comprising: a) providing a tRNA molecule and a mass
marker; b) aminoacylating said tRNA molecule with said mass marker
to create a misaminoacylated tRNA; c) introducing said
misaminoacylated tRNA into a translation system under conditions
such that said marker is incorporated into a nascent protein; d)
proteolyzing said nascent protein; and e) detecting said mass
marker by mass spectrometry.
11. The method of claim 10, wherein said proteolyzing generates one
or more polypeptide fragments comprising said mass marker.
12. The method of claim 11, wherein detection comprises identifying
a spectrometric peak which correspond to mass of said polypeptide
fragment plus the mass of the marker.
13. The method of claim 10, wherein said mass marker is selected
from the group consisting of photocleavable markers, coupling
agents and combinations thereof.
14. The method of claim 10, wherein said mass marker comprises a
cleavable marker attached to an amino acid of said nascent protein
such that cleavage results in an unmodified amino acid.
15. The method of claim 11, wherein said mass marker comprises a
cleavable marker attached to an amino acid of said nascent protein
such that cleavage results in a modified amino acid.
16. The method of claim 11, wherein said mass marker comprises an
amino acid-photocleavable biotin conjugate.
17. The method of claim 1, wherein the translation system comprises
a cellular or cell-free translation system.
18. A method, comprising: a) providing a tRNA molecule and a
photocleavable mass marker; b) aminoacylating said tRNA molecule
with said photocleavable mass marker to create a misaminoacylated
tRNA; c) introducing said misaminoacylated tRNA into a translation
system under conditions such that said photocleavable mass marker
is incorporated into a nascent protein; and d) exposing said
photocleavable mass marker to electromagnetic radiation.
19. The method of claim 18, wherein said photocleavable mass marker
is attached to an amino acid of said nascent protein such that said
exposing of step (d) results in photocleavage so as to generate an
unmodified amino acid.
20. The method of claim 18, wherein said photocleavable mass marker
is attached to an amino acid of said nascent protein such that said
exposing of step (d) results in photocleavage so as to generate a
modified amino acid.
Description
FIELD OF THE INVENTION
[0001] This invention relates to non-radioactive markers that
facilitate the detection and analysis of nascent proteins
translated within cellular or cell-free translation systems.
Nascent proteins containing these markers can be rapidly and
efficiently detected, isolated and analyzed without the handling
and disposal problems associated with radioactive reagents.
BACKGROUND OF THE INVENTION
[0002] Cells contain organelles, macromolecules and a wide variety
of small molecules. Except for water, the vast majority of the
molecules and macromolecules can be classified as lipids,
carbohydrates, proteins or nucleic acids. Proteins are the most
abundant cellular components and facilitate many of the key
cellular processes. They include enzymes, antibodies, hormones,
transport molecules and components for the cytoskeleton of the
cell.
[0003] Proteins are composed of amino acids arranged into linear
polymers or polypeptides. In living systems, proteins comprise over
twenty common amino acids. These twenty or so amino acids are
generally termed the native amino acids. At the center of every
amino acid is the alpha carbon atom (C.alpha.) which forms four
bonds or attachments with other molecules (FIG. 1). One bond is a
covalent linkage to an amino group (NH.sub.2) and another to a
carboxyl group (COOH) which both participate in polypeptide
formation. A third bond is nearly always linked to a hydrogen atom
and the fourth to a side chain which imparts variability to the
amino acid structure. For example, alanine is formed when the side
chain is a methyl group (--CH.sub.3) and a valine is formed when
the side chain is an isopropyl group (--CH(CH.sub.3).sub.2). It is
also possible to chemically synthesize amino acids containing
different side-chains, however, the cellular protein synthesis
system, with rare exceptions, utilizes native amino acids. Other
amino acids and structurally similar chemical compounds are termed
non-native and are generally not found in most organisms.
[0004] A central feature of all living systems is the ability to
produce proteins from amino acids. Basically, protein is formed by
the linkage of multiple amino acids via peptide bonds such as the
pentapeptide depicted in FIG. 1B. Key molecules involved in this
process are messenger RNA (mRNA) molecules, transfer RNA (tRNA)
molecules and ribosomes (rRNA-protein complexes). Protein
translation normally occurs in living cells and in some cases can
also be performed outside the cell in systems referred to as
cell-free translation systems. In either system, the basic process
of protein synthesis is identical. The extra-cellular or cell-free
translation system comprises an extract prepared from the
intracellular contents of cells. These preparations contain those
molecules which support protein translation and depending on the
method of preparation, post-translational events such as
glycosylation and cleavages as well. Typical cells from which
cell-free extracts or in vitro extracts are made are Escherichia
coli cells, wheat germ cells, rabbit reticulocytes, insect cells
and frog oocytes.
[0005] Both in vivo and in vitro syntheses involve the reading of a
sequence of bases on a mRNA molecule. The mRNA contains
instructions for translation in the form of triplet codons. The
genetic code specifies which amino acid is encoded by each triplet
codon. For each codon which specifies an amino acid, there normally
exists a cognate tRNA molecule which functions to transfer the
correct amino acid onto the nascent polypeptide chain. The amino
acid tyrosine (Tyr) is coded by the sequence of bases UAU and UAC,
while cysteine (Cys) is coded by UGU and UGC. Variability
associated with the third base of the codon is common and is called
wobble.
[0006] Translation begins with the binding of the ribosome to mRNA
(FIG. 2). A number of protein factors associate with the ribosome
during different phases of translation including initiation
factors, elongation factors and termination factors. Formation of
the initiation complex is the first step of translation. Initiation
factors contribute to the initiation complex along with the mRNA
and initiator tRNA (fmet and met) which recognizes the base
sequence UAG. Elongation proceeds with charged tRNAs binding to
ribosomes, translocation and release of the amino acid cargo into
the peptide chain. Elongation factors assist with the binding of
tRNAs and in elongation of the polypeptide chain with the help of
enzymes like peptidyl transferase. Termination factors recognize a
stop signal, such as the base sequence UGA, in the message
terminating polypeptide synthesis and releasing the polypeptide
chain and the mRNA from the ribosome.
[0007] The structure of tRNA is often shown as a cloverleaf
representation (FIG. 3A). Structural elements of a typical tRNA
include an acceptor stem, a D-loop, an anticodon loop, a variable
loop and a T.PSI.C loop. Aminoacylation or charging of tRNA results
in linking the carboxyl terminal of an amino acid to the 2'-(or
3'-) hydroxyl group of a terminal adenosine base via an ester
linkage. This process can be accomplished either using enzymatic or
chemical methods. Normally a particular tRNA is charged by only one
specific native amino acid. This selective charging, termed here
enzymatic aminoacylation, is accomplished by aminoacyl tRNA
synthetases. A tRNA which selectively incorporates a tyrosine
residue into the nascent polypeptide chain by recognizing the
tyrosine UAC codon will be charged by tyrosine with a
tyrosine-aminoacyl tRNA synthetase, while a tRNA designed to read
the UGU codon will be charged by a cysteine-aminoacyl tRNA
synthetase. These synthetases have evolved to be extremely accurate
in charging a tRNA with the correct amino acid to maintain the
fidelity of the translation process. Except in special cases where
the non-native amino acid is very similar structurally to the
native amino acid, it is necessary to use means other than
enzymatic aminoacylation to charge a tRNA.
[0008] Molecular biologists routinely study the expression of
proteins that are coded for by genes. A key step in research is to
express the products of these genes either in intact cells or in
cell-free extracts. Conventionally, molecular biologists use
radioactively labeled amino acid residues such as
.sup.35S-methionine as a means of detecting newly synthesized
proteins or so-called nascent proteins. These nascent proteins can
normally be distinguished from the many other proteins present in a
cell or a cell-free extract by first separating the proteins by the
standard technique of gel electrophoresis and determining if the
proteins contained in the gel possess the specific radioactively
labeled amino acids. This method is simple and relies on gel
electrophoresis, a widely available and practiced method. It does
not require prior knowledge of the expressed protein and in general
does not require the protein to have any special properties. In
addition, the protein can exist in a denatured or unfolded form for
detection by gel electrophoresis. Furthermore, more specialized
techniques such as blotting to membranes and coupled enzymatic
assays are not needed. Radioactive assays also have the advantage
that the structure of the nascent protein is not altered or can be
restored, and thus, proteins can be isolated in a functional form
for subsequent biochemical and biophysical studies.
[0009] Radioactive methods suffer from many drawbacks related to
the utilization of radioactively labeled amino acids. Handling
radioactive compounds in the laboratory always involves a health
risk and requires special laboratory safety procedures, facilities
and detailed record keeping as well as special training of
laboratory personnel. Disposal of radioactive waste is also of
increasing concern both because of the potential risk to the public
and the lack of radioactive waste disposal sites. In addition, the
use of radioactive labeling is time consuming, in some cases
requiring as much as several days for detection of the radioactive
label. The long time needed for such experiments is a key
consideration and can seriously impede research productivity. While
faster methods of radioactive detection are available, they are
expensive and often require complex image enhancement devices.
[0010] The use of radioactive labeled amino acids also does not
allow for a simple and rapid means to monitor the production of
nascent proteins inside a cell-free extract without prior
separation of nascent from preexisting proteins. However, a
separation step does not allow for the optimization of cell-free
activity. Variables including the concentration of ions and
metabolites and the temperature and the time of protein synthesis
cannot be adjusted.
[0011] Radioactive labeling methods also do not provide a means of
isolating nascent proteins in a form which can be further utilized.
The presence of radioactivity compromises this utility for further
biochemical or biophysical procedures in the laboratory and in
animals. This is clear in the case of in vitro expression when
proteins cannot be readily produced in vivo because the protein has
properties which are toxic to the cell. A simple and convenient
method for the detection and isolation of nascent proteins in a
functional form could be important in the biomedical field if such
proteins possessed diagnostic or therapeutic properties. Recent
research has met with some success, but these methods have had
numerous drawbacks.
[0012] Radioactive labeling methods also do not provide a simple
and rapid means of detecting changes in the sequence of a nascent
protein which can indicate the presence of potential disease
causing mutations in the DNA which code for these proteins or
fragments of these proteins. Current methods of analysis at the
protein level rely on the use of gel electrophoresis and
radioactive detection which are slow and not amenable to high
throughput analysis and automation. Such mutations can also be
detected by performing DNA sequence analysis on the gene coding for
a particular protein or protein fragment. However, this requires
large regions of DNA to be sequenced, which is time-consuming and
expensive. The development of a general method which allows
mutations to be detected at the nascent protein level is
potentially very important for the biomedical field.
[0013] Radioactive labeling methods also do not provide a simple
and rapid means of studying the interaction of nascent proteins
with other molecules including compounds which might be have
importance as potential drugs. If such an approach were available,
it could be extremely useful for screening large numbers of
compounds against the nascent proteins coded for by specific genes,
even in cases where the genes or protein has not yet been
characterized. In current technology, which is based on affinity
electrophoresis for screening of potential drug candidates, both in
natural samples and synthetic libraries, proteins must first be
labeled uniformly with a specific marker which often requires
specialized techniques including isolation of the protein and the
design of special ligand markers or protein engineering.
[0014] Special tRNAs, such as tRNAs which have suppressor
properties, suppressor tRNAs, have been used in the process of
site-directed non-native amino acid replacement (SNAAR) (C. Noren
et al., Science 244:182-188, 1989). In SNAAR, a unique codon is
required on the mRNA and the suppressor tRNA, acting to target a
non-native amino acid to a unique site during the protein synthesis
(PCT WO90/05785). However, the suppressor tRNA must not be
recognizable by the aminoacyl tRNA synthetases present in the
protein translation system (Bain et al., Biochemistry 30:5411-21,
1991). Furthermore, site-specific incorporation of non-native amino
acids is not suitable in general for detection of nascent proteins
in a cellular or cell-free protein synthesis system due to the
necessity of incorporating non-sense codons into the coding regions
of the template DNA or the mRNA.
[0015] Products of protein synthesis may also be detected by using
antibody based assays. This method is of limited use because it
requires that the protein be folded into a native form and also for
antibodies to have been previously produced against the nascent
protein or a known protein which is fused to the unknown nascent
protein. Such procedures are time consuming and again require
identification and characterization of the protein. In addition,
the production of antibodies and amino acid sequencing both require
a high level of protein purity.
[0016] In certain cases, a non-native amino acid can be formed
after the tRNA molecule is aminoacylated using chemical reactions
which specifically modify the native amino acid and do not
significantly alter the functional activity of the aminoacylated
tRNA (Promega Technical Bulletin No. 182; tRNA.sup.nscend.TM.:
Non-radioactive Translation Detection System, Sept. 1993). These
reactions are referred to as post-aminoacylation modifications. For
example, the .epsilon.-amino group of the lysine linked to its
cognate tRNA (tRNA.sup.LYS), could be modified with an amine
specific photoaffinity label (U. C. Krieg et al., Proc. Natl. Acad.
Sci. USA 83:8604-08, 1986). These types of post-aminoacylation
modifications, although useful, do not provide a general means of
incorporating non-native amino acids into the nascent proteins. The
disadvantage is that only those non-native amino acids that are
derivatives of normal amino acids can be incorporated and only a
few amino acid residues have side chains amenable to chemical
modification. More often, post-aminoacylation modifications can
result in the tRNA being altered and produce a non-specific
modification of the .alpha.-amino group of the amino acid (e.g. in
addition to the .epsilon.-amino group) linked to the tRNA. This
factor can lower the efficiency of incorporation of the non-native
amino acid linked to the tRNA. Non-specific, post-aminoacylation
modifications of tRNA structure could also compromise its
participation in protein synthesis. Incomplete chain formation
could also occur when the .alpha.-amino group of the amino acid is
modified.
[0017] In certain other cases, a nascent protein can be detected
because of its special and unique properties such as specific
enzymatic activity, absorption or fluorescence. This approach is of
limited use since most proteins do not have special properties with
which they can be easily detected. In many cases, however, the
expressed protein may not have been previously characterized or
even identified, and thus, its characteristic properties are
unknown.
SUMMARY OF THE INVENTION
[0018] The present invention overcomes the problems and
disadvantages associated with current strategies and designs and
provides methods for the labeling, detection, quantitation,
analysis and isolation of nascent proteins produced in a cell-free
or cellular translation system without the use of radioactive amino
acids or other radioactive labels. One embodiment of the invention
is directed to methods for detecting nascent proteins translated in
a translation system. A tRNA molecule is aminoacylated with a
fluorescent marker to create a misaminoacylated tRNA. The
misaminoacylated, or charged, tRNA can be formed by chemical,
enzymatic or partly chemical and partly enzymatic techniques which
place a fluorescent marker into a position on the tRNA molecule
from which it can be transferred into a growing peptide chain.
Markers may comprise native or non-native amino acids with
fluorescent moeities, amino acid analogs or derivatives with
fluorescent moities, detectable labels, coupling agents or
combinations of these components with fluoresecent moieties. The
misaminoacylated tRNA is introduced to the translation system such
as a cell-free extract, the system is incubated and the fluorescent
marker incorporated into nascent proteins.
[0019] It is not intended that the present invention be limited to
the nature of the particular fluorescent moeity. A variety of
fluorescent compounds are contemplated, including fluorescent
compounds that have been derivatized (e.g. with NHS) to be soluble
(e.g. NHS-derivatives of coumarin). Nonetheless, compared to many
other fluorophores with high quantum yields, several BODIPY
compounds and reagents have been empirically found to have the
additional important and unusual property that they can be
incorporated with high efficiency into nascent proteins for both UV
and visible excited fluorescence detection. These methods,
utilitzing fluorescent moeities may be used to detect, isolate and
quantitate such nascent proteins as recombinant gene products, gene
fusion products, truncated proteins caused by mutations in human
genes, enzymes, cytokines, hormones, immunogenic proteins, human
proteins, carbohydrate and lipid binding proteins, nucleic acid
binding proteins, viral proteins, bacterial proteins, parasitic
proteins and fragments and combinations thereof.
[0020] Another embodiment of the invention is directed to methods
for labeling nascent proteins at their amino terminus. An initiator
tRNA molecule, such as methionine-initiator tRNA or
formylmethionine-initiator tRNA is misaminoacylated with a
fluorescent moeity (e.g. a BODIPY moiety) and introduced to a
translation system. The system is incubated and marker is
incorporate at the amino terminus of the nascent proteins. Nascent
proteins containing marker can be detected, isolated and
quantitated. Markers or parts of markers may be cleaved from the
nascent proteins which substantially retain their native
configuration and are functionally active.
[0021] Thus, the present invention contemplates compositions,
methods and systems. In terms of compositions, the present
invention specifically contemplates a tRNA molecule
misaminoacylated with a BODIPY marker.
[0022] In one embodiment, the present invention contemplates a
method, comprising: a) providing a tRNA molecule and a BODIPY
marker; and b) aminoacylating said tRNA molecule with said BODIPY
marker to create a misaminoacylated tRNA. In a particular
embodiment, the method further comprises c) introducing said
misaminoacylated tRNA into a translation system under conditions
such that said marker is incorporated into a nascent protein. In
yet another embodiment, the method further comprises d) detecting
said nascent protein containing said marker. In still another
embodiment, the method further comprises e) isolating said detected
nascent protein.
[0023] The present invention contemplates aminoacylation of the
tRNA molecule by chemical or enzymatic misaminoacylation. The
present invention also contemplates embodiments wherein two or more
different misaminoacylated tRNAs are introduced into the
translation system. In a preferred embodiment, the nascent protein
detected (by virtue of the incorporated marker) is functionally
active.
[0024] It is not intended that the present invention be limited by
the particular nature of the nascent protein. In one embodiment,
the present invention contemplates a method for detecting nascent
proteins which are conjugated to the mRNA message which codes for
all or part of the nascent protein. In general, a variety of
modifications of the nascent protein are envisioned including
post-translational modifications, proteolysis, attachment of an
oligonucleotide through a puromycin linker to the C-terminus of the
protein, and interaction of the nascent protein with other
components of the translation system including those which are
added exogenously.
[0025] It is not intended that the present invention be limited by
the particular nature of the tRNA molecule. In one embodiment, the
tRNA molecule is an initiator tRNA molecule. In another embodiment,
the tRNA molecule is a suppressor tRNA molecule.
[0026] The present invention also contemplates kits. In one
embodiment, the kit comprises a) a first containing means (e.g.
tubes, vials, etc) containing at least one component of a protein
synthesis system; and b) a second containing means containing a
misaminoacylated tRNA, wherein said tRNA is misaminoacylated with a
BODIPY marker. Such kits may include initiator tRNA and/or
suppressor tRNA. Importantly, the kit is not limited to the
particular components of said protein synthesis system; a variety
of components are contemplated (e.g. ribosomes).
[0027] Another embodiment of the invention is directed to methods
for detecting nascent proteins translated in a translation system.
A tRNA molecule is aminoacylated with one component of a binary
marker system. The misaminoacylated, or charged, tRNA can be formed
by chemical, enzymatic or partly chemical and partly enzymatic
techniques which place a component of a binary marker system into a
position on the tRNA molecule from which it can be transferred into
a growing peptide chain. The component of the binary marker system
may comprise native or non-native amino acids, amino acid analogs
or derivatives, detectable labels, coupling agents or combinations
of these components. The misaminoacylated tRNA is introduced to the
translation system such as a cell-free extract, the system is
incubated and the marker incorporated into nascent proteins. The
second component of the binary marker system is then introduced
making the first component incorporated into the nascent protein
specifically detectable. These methods may be used to detect,
isolate and quantitate such nascent proteins as recombinant gene
products, gene fusion products, enzymes, cytokines, hormones,
immunogenic proteins, human proteins, carbohydrate and lipid
binding proteins, nucleic acid binding proteins, viral proteins,
bacterial proteins, parasitic proteins and fragments and
combinations thereof.
[0028] It is not intended that the present invention be limited to
a particular translation system. In one embodiment, a cell-free
translation system is selected from the group consisting of
Escherichia coli lysates, wheat germ extracts, insect cell lysates,
rabbit reticulocyte lysates, frog oocyte lysates, dog pancreatic
lysates, human cell lysates, mixtures of purified or semi-purified
translation factors and combinations thereof. It is also not
intended that the present invention be limited to the particular
reaction conditions employed. However, typcially the cell-free
translation system is incubated at a temperature of between about
25.degree. C. to about 45.degree. C. The present invention
contemplates both continuous flow systems or dialysis systems.
[0029] Another embodiment of the invention is directed to methods
for the detection of nascent proteins translated in a cellular or
cell-free translation system using non-radioactive markers which
have detectable electromagnetic spectral properties. As before, a
non-radioactive marker is misaminoacylated to a tRNA molecule and
the misaminoacylated tRNA is added to the translation system. The
system is incubated to incorporate marker into the nascent
proteins. Nascent proteins containing marker can be detected from
the specific electromagnetic spectral property of the marker.
Nascent proteins can also be isolated by taking advantage of unique
properties of these markers or by conventional means such as
electrophoresis, gel filtration, high-pressure or fast-pressure
liquid chromatography, affinity chromatography, ion exchange
chromatography, chemical extraction, magnetic bead separation,
precipitation or combinations of these techniques.
[0030] Another embodiment of the invention is directed to the
synthesis of nascent proteins containing markers which have
reporter properties when the reporter is brought into contact with
a second agent. Reporter markers are chemical moieties which have
detectable electromagnetic spectral properties when incorporated
into peptides and whose spectral properties can be distinguished
from unincorporated markers and markers attached to tRNA molecules.
As before, tRNA molecules are misaminoacylated, this time using
reported markers. The misaminoacylated tRNAs are added to a
translation system and incubated to incorporate marker into the
peptide. Reporter markers can be used to follow the process of
protein translation and to detect and quantitate nascent proteins
without prior isolation from other components of the protein
synthesizing system.
[0031] Another embodiment of the invention is directed to
compositions comprised of nascent proteins translated in the
presence of markers, isolated and, if necessary, purified in a
cellular or cell-free translation system. Compositions may further
comprise a pharmaceutically acceptable carrier and be utilized as
an immunologically active composition such as a vaccine, or as a
pharmaceutically active composition such as a drug, for use in
humans and other mammals.
[0032] Another embodiment of the invention is directed to methods
for detecting nascent proteins translated in a translation system
by using mass spectrometry. A non-radioactive marker of known mass
is misaminoacylated to a tRNA molecule and the misaminoacylated
tRNA is added to the translation system. The system is incubated to
incorporate the mass marker into the nascent proteins. The mass
spectrum of the translation system is then measured. The presence
of the nascent protein can be directly detected by identifying
peaks in the mass spectrum of the protein synthesis system which
correspond to the mass of the unmodified protein and a second band
at a higher mass which corresponds to the mass of the nascent
protein plus the modified amino acid containing the mass of the
marker. When the mass marker is photocleavable, the assignment of
the second band to a nascent protein containing the mass marker can
be verified by removing the marker with light.
[0033] Another embodiment of the invention is directed to methods
for detecting nascent proteins with mutations which are translated
in a translation system. RNA or DNA coding for the protein which
may contain a possible mutation is added to the translation system.
The system is incubated to synthesize the nascent proteins. The
nascent protein is then separated from the translation system using
an affinity marker located at or close to the N-terminal end of the
protein. The protein is then analyzed for the presence of a
detectable marker located at or close to the N-terminal of the
protein (N-terminal marker). A separate measurement is then made on
a sequence dependent detectable marker located at or close to the
C-terminal end of the protein (C-terminal marker). A comparison is
then made of the level of incorporation of the N-terminal and
C-terminal markers in the nascent protein. It is not intended that
the present invention be limited by the nature of the N- and
C-terminal markers, or the type of affinity marker utilized. A
variety of markers are contemplated. In one embodiment, the
affinity marker comprises an epitope recognized by an antibody or
other binding molecule. In one embodiment, the N-terminal marker
comprises a fluorescent marker (e.g. a BODIPY marker), while the
C-terminal marker comprises a metal binding region (e.g. His
tag).
[0034] The present invention contemplates a variety of methods
wherein the three markers (e.g. the N- and C-terminal markers and
the affinity markers) are introduced into a nascent protein. In one
embodiment, the method comprises: a) providing i) a
misaminoacylated initiator tRNA molecule which only recognizes the
first AUG codon that serves to initiate protein synthesis, said
misaminoacylated initiator tRNA molecule comprising a first marker,
and ii) a nucleic acid template encoding a protein, said protein
comprising a C-terminal marker and (in some embodiments) an
affinity marker; b) introducing said misaminoacylated initiator
tRNA to a translation system comprising said template under
conditions such that a nascent protein is generated, said protein
comprising said first marker, said C-terminal marker and (in some
embodiments) said affinity marker. In one embodiment, the method
further comprises, after step b), isolating said nascent
protein.
[0035] In another embodiment, the method comprises: a) providing i)
a misaminoacylated initiator tRNA molecule which only recognizes
the first AUG codon that serves to initiate protein synthesis, said
misaminoacylated initiator tRNA molecule comprising a first marker,
and ii) a nucleic acid template encoding a protein, said protein
comprising a C-terminal marker and (in some embodiments) an
affinity marker; b) introducing said misaminoacylated initiator
tRNA to a translation system comprising said template under
conditions such that a nascent protein is generated, said protein
comprising said first marker at the N-terminus of said protein, a
C-terminal marker, and (in some embodiments) said affinity marker
adjacent to said first marker. In one embodiment, the method
further comprises, after step b), isolating said nascent
protein.
[0036] In yet another embodiment, the method comprises: a)
providing i) a misaminoacylated tRNA molecule which only recognizes
the first codon designed to serve to initiate protein synthesis,
said misaminoacylated initiator tRNA molecule comprising a first
marker, and ii) a nucleic acid template encoding a protein, said
protein comprising a C-terminal marker and (in some embodiments) an
affinity marker; b) introducing said misaminoacylated initiator
tRNA to a translation system comprising said template under
conditions such that a nascent protein is generated, said protein
comprising said first marker, said C-terminal marker and (in some
embodiments) said affinity marker. In one embodiment, the method
further comprises, after step b), isolating said nascent
protein.
[0037] In still another embodiment, the method comprises: a)
providing i) a misaminoacylated tRNA molecule which only recognizes
the first codon designed to serve to initiate protein synthesis,
said misaminoacylated initiator tRNA molecule comprising a first
marker, and ii) a nucleic acid template encoding a protein, said
protein comprising a C-terminal marker and (in some embodiments) an
affinity marker; b) introducing said misaminoacylated initiator
tRNA to a translation system comprising said template under
conditions such that a nascent protein is generated, said protein
comprising said first marker at the N-terminus of said protein, a
C-terminal marker, and (in some embodiments) said affinity marker
adjacent to said first marker. In one embodiment, the method
further comprises, after step b), isolating said nascent
protein.
[0038] The present invention also contemplates embodiments where
only two markers are employed (e.g. a marker at the N-terminus and
a marker at the C-terminus). In one embodiment, the nascent protein
is non-specifically bound to a solid support (e.g. beads,
microwells, strips, etc.), rather than by the specific interaction
of an affinity marker. In this context, "non-specific" binding is
meant to indicate that binding is not driven by the uniqueness of
the sequence of the nascent protein. Instead, binding can be by
charge interactions. In one embodiment, the present invention
contemplates that the solid support is modified (e.g.
functionalized to change the charge of the surface) in order to
capture the nascent protein on the surface of the solid support. In
one embodiment, the solid support is poly-L-lysine coated. In yet
another embodiment, the solid support is nitrocellulose (e.g.
strips, nicrocellulose containing microwells, etc.). Regardless of
the particular nature of the solid support, the present invention
contemplates that the nascent protein containing the two markers is
captured under conditions that permit the ready detection of the
markers.
[0039] In both the two marker and three marker embodiments
described above, the present invention contemplates that one or
more of the markers will be introduced into the nucleic acid
template by primer extension or PCR. In one embodiment, the present
invention contemplates a primer comprising (on or near the 5'-end)
a promoter, a ribosome binding site ("RBS"), and a start codon
(e.g. ATG), along with a region of complementarity to the template.
In another embodiment, the present invention contemplates a primer
comprising (on or near the 5'-end) a promoter, a ribosome binding
site ("RBS"), a start codon (e.g. ATG), a region encoding an
affinity marker, and a region of complementarity to the template.
It is not intended that the present invention be limited by the
length of the region of complementarity; preferably, the region is
greater than 8 bases in length, more preferably greater than 15
bases in length, and still more preferably greater than 20 bases in
length.
[0040] It is also not intended that the present invention be
limited by the ribosome binding site. In one embodiment, the
present invention contemplates primers comprising the Kozak
sequence, a string of non-random nucleotides (consensus sequence
5'-GCCA/GCCATGG-3') which are present before the translation
initiating first ATG in majority of the mRNAs which are transcribed
and translated in an eukarytic cells. See M. Kozak, Cell 44:283-292
(1986). In another embodiment, the present invention contemplates a
primer comprising the the prokaryotic mRNA ribosome binding site,
which usually contains part or all of a polypurine domain UAAGGAGGU
known as the Shine-Dalgarno (SD) sequence found just 5' to the
translation initiation codon: mRNA
5'-UAAGGAGGU--N.sub.5-10-AUG.
[0041] For PCR, two primers are used. In one embodiment, the
present invention contemplates as the forward primer a primer
comprising (on or near the 5'-end) a promoter, a ribosome binding
site ("RBS"), and a start codon (e.g. ATG), along with a region of
complementarity to the template. In another embodiment, the present
invention contemplates as the forward primer a primer comprising
(on or near the 5'-end) a promoter, a ribosome binding site
("RBS"), a start codon (e.g. ATG), a region encoding an affinity
marker, and a region of complementarity to the template. The
present invention contemplates that the reverse primer, in one
embodiment, comprises (at or near the 5'-end) one or more stop
condons and a region encoding a C-terminus marker (such as a
HIS-tag).
[0042] Another embodiment of the invention is directed to methods
for detecting by electrophoresis (e.g. capillary electrophoresis)
the interaction of molecules with nascent proteins which are
translated in a translation system. A tRNA misaminoacylated with a
detectable marker is added to the protein synthesis system. The
system is incubated to incorporate the detectable marker into the
nascent proteins. One or more specific molecules are then combined
with the nascent proteins (either before or after isolation) to
form a mixture containing nascent proteins/molecule conjugates.
Aliquots of the mixture are then sujected to capillarly
electrophoresis. Nascent proteins/molecule conjugates are
identified by detecting changes in the electrophoretic mobility of
nascent proteins with incorporated markers.
[0043] Other embodiments and advantages of the invention are set
forth, in part, in the description which follows and, in part, will
be obvious from this description, or may be learned from the
practice of the invention.
[0044] Definitions p To facilitate understanding of the invention,
a number of terms are defined below.
[0045] The term "gene" refers to a DNA sequence that comprises
control and coding sequences necessary for the production of a
polypeptide or precursor. The polypeptide can be encoded by a full
length coding sequence or by any portion of the coding sequence so
long as the desired enzymatic activity is retained.
[0046] The term "wild-type" refers to a gene or gene product which
has the characteristics of that gene or gene product when isolated
from a naturally occurring source. A wild-type gene is that which
is most frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene. In contrast,
the term "modified" or "mutant" refers to a gene or gene product
which displays modifications in sequence and or functional
properties (i.e., altered characteristics) when compared to the
wild-type gene or gene product. It is noted that
naturally-occurring mutants can be isolated; these are identified
by the fact that they have altered characteristics when compared to
the wild-type gene or gene product.
[0047] The term "oligonucleotide" as used herein is defined as a
molecule comprised of two or more deoxyribonucleotides or
ribonucleotides, preferably more than three, and usually more than
ten. The exact size will depend on many factors, which in turn
depends on the ultimate function or use of the oligonucleotide. The
oligonucleotide may be generated in any manner, including chemical
synthesis, DNA replication, reverse transcription, or a combination
thereof.
[0048] Because mononucleotides are reacted to make oligonucleotides
in a manner such that the 5' phosphate of one mononucleotide
pentose ring is attached to the 3' oxygen of its neighbor in one
direction via a phosphodiester linkage, an end of an
oligonucleotide is referred to as the "5' end" if its 5' phosphate
is not linked to the 3' oxygen of a mononucleotide pentose ring and
as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of
a subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide, also
may have 5' and 3' ends.
[0049] The term "primer" refers to an oligonucleotide which is
capable of acting as a point of initiation of synthesis when placed
under conditions in which primer extension is initiated. An
oligonucleotide "primer" may occur naturally, as in a purified
restriction digest or may be produced synthetically.
[0050] A primer is selected to have on its 3' end a region that is
"substantially" complementary to a strand of specific sequence of
the template. A primer must be sufficiently complementary to
hybridize with a template strand for primer elongation to occur. A
primer sequence need not reflect the exact sequence of the
template. For example, a non-complementary nucleotide fragment may
be attached to the 5' end of the primer, with the remainder of the
primer sequence being substantially complementary to the strand.
Non-complementary bases or longer sequences can be interspersed
into the primer, provided that the primer sequence has sufficient
complementarity with the sequence of the template to hybridize and
thereby form a template primer complex for synthesis of the
extension product of the primer.
[0051] As used herein, the terms "hybridize" and "hybridization"
refers to the annealing of a complementary sequence to the target
nucleic acid. The ability of two polymers of nucleic acid
containing complementary sequences to find each other and anneal
through base pairing interaction is a well-recognized phenomenon.
Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty
et al., Proc. Natl. Acad. Sci. USA 46:461 (1960). The terms
"annealed" and "hybridized" are used interchangeably throughout,
and are intended to encompass any specific and reproducible
interaction between an oligonucleotide and a target nucleic acid,
including binding of regions having only partial
complementarity.
[0052] The complement of a nucleic acid sequence as used herein
refers to an oligonucleotide which, when aligned with the nucleic
acid sequence such that the 5' end of one sequence is paired with
the 3' end of the other, is in "antiparallel association." Certain
bases not commonly found in natural nucleic acids may be included
in the nucleic acids of the present invention and include, for
example, inosine and 7-deazaguanine. Complementarity need not be
perfect; stable duplexes may contain mismatched base pairs or
unmatched bases. Those skilled in the art of nucleic acid
technology can determine duplex stability empirically considering a
number of variables including, for example, the length of the
oligonucleotide, base composition and sequence of the
oligonucleotide, ionic strength and incidence of mismatched base
pairs.
[0053] The stability of a nucleic acid duplex is measured by the
melting temperature, or "T.sub.m." The T.sub.m of a particular
nucleic acid duplex under specified conditions is the temperature
at which on average half of the base pairs have disassociated.
[0054] The term "probe" as used herein refers to an oligonucleotide
which forms a duplex structure or other complex with a sequence in
another nucleic acid, due to complementarity or other means of
reproducible attractive interaction, of at least one sequence in
the probe with a sequence in the other nucleic acid.
[0055] "Oligonucleotide primers matching or complementary to a gene
sequence" refers to oligonucleotide primers capable of facilitating
the template-dependent synthesis of single or double-stranded
nucleic acids. Oligonucleotide primers matching or complementary to
a gene sequence may be used in PCRs, RT-PCKs and the like. As noted
above, an oligonucleotide primer need not be perfectly
complementary to a target or template sequence. A primer need only
have a sufficient interaction with the template that it can be
extended by template-dependent synthesis.
[0056] As used herein, the term "poly-histidine tract" or (HIS-tag)
refers to the presence of two to ten histidine residues at either
the amino- or carboxy-terminus of a nascent protein A
poly-histidine tract of six to ten residues is preferred. The
poly-histidine tract is also defined functionally as being a number
of consecutive histidine residues added to the protein of interest
which allows the affinity purification of the resulting protein on
a nickel-chelate column, or the indentification of a protein
terminus through the interaction with another molecule (e.g. an
antibody reactive with the HIS-tag).
DESCRIPTIONS OF THE DRAWINGS
[0057] FIG. 1 shows the structure of (A) an amino acid and (B) a
peptide.
[0058] FIG. 2 provides a description of the molecular steps that
occur during protein synthesis in a cellular or cell-free
system.
[0059] FIG. 3 shows a structure of (A) a tRNA molecule and (B)
approaches involved in the aminoacylation of tRNAs.
[0060] FIG. 4 is a schematic representation of the method of
detecting nascent proteins using fluorescent marker amino
acids.
[0061] FIG. 5 shows schemes for synthesis and misaminoacylation to
tRNA of two different marker amino acids, dansyllysine (scheme 1)
and coumarin (scheme 2), with fluorescent properties suitable for
the detection of nascent proteins using gel electrophoresis and UV
illumination.
[0062] FIG. 6(A) shows chemical compounds containing the
2-nitrobenzyl moiety, and FIG. 6(B) shows cleavage of substrate
from a nitrobenzyl linkage.
[0063] FIG. 7 provides examples of photocleavable markers.
[0064] FIG. 8(A) shows chemical variations of PCB, and FIG. 8(B)
depicts possible amino acid linkages.
[0065] FIG. 9 shows the photolysis of PCB.
[0066] FIG. 10 is a schematic representation of the method for
monitoring the production of nascent proteins in a cell-free
protein expression systems without separating the proteins.
[0067] FIG. 11(A) provides examples of non-native amino acids with
reporter properties, FIG. 11(B) shows participation of a reporter
in protein synthesis, and FIG. 11(C) shows synthesis of a
reporter.
[0068] FIG. 12 shows structural components of photocleavable
biotin.
[0069] FIG. 13 is a schematic representation of the method for
introduction of markers at the N-termini of nascent proteins.
[0070] FIG. 14 provides a description of the method of detection
and isolation of marker in nascent proteins.
[0071] FIG. 15 shows the steps in one embodiment for the synthesis
of PCB-lysine.
[0072] FIG. 16 provides an experimental strategy for the
misaminoacylation of tRNA.
[0073] FIG. 17 illustrates dinucleotide synthesis including (i)
deoxycytidine protection, (ii) adenosine protection, and (iii)
dinucleotide synthesis.
[0074] FIG. 18 depicts aminoacylation of a dinucleotide using
marker amino acids.
[0075] FIG. 19 shows the structure of dipyrrometheneboron
difluoride (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)dyes.
[0076] FIG. 20 is a photograph of a gel showing the incorporation
of various fluorsecent molecules into hemolysin during
translation.
[0077] FIG. 21 shows the incorporation of BODIPY-FL into various
proteins. FIG. 21A shows the results visualized using laser based
Molecular Dynamics FluorImager 595, while FIG. 21B shows the
results visualized using a UV-transilluminator.
[0078] FIG. 22A shows a time course of fluorescence labeling. FIG.
22B shows the SDS-PAGE results of various aliquotes of the
translation mixture, demonstrating the sensitivity of the
system.
[0079] FIG. 23A is a bar graph showing gel-free quantitation of an
N-terminal marker introduced into a nascent protein in accordance
with the method of the present invention. FIG. 23B is a bar graph
showing gel-free quantitation of an C-terminal marker of a nascent
protein quantitated in accordance with the method of the present
invention.
[0080] FIG. 24 shows gel results for protease treated and untreated
protein.
[0081] FIG. 25 shows gel results for protein treated with RBCs and
untreated protein.
[0082] FIG. 26A is a gel showing the incorporation of various
fluorescent molecules into .alpha.-hemolysin in E. coli translation
system using misaminoacylated lysyl-tRNA.sup.lys.
[0083] FIG. 26B shows the incorporation of various fluorescent
molecules into luciferase in a TnT wheat germ system using
misaminoacylated lysyl-tRNA.sup.lys.
[0084] FIG. 27 shows gel results of in vitro translation of
.alpha.-HL carried out in the presence of various
fluorescent-tRNAs, including a tRNA-coumarin derivative.
[0085] FIGS. 28A and 28B show mobility shift results by capillary
electrophoresis.
[0086] FIG. 29 are gel results of in vitro translation results
wherein three markers were introduced into a nascent protein.
DESCRIPTION OF THE INVENTION
[0087] As embodied and described herein, the present invention
comprises methods for the non-radioactive labeling and detection of
the products of new or nascent protein synthesis, and methods for
the isolation of these nascent proteins from preexisting proteins
in a cellular or cell-free translation system. As radioactive
labels are not used, there are no special measures which must be
taken to dispose of waste materials. There is also no radioactivity
danger or risk which would prevent further utilization of the
translation product as occurs when using radioactive labels and the
resulting protein product may be used directly or further purified.
In addition, no prior knowledge of the protein sequence or
structure is required which would involve, for example, unique
suppressor tRNAs. Further, the sequence of the gene or mRNA need
not be determined. Consequently, the existence of non-sense codons
or any specific codons in the coding region of the mRNA is not
necessary. Any tRNA can be used, including specific tRNAs for
directed labeling, but such specificity is not required. Unlike
post-translational labeling, nascent proteins are labeled with
specificity and without being subjected to post-translational
modifications which may effect protein structure or function.
[0088] One embodiment of the invention is directed to a method for
labeling nascent proteins synthesized in a translation system.
These proteins are labeled while being synthesized with detectable
markers which are incorporated into the peptide chain. Markers
which are aminoacylated to tRNA molecules, may comprise native
amino acids, non-native amino acids, amino acid analogs or
derivatives, or chemical moieties. These markers are introduced
into nascent proteins from the resulting misaminoacylated tRNAs
during the translation process. Aminoacylation is the process
whereby a tRNA molecule becomes charged. When this process occurs
in vivo, it is referred to as natural aminoacylation and the
resulting product is an aminoacylated tRNA charged with a native
amino acid. When this process occurs through artificial means, it
is called misaminoacylation and a tRNA charged with anything but a
native amino acid molecule is referred to as a misaminoacylated
tRNA.
[0089] According to the present method, misaminoacylated tRNAs are
introduced into a cellular or cell-free protein synthesizing
system, the translation system, where they function in protein
synthesis to incorporate detectable marker in place of a native
amino acid in the growing peptide chain. The translation system
comprises macromolecules including RNA and enzymes, translation,
initiation and elongation factors, and chemical reagents. RNA of
the system is required in three molecular forms, ribosomal RNA
(rRNA), messenger RNA (mRNA) and transfer RNA (tRNA). mRNA carries
the genetic instructions for building a peptide encoded within its
codon sequence. tRNAs contain specific anti-codons which decode the
mRNA and individually carry amino acids into position along the
growing peptide chain. Ribosomes, complexes of rRNA and protein,
provide a dynamic structural framework on which the translation
process, including translocation, can proceed. Within the cell,
individualized aminoacyl tRNA synthetases bind specific amino acids
to tRNA molecules carrying the matching anti-codon creating
aminoacylated or charged tRNAs by the process of aminoacylation.
The process of translation including the aminoacylation or charging
of a tRNA molecule is described in Molecular Cell Biology (J.
Darnell et al. editors, Scientific American Books, N.Y., N.Y.
1991), which is hereby specifically incorporated by reference.
Aminoacylation may be natural or by artificial means utilizing
native amino acids, non-native amino acid, amino acid analogs or
derivatives, or other molecules such as detectable chemicals or
coupling agents. The resulting misaminoacylated tRNA comprises a
native amino acid coupled with a chemical moiety, non-native amino
acid, amino acid derivative or analog, or other detectable
chemicals. These misaminoacylated tRNAs incorporate their markers
into the growing peptide chain during translation forming labeled
nascent proteins which can be detected and isolated by the presence
or absence of the marker.
[0090] Any proteins that can be expressed by translation in a
cellular or cell-free translation system may be nascent proteins
and consequently, labeled, detected and isolated by the methods of
the invention. Examples of such proteins include enzymes such as
proteolytic proteins, cytokines, hormones, immunogenic proteins,
carbohydrate or lipid binding proteins, nucleic acid binding
proteins, human proteins, viral proteins, bacterial proteins,
parasitic proteins and fragments and combinations. These methods
are well adapted for the detection of products of recombinant genes
and gene fusion products because recombinant vectors carrying such
genes generally carry strong promoters which transcribe mRNAs at
fairly high levels. These mRNAs are easily translated in a
translation system.
[0091] Translation systems may be cellular or cell-free, and may be
prokaryotic or eukaryotic. Cellular translation systems include
whole cell preparations such as permeabilized cells or cell
cultures wherein a desired nucleic acid sequence can be transcribed
to mRNA and the mRNA translated.
[0092] Cell-free translation systems are commercially available and
many different types and systems are well-known. Examples of
cell-free systems include prokaryotic lysates such as Escherichia
coli lysates, and eukaryotic lysates such as wheat germ extracts,
insect cell lysates, rabbit reticulocyte lysates, frog oocyte
lysates and human cell lysates. Eukaryotic extracts or lysates may
be preferred when the resulting protein is glycosylated,
phosphorylated or otherwise modified because many such
modifications are only possible in eukaryotic systems. Some of
these extracts and lysates are available commercially (Promega;
Madison, Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington
Heights, Ill.; GIBCO/BRL; Grand Island, N.Y.). Membranous extracts,
such as the canine pancreatic extracts containing microsomal
membranes, are also available which are useful for translating
secretory proteins. Mixtures of purified translation factors have
also been used successfully to translate mRNA into protein as well
as combinations of lysates or lysates supplemented with purified
translation factors such as initiation factor-1 F-1), IF-2, IF-3
(.alpha. or .beta.), elongation factor T (EF-Tu), or termination
factors.
[0093] Cell-free systems may also be coupled
transcription/translation systems wherein DNA is introduced to the
system, transcribed into mRNA and the mRNA translated as described
in Current Protocols in Molecular Biology (F. M. Ausubel et al.
editors, Wiley Interscience, 1993), which is hereby specifically
incorporated by reference. RNA transcribed in eukaryotic
transcription system may be in the form of heteronuclear RNA
(hnRNA) or 5'-end caps (7-methyl guanosine) and 3'-end poly A
tailed mature mRNA, which can be an advantage in certain
translation systems. For example, capped mRNAs are translated with
high efficiency in the reticulocyte lysate system.
[0094] tRNA molecules chosen for misaminoacylation with marker do
not necessarily possess any special properties other than the
ability to function in the protein synthesis system. Due to the
universality of the protein translation system in living systems, a
large number of tRNAs can be used with both cellular and cell-free
reaction mixtures. Specific tRNA molecules which recognize unique
codons, such as nonsense or amber codons (UAG), are not
required.
[0095] Site-directed incorporation of the nonnative analogs into
the protein during translation is also not required. Incorporation
of markers can occur anywhere in the polypeptide and can also occur
at multiple locations. This eliminates the need for prior
information about the genetic sequence of the translated mRNA or
the need for modifying this genetic sequence.
[0096] In some cases, it may be desirable to preserve the
functional properties of the nascent protein. A subset of tRNAs
which will incorporate markers at sites which do not interfere with
protein function or structure can be chosen. Amino acids at the
amino or carboxyl terminus of a polypeptide do not alter
significantly the function or structure. tRNA molecules which
recognize the universal codon for the initiation of protein
translation (AUG), when misaminoacylated with marker, will place
marker at the amino terminus. Prokaryotic protein synthesizing
systems utilize initiator tRNA.sup.fMet molecules and eukaryotic
systems initiator tRNA.sup.Met molecules. In either system, the
initiator tRNA molecules are aminoacylated with markers which may
be non-native amino acids or amino acid analogs or derivatives that
possess marker, reporter or affinity properties. The resulting
nascent proteins will be exclusively labeled at their amino
terminus, although markers placed internally do not necessarily
destroy structural or functional aspects of a protein. For example,
a tRNA.sup.LYS may be misaminoacylated with the amino acid
derivative dansyllysine which does not interfere with protein
function or structure. In addition, using limiting amounts of
misaminoacylated tRNAs, it is possible to detect and isolate
nascent proteins having only a very small fraction labeled with
marker which can be very useful for isolating proteins when the
effects of large quantities of marker would be detrimental or are
unknown.
[0097] tRNAs molecules used for aminoacylation are commercially
available from a number of sources and can be prepared using
well-known methods from sources including Escherichia coli, yeast,
calf liver and wheat germ cells (Sigma Chemical; St. Louis, Mo.;
Promega; Madison, Wis.; Boehringer Mannheim Biochemicals;
Indianapolis, Ind.). Their isolation and purification mainly
involves cell-lysis, phenol extraction followed by chromatography
on DEAE-cellulose. Amino-acid specific tRNA, for example
tRNA.sup.fMet, can be isolated by expression from cloned genes and
overexpressed in host cells and separated from total tRNA by
techniques such as preparative polyacrylamide gel electrophoresis
followed by band excision and elution in high yield and purity
(Seong and RajBhandary, Proc. Natl. Acad. Sci. USA 84:334-338,
1987). Run-off transcription allows for the production of any
specific tRNA in high purity, but its applications can be limited
due to lack of post-transcriptional modifications (Bruce and
Uhlenbeck, Biochemistry 21:3921, 1982).
[0098] Misaminoacylated tRNAs are introduced into the cellular-or
cell-free protein synthesis system. In the cell-free protein
synthesis system, the reaction mixture contains all the cellular
components necessary to support protein synthesis including
ribosomes, tRNA, rRNA, spermidine and physiological ions such as
magnesium and potassium at appropriate concentrations and an
appropriate pH. Reaction mixtures can be normally derived from a
number of different sources including wheat germ, E. coli (S-30),
red blood cells (reticulocyte lysate,) and oocytes, and once
created can be stored as aliquots at about +4.degree. C. to
-70.degree. C. The method of preparing such reaction mixtures is
described by J. M. Pratt (Transcription and Translation, B. D.
Hames and S. J. Higgins, Editors, p. 209, IRL Press, Oxford, 1984)
which is hereby incorporated by reference. Many different
translation systems are commercially available from a number of
manufacturers.
[0099] The misaminoacylated tRNA is added directly to the reaction
mixture as a solution of predetermined volume and concentration.
This can be done directly prior to storing the reaction mixture at
-70.degree. C. in which case the entire mixture is thawed prior to
initiation of protein synthesis or prior to the initiation of
protein synthesis. Efficient incorporation of markers into nascent
proteins is sensitive to the final pH and magnesium ion
concentration. Reaction mixtures are normally about pH 6.8 and
contain a magnesium ion concentration of about 3 mM. These
conditions impart stability to the base-labile aminoacyl linkage of
the misaminoacylated tRNA. Aminoacylated tRNAs are available in
sufficient quantities from the translation extract.
Misaminoacylated tRNAs charged with markers are added at between
about 1.0 .mu.g/ml to about 1.0 mg/ml, preferably at between about
10 .mu.g/ml to about 500 .mu.g/ml, and more preferably at about 150
.mu.g/ml.
[0100] Initiation of protein synthesis occurs upon addition of a
quantity of mRNA or DNA to the reaction mixture containing the
misaminoacylated tRNAs. mRNA molecules may be prepared or obtained
from recombinant sources, or purified from other cells by procedure
such as poly-dT chromatography. One method of assuring that the
proper ratio of the reaction mixture components is to use
predetermined volumes that are stored in convenient containers such
as vials or standard microcentrifuge tubes. For example, DNA and/or
mRNA coding for the nascent proteins and the misaminoacylated tRNA
solution are premixed in proper amounts and stored separately in
tubes. Tubes are mixed when needed to initiate protein
synthesis.
[0101] Translations in cell-free systems generally require
incubation of the ingredients for a period of time. Incubation
times range from about 5 minutes to many hours, but is preferably
between about thirty minutes to about five hours and more
preferably between about one to about three hours. Incubation may
also be performed in a continuous manner whereby reagents are
flowed into the system and nascent proteins removed or left to
accumulate using a continuous flow system (A. S. Spirin et al.,
Sci. 242:1162-64, 1988). This process may be desirable for large
scale production of nascent proteins. Incubations may also be
performed using a dialysis system where consumable reagents are
available for the translation system in an outer reservoir which is
separated from larger components of the translation system by a
dialysis membrane [Kim, D., and Choi, C. (1996) Biotechnol Prog 12,
645-649]. Incubation times vary significantly with the volume of
the translation mix and the temperature of the incubation.
Incubation temperatures can be between about 4.degree. C. to about
60.degree. C., and are preferably between about 15.degree. C. to
about 50.degree. C., and more preferably between about 25.degree.
C. to about 45.degree. C. and even more preferably at about
25.degree. C. or about 37.degree. C. Certain markers may be
sensitive to temperature fluctuations and in such cases, it is
preferable to conduct those incubations in the non-sensitive
ranges. Translation mixes will typically comprise buffers such as
Tris-HCl, Hepes or another suitable buffering agent to maintain the
pH of the solution between about 6 to 8, and preferably at about 7.
Again, certain markers may be pH sensitive and in such cases, it is
preferable to conduct incubations outside of the sensitive ranges
for the marker. Translation efficiency may not be optimal, but
marker utility will be enhanced. Other reagents which may be in the
translation system include dithiothreitol (DTT) or
2-mercaptoethanol as reducing agents, RNasin to inhibit RNA
breakdown, and nucleoside triphosphates or creatine phosphate and
creatine kinase to provide chemical energy for the translation
process.
[0102] In cellular protein synthesis, it is necessary to introduce
misaminoacylated tRNAs or markers into intact cells, cell
organelles, cell envelopes and other discrete volumes bounded by an
intact biological membrane, which contain a protein synthesizing
system. This can be accomplished through a variety of methods that
have been previously established such as sealing the tRNA solution
into liposomes or vesicles which have the characteristic that they
can be induced to fuse with cells. Fusion introduces the liposome
or vesicle interior solution containing the tRNA into the cell.
Alternatively, some cells will actively incorporate liposomes into
their interior cytoplasm through phagocytosis. The tRNA solution
could also be introduced through the process of cationic detergent
mediated lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA
84:7413-17, 1987), or injected into large cells such as oocytes.
Injection may be through direct perfusion with micropipettes or
through the method of electroporation.
[0103] Alternatively, cells can be permeabilized by incubation for
a short period of time in a solution containing low concentrations
of detergents in a hypotonic media. Useful detergents include
Nonidet-P 40 (NP40), Triton X-100 (TX-100) or deoxycholate at
concentrations of about 0.01 nM to 1.0 mM, preferably between about
0.1 .mu.M to about 0.01 mM, and more preferably about 1 .mu.M.
Permeabilized cells allow marker to pass through cellular membranes
unaltered and be incorporated into nascent proteins by host cell
enzymes. Such systems can be formed from intact cells in culture
such as bacterial cells, primary cells, immortalized cell lines,
human cells or mixed cell populations. These cells may, for
example, be transfected with an appropriate vector containing the
gene of interest, under the control of a strong and possibly
regulated promoter. Messages are expressed from these vectors and
subsequently translated within cells. Intact misaminoacylated tRNA
molecules, already charged with a non-radioactive marker could be
introduced to cells and incorporated into translated product.
[0104] One example of the use of misaminoacylation to detect
nascent protein is schematically represented in FIG. 4. A tRNA
molecule is misaminoacylated with the marker which is highly
fluorescent when excited with UW (ultraviolet) radiation. The
misaminoacylated tRNA is then introduced into a cell-free protein
synthesis extract and the nascent proteins containing the marker
analog produced. Proteins in the cell-free extract are separated by
polyacrylamide gel electrophoresis (PAGE). The resulting gel
contains bands which correspond to all of the proteins present in
the cell-free extract. The nascent protein is identified upon UV
illumination of the gel by detection of fluorescence from the band
corresponding to proteins containing marker. Detection can be
through visible observation or by other conventional means of
fluorescence detection.
[0105] The misaminoacylated tRNA can be formed by natural
aminoacylation using cellular enzymes or misaminoacylation such as
chemical misaminoacylation. One type of chemical misaminoacylation
involves truncation of the tRNA molecule to permit attachment of
the marker or marker precursor. For example, successive treatments
with periodate plus lysine, pH 8.0, and alkaline phosphatase
removes 3'-terminal residues of any tRNA molecule generating
tRNA-OH-3' with a mononucleotide or dinucleotide deletion from the
3'-terminus (Neu and Heppel, J. Biol. Chem. 239:2927-34, 1964).
Alternatively, tRNA molecules may be genetically manipulated to
delete specific portions of the tRNA gene. The resulting gene is
transcribed producing truncated tRNA molecules (Sampson and
Uhlenbeck, Proc. Natl. Acad. Sci. USA 85:1033-37, 1988). Next, a
dinucleotide is chemically linked to a modified amino acid or other
marker by, for example, acylation. Using this procedure, markers
can be synthesized and acylated to dinucleotides in high yield
(Hudson, J. Org. Chem. 53:617-624, 1988; Happ et al., J. Org. Chem.
52:5387-91, 1987). These modified groups are bound together and
linked via the dinucleotide to the truncated tRNA molecules in a
process referred to as ligase coupling (FIG. 3B).
[0106] A different bond is involved in misaminoacylation (FIG. 3B,
link B) than the bond involved with activation of tRNA by aminoacyl
tRNA synthetase (FIG. 3B, link A). As T4 RNA ligase does not
recognize the acyl substituent, tRNA molecules can be readily
misaminoacylated with few chemical complications or side reactions
(link B, FIG. 3B) (T. G. Heckler et al., Biochemistry 23:1468-73,
1984; and T. G. Heckler et al., Tetrahedron 40:87-94, 1984). This
process is insensitive to the nature of the attached amino acid and
allows for misaminoacylation using a variety of non-native amino
acids. In contrast, purely enzymatic aminoacylation (link A) is
highly sensitive and specific for the structures of substrate tRNA
and amino acids.
[0107] Markers are basically molecules which will be recognized by
the enzymes of the translation process and transferred from a
charged tRNA into a growing peptide chain. To be useful, markers
must also possess certain physical and physio-chemical properties.
Therefore, there are multiple criteria which can be used to
identify a useful marker. First, a marker must be suitable for
incorporation into a growing peptide chain. This may be determined
by the presence of chemical groups which will participate in
peptide bond formation. Second, markers should be attachable to a
tRNA molecule. Attachment is a covalent interaction between the
3'-terminus of the tRNA molecule and the carboxy group of the
marker or a linking group attached to the marker and to a truncated
tRNA molecule. Linking groups may be nucleotides, short
oligonucleotides or other similar molecules and are preferably
dinucleotides and more preferably the dinucleotide CA. Third,
markers should have one or more physical properties that facilitate
detection and possibly isolation of nascent proteins. Useful
physical properties include a characteristic electromagnetic
spectral property such as emission or absorbance, magnetism,
electron spin resonance, electrical capacitance, dielectric
constant or electrical conductivity.
[0108] Useful markers are native amino acids coupled with a
detectable label, detectable non-native amino acids, detectable
amino acid analogs and detectable amino acid derivatives. Labels
and other detectable moieties may be ferromagnetic, paramagnetic,
diamagnetic, luminescent, electrochemiluminescent, fluorescent,
phosphorescent, chromatic or have a distinctive mass. Fluorescent
moieties which are useful as markers include dansyl fluorophores,
coumarins and coumarin derivatives, fluorescent acridinium moieties
and benzopyrene based fluorophores. Preferably, the fluorescent
marker has a high quantum yield of fluorescence at a wavelength
different from native amino acids and more preferably has high
quantum yield of fluoresence can be excited in both the UV and
visible portion of the spectrum. Upon excitation at a preselected
wavelength, the marker is detectable at low concentrations either
visually or using conventional fluorescence detection methods.
Electrochemiluminescent markers such as ruthenium chelates and its
derivatives or nitroxide amino acids and their derivatives are
preferred when extreme sensitivity is desired (J. DiCesare et al.,
BioTechniques 15:152-59, 1993). These markers are detectable at the
femtomolar ranges and below.
[0109] In addition to fluorescent markers, a variety of markers
possessing other specific physical properties can be used to detect
nascent protein production. In general, these properties are based
on the interaction and response of the marker to electromagnetic
fields and radiation and include absorption in the UV, visible and
infrared regions of the electromagnetic spectrum, presence of
chromophores which are Raman active, and can be further enhanced by
resonance Raman spectroscopy, electron spin resonance activity and
nuclear magnetic resonances and use of a mass spectrometer to
detect presence of a marker with a specific molecular mass. These
electromagnetic spectroscopic properties are preferably not
possessed by native amino acids or are readily distinguishable from
the properties of native amino acids. For example, the amino acid
tryptophan absorbs near 290 nm, and has fluorescent emission near
340 nm when excited with light near 290 nm. Thus, tryptophan
analogs with absorption and/or fluorescence properties that are
sufficiently different from tryptophan can be used to facilitate
their detection in proteins.
[0110] Many different modified amino acids which can be used as
markers are commercially available (Sigma Chemical; St. Louis, Mo.;
Molecular Probes; Eugene, Oreg.). One such marker is
N.epsilon.-dansyllysine created by the misarninoacylation of a
dansyl fluorophore to a tRNA molecule (FIG. 5; scheme 1). The
.alpha.-amino group of N.epsilon.-dansyllysine is first blocked
with NVOC (ortho-nitro veratryl oxycarbonyl chloride) and the
carboxyl group activated with cyanomethyl ester. Misaminoacylation
is performed as described. The misaminoacylated tRNA molecules are
then introduced into the protein synthesis system, whereupon the
dansyllysine is incorporated directly into the newly synthesized
proteins.
[0111] Another such marker is a fluorescent amino acid analog based
on the highly fluorescent molecule coumarin (FIG. 5; scheme 2).
This fluorophore has a much higher fluorescence quantum yield than
dansyl chloride and can facilitate detection of much lower levels
of nascent protein. In addition, this coumarin derivative has a
structure similar to the native amino acid tryptophan. These
structural similarities are useful where maintenance of the nascent
proteins' native structure or function are important or desired.
Coumarin is synthesized as depicted in FIG. 5 (scheme 2).
Acetamidomalonate is alkylated with a slight excess of
4-bromomethyl coumarin (Aldrich Chemicals; Milwaukee; Wis.) in the
presence of sodium ethoxide followed by acid hydrolysis. The
corresponding amino acid as a hydrochloride salt that can be
converted to the free amino acid analog.
[0112] The coumarin derivative can be used most advantageously if
it misamino-acylates the tryptophan-tRNA, either enzymatically or
chemically. When introduced in the form of the misaminoacylated
tryptophan-tRNA, the coumarin amino acid will be incorporated only
into tryptophan positions. By controlling the concentration of
misaminoacylated tRNAs or free coumarin derivatives in the
cell-free synthesis system, the number of coumarin amino acids
incorporated into the nascent protein can also be controlled. This
procedure can be utilized to control the amount of most any markers
in nascent proteins.
[0113] Markers can be chemically synthesized from a native amino
acid and a molecule with marker properties which cannot normally
function as an amino acid. For example a highly fluorescent
molecule can be chemically linked to a native amino acid group. The
chemical modification can occur on the amino acid side-chain,
leaving the carboxyl and amino functionalities free to participate
in a polypeptide bond formation. Highly fluorescent dansyl chloride
can be linked to the nucleophilic side chains of a variety of amino
acids including lysine, arginine, tyrosine, cysteine, histidine,
etc., mainly as a sulfonamide for amino groups or sulfate bonds to
yield fluorescent derivatives. Such derivatization leaves the
ability to form peptide bond intact, allowing the normal
incorporation of dansyllysine into a protein.
[0114] A number of factors determine the usefulness of a marker
which is to be incorporated into nascent proteins through
misaininoacylated tRNAs. These include the ability to incorporate
the marker group into the protein through the use of a
misaminoacylated tRNA in a cell-free or cellular protein synthesis
system and the intrinsic detectability of the marker once it is
incorporated into the nascent protein. In general, markers with
superior properties will allow shorter incubation times and require
smaller samples for the accurate detection of the nascent proteins.
These factors directly influence the usefulness of the methods
described. In the case of fluorescent markers used for the
incorporation into nascent proteins, favorable properties can be
but are not limited to, small size, high quantum yield of
fluorescence, and stability to prolonged light exposure (bleach
resistance).
[0115] Even with knowledge of the above factors, the ability to
incorporate a specific marker into a protein using a specific
cell-free or cellular translation system is difficult to determine
a priori since it depends on the detailed interaction of the marker
group with components of the protein translational synthesis system
including the tRNA, initiation or elongation factors and components
of the ribosome. While it is generally expected that markers with
smaller sizes can be accommodated more readily into the ribosome,
the exact shape of the molecule and its specific interactions in
the ribosomal binding site will be the most important determinant.
For this reason, it is possible that some markers which are larger
in size can be more readily incorporated into nascent proteins
compared to smaller markers. For example, such factors are very
difficult to predict using known methods of molecular modeling.
[0116] One group of fluorophores with members possessing several
favorable properties (including favorable interactions with
components of the protein translational synthesis system) is the
group derived from dipyrrometheneboron difluoride derivatives
(BODIPY) (FIG. 19). Compared to a variety of other commonly used
fluorophores with advantageous properties such as high quantum
yields, some BODIPY compounds have the additional unusual property
that they are highly compatible with the protein synthesis system.
The core structure of all BODIPY fluorophores is
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. See U.S. Pat. Nos.
4,774,339; 5,187,288; 5,248,782; 5,274,113; 5,433,896; 5,451,663,
all hereby incorporated by reference. A central feature is a
difluoroboron as shown in FIG. 19. All BODIPY fluorophores have
several desirable properties for a marker (Molecular Probes
Catalog, pages 13-18) including a high extinction coefficient, high
fluorescence quantum yield, spectra that are insensitive to solvent
polarity and pH, narrow emission bandwidth resulting in a higher
peak intensity compared to other dyes such as fluoresceine, absence
of ionic charge and enhanced photostability compared to
fluorosceine. The addition of substituents to the basic BODIPY
structure which cause additional conjugation can be used to shift
the wavelength of excitation or emission to convenient wavelengths
compatible with the means of detection.
[0117] These dyes were described for the first time by Vos de Waal
et al. (1977) and its fluorescence properties subsequently
described by Wories [See Wories et al., "A novel water-soluble
fluorescent probe: Synthesis, luminescence and biological
properties of the sodium salt of the 4-sulfonato-3,3',
5',5-tetramethyl-2,2'-pyrromethen-1,1'-BF.sub.2 complex," Recl.
Trav. Chim. PAYSBAS 104, 288 (1985). Dyes derived from
dipyrrometheneboron difluoride have additional characteristics that
make them suitable for incorporation into nascent proteins. Simple
alkyl derivatives of the fluorophore
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene have been described by
Treibs & Kreuzer, [Difluorboryl-komplexe von di- und
tripyrrylmethenen, LIEBIGS ANNALEN CHEM. 718,208 (1968)] and by
Worries, Kopek, Lodder, & Lugtenburg, [A novel water-soluble
fluorescent probe: Synthesis, luminescence and biological
properties of the sodium salt of the
4-sulfonato-3,3',5,5'-tetramethyl-2,2'-pyrromethen-1,1'-BF.su- b.2
complex, RECL. TRAV. CHIM. PAYS-BAS 104, 288 (1985)] as being
highly fluorescent with spectral properties that are similar to
fluorescein, with maximum absorbance at about 490 to 510 nm and
maximum emission at about 500 to 530 nm. U.S. Pat. No. 4,774,339 to
Haugland et al. (1988) ('339 patent) (hereby incorporated by
reference) describes 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
(dipyrrometheneboron difluoride) dyes including hydrogen, halogen,
alkyl, cycloalkyl, aryl, arylalkyl, acyl, and sulfo-substituted
derivatives that contain reactive groups suitable for conjugation
to biomolecules, that have good photostability, and which have
fluorescein-like spectra. As described in the '339 patent, and by
Pavlopoulos, et al., [Laser action from a
tetramethylpyrromethene-BF.sub.2 complex, APP. OPTICS 27, 4998
(1988)], the emission of the alkyl derivatives of
4,4-difluoro-4-bora-3a,4a-diaza-- s-indacene fluorescent dyes
clearly overlaps that of fluorescein. The overlap allows the alkyl
derivatives of dipyrrometheneboron difluoride to be used with the
same optical equipment as used with fluorescein-based dyes without
modification of the excitation sources or optical filters.
Similarly, aryl/heteroaryl substituents in the dipyrrometheneboron
difluoride cause the maximum of absorbance/emission to shift into
longer wavelengths (See U.S. Pat. No. 5,451,663 hereby incorporated
by reference).
[0118] A variety of BODIPY molecules are commercially available in
an amine reactive form which can be used to derivitize
aminoacylated tRNAs to yield a misaminoacylated tRNA with a BODIPY
marker moiety. One example of a compound from this family which
exhibits superior properties for incorporation of a detectable
marker into nascent proteins is
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene
(BODIPY-FL). When the sulfonated N-hydroxysuccinimide (NHS)
derivative of BODIPY-FL is used to misaminoacylate an E. coli
initiator tRNA.sup.fmet, the nascent protein produced can be easily
detected on polyacyrlamide gels after electrophoresis using a
standard UV-transilluminator and photographic or CCD imaging
system. This can be accomplished by using purified tRNA.sup.fmet
which is first aminoacylated with methionine and then the
.alpha.-amino group of methionine is specifically modified using
N-hydroxysuccinimide BODIPY. Before the modification reaction, the
tRNA.sup.fmet is charged maximally (>90%) and confirmed by using
.sup.35S-methionine and acid-urea gels [Varshney, U., Lee, C. P.,
and RajBhandary, U. L. 1991. Direct analysis of aminoacylation
levels of tRNA in vitro. J. Biol. Chem. 266:24712-24718].
[0119] Less than 10 nanoliters of a commercially available E. coli
extract (E. coli T7 translation system, Promega, Madison, Wis.) are
needed for analysis corresponding to less than 1 ng of synthesized
protein. Incubation times required to produce detectable protein is
approximately 1 hour but can be as little as 5 minutes. BODIPY-FL
can also be detected with higher sensitivity using commercially
available fluorescent scanners with 488 nm excitation and emission
measurement above 520 nm. Similar tests using other commercially
available dyes including NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), and
Pyrine-PyMPO show approximately an order of magnitude reduction in
fluorescence making them difficult to detect using standard
laboratory equipment such as a UV-transilluminator or fluorescent
scanner. It has previously been shown that fluorescent markers such
as 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3,-diaminopropr- ionic
acid (NBD-DAP) and coumarin could be incorporated into proteins
using misaminoacylated tRNAs. However, detection of nascent
proteins containing these markers was only demonstrated using
highly sensitive instrumentation such a fluorescent spectrometer or
a microspectrofluorimeter and often require indirect methods such
as the use of fluorescence resonance energy transfer (FRET)
(Turcatti, G., Nemeth, K., Edgerton, M. D., Meseth, U., Talabot,
F., Peitsch, M., Knowles, J., Vogel, H., and Chollet, A. (1996) J
Biol Chem 271(33), 19991-8; Kudlicki, W., Odom, O. W., Kramer, G.,
and Hardesty, B. (1994) J Mol Biol 244(3), 319-31). Such
instruments are generally not available for routine use in a
molecular biology laboratory and only with special adaptation can
be equipped for measurement of fluorescent bands on a gel.
[0120] An additional advantage of BODIPY-FL as a marker is the
availability of monoclonal antibodies directed against it which can
be used to affinity purify nascent proteins containing said marker.
One example of such a monoclonal antibody is anti-BODIPY-FL
antibody (Cat# A-5770, Molecular Probes, Eugene, Oreg.). This
combined with the ability incorporate BODIPY-FL into nascent
proteins with high efficiency relative to other commercially
available markers using misaminoacylated tRNAs facilitates more
efficient isolation of the nascent protein. These antibodies
against BODIPY-FL can be used for quantitation of incorporation of
the BODIPY into the nascent protein.
[0121] A marker can also be modified after the tRNA molecule is
aminoacylated or misaminoacylated using chemical reactions which
specifically modify the marker without significantly altering the
functional activity of the aminoacylated tRNA. These types of
post-aminoacylation modifications may facilitate detection,
isolation or purification, and can sometimes be used where the
modification allow the nascent protein to attain a native or more
functional configuration.
[0122] Fluorescent and other markers have detectable
electromagnetic spectral properties that can be detected by
spectrometers and distinguished from the electromagnetic spectral
properties of native amino acids. Spectrometers which are most
useful include fluorescence, Raman, absorption, electron spin
resonance, visible, infrared and ultraviolet spectrometers. Other
markers, such as markers with distinct electrical properties can be
detected by an apparatus such as an ammeter, voltmeter or other
spectrometer. Physical properties of markers which relate to the
distinctive interaction of the marker with an electromagnetic field
is readily detectable using instruments such as fluorescence,
Raman, absorption, electron spin resonance spectrometers. Markers
may also undergo a chemical, biochemical, electrochemical or
photochemical reaction such as a color change in response to
external forces or agents such as an electromagnetic field or
reactant molecules which allows its detection.
[0123] One class of fluorescent markers contemplated by the present
invention is the class of small peptides that can specifically bind
to molecules which, upon binding, are detectable. One example of
this approach is the peptide having the sequence of
WEAAAREACCRECCARA. This sequence (which contains four cysteine
residues) allows the peptide to specifically bind the
non-fluorescent dye molecule
4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FLASH, which stands
for fluorescein arsenic helix binder). This dye has the interesting
property that, upon binding, it becomes fluorescent. In other
words, fluorescence is observed only when this specific peptide
sequence is present in the nascent protein. So by putting the
peptide sequence at the N- or C-terminal, one can easily monitor
the amount of protein synthesized. This peptide sequence can be
introduced by designing the nucleic acid primers such that they
carry a region encoding the peptide sequence.
[0124] Regardless of which class of fluorescent compounds is used,
detection normally first involves physical separation of the
nascent proteins from other biomolecules present in the cellular or
cell-free protein synthesis system. Protein separation can be
performed using, for example, gel electrophoresis or column
chromatography and can be further facilitated with affinity markers
which uniquely bind acceptor groups. Detection of a marker
containing a fluorophore by gel electrophoresis can be accomplished
using conventional fluorescence detection methods.
[0125] After protein synthesis in a cell-free system, the reaction
mixture, which contains all of the biomolecules necessary for
protein synthesis as well as nascent proteins, is loaded onto a gel
which may be composed of polyacrylamide or agarose (R. C. Allen et
al., Gel Electrophoresis and Isoelectric Focusing of Proteins,
Walter de Gruyter, New York 1984). This mixture also contains the
misaminoacylated tRNAs bearing the marker as well as uncharged
tRNAs. Subsequent to loading the reaction mixture, a voltage is
applied which spatially separates the proteins on the gel in the
direction of the applied electric field. The proteins separate and
appear as a set of discrete or overlapping bands which can be
visualized using a pre- or post-gel staining technique such as
Coomasie blue staining. The migration of the protein band on the
gel is a function of the molecular weight of the protein with
increasing distance from the loading position being a function of
decreasing molecular weight. Bands on the gel which contain nascent
proteins will exhibit fluorescence when excited at a suitable
wavelength. These bands can be detected visually, photographically
or spectroscopically and, if desired, the nascent proteins purified
from gel sections.
[0126] For example, if BODIPY-FL is used as a marker, nascent
proteins will fluoresce at 510 nm when excited by UV illumination.
This fluorescence can be detected visually by simply using a
standard hand-held UV illuminator or a transilluminator.
Approximately 10 nanograms (ng) of the protein alpha-hemolysin is
detectable using this method. Also useful are electronic imaging
devices which can rapidly screen and identify very low
concentrations of markers such as a fluorescent scanner based on a
low-temperature CCD imager. In this case as low as 0.3 ng of
protein can be detected.
[0127] The molecular weight and quantity of the nascent protein can
be determined by comparison of its band-position on the gel with a
set of bands of proteins of predetermined molecular weight which
are fluorescently labeled. For example, a nascent protein of
molecular weight 25,000 could be determined because of its relative
position on the gel relative to a calibration gel containing the
commercially available standard marker proteins of known quantities
and with known molecular weights (bovine serum albumin, 66 kD;
porcine heart fumarase, 48.5 kD; carbonic anhydrase, 29 kD,
.beta.-lactoglobulin, 18.4 kD; .alpha.-lactoglobulin, 14.2 kD;
Sigma Chemical; St. Louis, Mo.).
[0128] Calibration proteins may also contain a similar markers for
convenient detection using the same method as the gel bearing the
nascent protein. This can be accomplished in many cases by directly
reacting the calibration proteins with a molecule similar to the
marker. For example, the calibration proteins can be modified with
dansyl chloride so as to obtain their fluorescent derivatives (R.
E. Stephens, Anal. Biochem. 65, 369-79, 1975). Alternatively, the
proteins could be labeled with an NHS derivitive of BODIPY-FL.
These fluorescent proteins can be analyzed using PAGE. Combined
detection of these fluorescent calibration proteins along with that
of nascent protein which contains fluorescent marker analog will
accurately determine both the molecular weight and quantity of the
nascent protein synthesized. If necessary, the amounts of marker
within each calibration and nascent protein can be determined to
provide an accurate quantitation. Proteins with predetermined
levels of fluorescent markers can be used advantageously to provide
for quantitation of the gel bearing the nascent protein. This could
be accomplished by genetically engineering a calibration protein so
that it contains a specific reactive residue such as cysteine so
that only one fluorescent dye will be attached per protein.
[0129] Other methods of protein separation are also useful for
detection and subsequent isolation and purification of nascent
proteins containing markers. For example, proteins can be separated
using capillary electrophoresis, isoelectric focusing, low pressure
chromatography and high-performance or fast-pressure liquid
chromatography (HPLC or FPLC). In these cases, the individual
proteins are separated into fractions which can be individually
analyzed by fluorescent detectors at the emission wavelengths of
the markers. Alternatively, on-line fluorescence detection can be
used to detect nascent proteins as they emerge from the column
fractionation system. A graph of fluorescence as a function of
retention time provides information on both the quantity and purity
of nascent proteins produced.
[0130] Another embodiment of the invention is directed to a method
for labeling, detecting and, if desired, isolating and purifying
nascent proteins, as described above, containing cleavable markers.
Cleavable markers comprise a chemical structure which is sensitive
to external effects such as physical or enzymatic treatments,
chemical or thermal treatments, electromagnetic radiation such as
gamma rays, x-rays, ultraviolet light, visible light, infrared
light, microwaves, radio waves or electric fields. The marker is
aminoacylated to tRNA molecules as before using conventional
technology or misaminoacylated and added to a translation system.
After incubation and production of nascent proteins, marker can be
cleaved by the application of specified treatments and nascent
proteins detected. Alternatively, nascent proteins may also be
detected and isolated by the presence or absence of the cleaved
marker or the chemical moiety removed from the marker.
[0131] One example of a cleavable marker is a photocleavable marker
such as chemical compounds which contain the 2-nitrobenzyl moiety
(FIG. 6A). Upon illumination, these aromatic nitro compounds
undergo an internal oxidation-reduction reaction (Pillai, Synthesis
1-26, 1980; Patchornik et al., J. Am. Chem. Soc. 92:6333-35, 1970).
As a result, the nitro group is reduced to a nitroso group and an
oxygen is inserted into the benzylic carbon-hydrogen bond at the
ortho position. The primary photochemical process is the
intramolecular hydrogen abstraction by the excited nitro group.
This is followed by an electron-redistribution process to the
aci-nitro form which rearranges to the nitroso product. Subsequent
thermal reaction leads to the cleavage of substrate from the
nitrobenzyl linkage (FIG. 6B). Examples of other cleavable markers
are shown in FIG. 7.
[0132] It may sometimes be desirable to create a distance between
the substrate and the cleavable moiety. To accomplish this,
cleavable moieties may be separated from substrates by cross-linker
arms. Cross-linkers increase substrate access and stabilize the
chemical structure, and can be constructed using, for example, long
alkyl chains or multiple repeat units of caproyl moieties linked
via amide linkages.
[0133] There are as many methods to synthesize cleavable markers as
there are different markers. One example for the synthesis of
photocleavable biotins based on nitrobenzyl alcohols involves four
major steps. 2-bromo-2'-nitroacetphenone, a precursor of the
photoreactive moiety, is first converted into an .alpha.- or
.omega.-amino acid like .epsilon.-aminocaprylic acid. The resulting
acid and amino functionality of the photoreactive group is coupled
using dicyclohexyl carbodimide (DCC). The benzoyl carbonyl group of
the resulting amide is reduced using sodium borohydride. The
resulting derivative of nitrobenzyl alcohol is derivatized to
obtain the final component which is able to react with biomolecular
substrates, for example by the reaction with phosgene gas, to
introduce the chloroformate functionality. The resulting compound
is depicted in FIG. 8A along with alternative derivatives of PCB.
Possible linkages to amino acids are depicted in FIG. 8B.
[0134] Cleavable markers can facilitate the isolation of nascent
proteins. For example, one type of a cleavable marker is
photocleavable biotin coupled to an amino acid. This marker can be
incorporated into nascent proteins and the proteins purified by the
specific interaction of biotin with avidin or streptavidin. Upon
isolation and subsequent purification, the biotin is removed by
application of electromagnetic radiation and nascent proteins
utilized in useful applications without the complications of an
attached biotin molecule. Other examples of cleavable markers
include photocleavable coumarin, photocleavable dansyl,
photocleavable dinitrophenyl and photocleavable coumarin-biotin.
Photocleavable markers are cleaved by electromagnetic radiation
such as UV light, peptidyl markers are cleaved by enzymatic
treatments, and pyrenyl fluorophores linked by disulfide bonds are
cleaved by exposure to certain chemical treatments such as thiol
reagents.
[0135] Cleavage of photocleavable markers is dependent on the
structure of the photoreactive moiety and the wavelength of
electromagnetic radiation used for illumination. Other wavelengths
of electromagnetic radiation should not damage the proteins or
other chemical moieties. In the case of unsubstituted 2-nitrobenzyl
derivatives, the yield of photolysis and recovery of the substrate
are significantly decreased by the formation of side products which
act as internal light filters and are capable to react with amino
groups of the substrate. Typical illumination times vary from 1 to
about 24 hours and yields are 1-95%. Radiation sources are placed
within about 10 cm of the substrate proteins and set on low power
so as to minimize side reactions, if any, which may occur in the
nascent proteins. In the case of alpha-substituted 2-nitrobenzyl
derivatives (methyl, phenyl, etc.), a considerable increase in rate
of photo-removal as well as yield of the released substrate are
observed. The introduction of other electron donor groups into
phenyl rings of photoreactive moieties increases the yield of
substrate. The general reaction for the photolysis of PCB is
depicted in FIG. 9.
[0136] For enzymatic cleavage, markers introduced contain specific
bonds which are sensitive to unique enzymes of chemical substances.
Introduction of the enzyme or chemical into the protein mixture
cleaves the marker from the nascent protein. When the marker is a
modified amino acid, this can result in the production of native
protein forms. Thermal treatments of, for example, heat sensitive
chemical moieties operate in the same fashion. Mild application of
thermal energy, such as with microwaves or radiant heat, cleaves
the sensitive marker from the protein without producing any
significant damage to the nascent proteins.
[0137] Description of Preferred Embodiments
[0138] A. Detection of Mutations
[0139] Detection of mutations is an increasingly important area in
clinical diagnosis, including but not limited to the diagnosis of
cancer and/or individuals disposed to cancer. The protein
truncation test (PTT) is a technique for the detection of nonsense
and frameshift mutations which lead to the generation of truncated
protein products. Genes associated with Duchenne muscular
dystrophy, adenomatous polyposis coli, human mutL homologue and
human nutS homologue (both involved in colon cancer), and BRAC1
(involved in familial breast cancer) can now be screened for
mutations in this manner, along with others (see Table 1).
[0140] Typically, the PTT technique involves the incorproation of a
T7 promoter site, ribosome binding site, and an artificial
methionine start site into a PCR product covering the region of the
gene to be investigated. The PCR product is then transcribed and
translated using either an in vitro rabbit reticulocyte lysate or
wheat germ lysate system, to generate a protein corresponding to
the region of the gene amplified. The presence of a stop codon in
the sequence, generated by a nonsense mutation or a frameshift,
will result in the premature termination of protein translation,
producing a truncated protein that can be detected by standard gel
electrophoresis (e.g. SDS-PAGE) analysis combined with radioactive
dection.
[0141] There are drawbacks to the technique as currently practiced.
One of the most important problems involves the identification of
the product of interest. This is made difficult because of
nonspecific radiolabeled products. Attempts to address these
problems have been made. One approach is to introduce an affinity
tag after the start site and before the region encoding the gene of
interest. See Rowan and Bodmer, "Introduction of a myc Reporter Taq
to Improve the Quality of Mutation Detection Using the Protein
Truncation Test," Human Mutation 9:172 (1997). However, such
approaches still have the disadvantage that they rely on
electrophoresis.
1TABLE 1 Applications of PTT in Human Molecular Genetics Disease
References % Truncating Mutations Gene Familial Adenomatous 95% APC
Polyposis Hereditary desmold disease 100% APC Ataxia telangiectasia
90% ATM Hereditary Breast and 90% BRCA1 Ovarian Cancer 90% BRCA2
Cystic Fibrosis 15% CFTR Duchenne Muscular 95% DMD Dystrophy
Emery-Dreifuss Muscular 80% EMD Dystrophy Fanconi anaemia 80% FAA
Hunter Syndrome -50% IDS Hereditary non-polyposis -80% hMSH2
colorectal cancer -70% hMLH1 Neurofibromatosis type 1 50% NF1
Neurofibromatosis type 2 65% NF2 Polycystic Kidney Disease 95% PKD1
Rubinstein-Taybi Syndrome 10% RTS The percentage of truncating
mutations reported which should be detectable using PTT.
[0142] The present invention contemplates a gel-free truncation
test (GFTT), wherein two or three markers are introduced into the
nascent protein. The present invention contemplates both pre-natal
and post-natal testing to determine predisposition to disease. In a
preferred embodiment of the invention, the novel compositions and
methods are directed to the detection of frameshift or chain
terminating mutations. In order to detect such mutations, a nascent
protein is first synthesized in a cell-free or cellular translation
system from message RNA or DNA coding for the protein which may
contain a possible mutation. The nascent protein is then separated
from the cell-free or cellular translation system using an affinity
marker located at or close to the N-terminal end of the protein.
The protein is then analyzed for the presence of a detectable
marker located at or close to the N-terminal of the protein
(N-terminal marker). A separate measurement is then made on a
sequence dependent detectable marker located at or close to the
C-terminal end of the protein (C-terminal marker).
[0143] A comparison of the measurements from the C-terminal marker
and N-terminal marker provides information about the fraction of
nascent proteins containing frameshift or chain terminating
mutations in the gene sequence coding for the nascent protein. The
level of sequence dependent marker located near the C-terminal end
reflects the fraction of protein which did not contain chain
terminating or out-of-frame mutations. The measurement of the
N-terminal marker provides an internal control to which measurement
of the C-terminal marker is normalized. Normalizing the level of
the C-terminal marker to the N-terminal marker eliminates the
inherent variabilities such as changes in the level of protein
expression during translation that can undermine experimental
accuracy. Separating the protein from the translation mixture using
an using an affinity marker located at or close to the N-terminal
end of the protein eliminates the occurrence of false starts which
can occur when the protein is initiated during translation from an
internal AUG in the coding region of the message. A false start can
lead to erroneous results since it can occurs after the chain
terminating or out-of-frame mutation. This is especially true if
the internal AUG is in-frame with the message. In this case, the
peptide C-terminal marker will still be present even if message
contains a mutation.
[0144] In one example, a detectable marker comprising a non-native
amino acid or amino acid derivative is incorporated into the
nascent protein during its translation at the amino terminal
(N-terminal end) using a misaminoacylate initiator tRNA which only
recognizes the AUG start codon signaling the initiation of protein
synthesis. One example of a detectable marker is the highly
fluorescent compound BODIPY FL. The marker might also be
photocleavable such as photocleavable coumarin or photocleavable
biotin. The nascent protein is then separated from the cell-free or
cellular translation system by using a coupling agent which binds
to an affinity marker located adjacent to the N-terminal of the
protein. One such affinity marker is a specific protein sequence
known as an epitope. An epitope has the property that it
selectively interacts with molecules and/or materials containing
acceptor groups. There are many epitope sequences reported in the
literature including His.times.6 (HHHHHH) described by ClonTech and
C-myc (-EQKLISEEDL) described by Roche-BM, Flag (DYKDDDDK)
described by Stratagene), SteptTag (WSHPQFEK) described by
Sigma-Genosys and HA Tag (YPYDVPDYA) described by Roche-BM.
[0145] Once the nascent protein is isolated from the translation
system, it is analyzed for presence of the detectable marker
incorporated at the N-terminal of the protein. In the case of
BODIPY FL, it can be detected by measuring the level of
fluorescence using a variety of commercially available instrument
such as a Molecular Dynamics Model 595 fluorscence scanner that is
equipped with excitation at 488 nm and an emission filter that
allows light above 520 nm to be transmitted to the detector.
[0146] The protein is then analyzed for the presence of a sequence
specific marker located near the C-terminal end of the protein. In
normal practice, such a sequence specific marker will consist of a
specific sequence of amino acids located near the C-terminal end of
the protein which is recognized by a coupling agent. For example,
an antibody can be utilized which is directed against an amino acid
sequence located at or near C-terminal end of the nascent protein
can be utilized. Such antibodies can be labeled with a variety of
markers including fluorscent dyes that can be easily detected and
enzymes which catalzye detectable reactions that lead to easily
detectable substrates. The marker chosen should have a different
detectable property than that used for the N-terminal marker. An
amino acid sequence can also comprise an epitope which is
recognized by coupling agents other than antibodies. One such
sequence is 6 histidines sometimes referred to as a his-tag which
binds to cobalt complex coupling agent.
[0147] A variety of N-terminal markers, affinity markers and
C-terminal markers are available which can be used for this
embodiment. The N-terminal marker could be BODIPY, affinity marker
could be StrepTag and C-terminal marker could be a His.times.6 tag.
In this case, after translation, the reaction mixture is incubated
in streptavidin coated microtiter plate or with streptavidin coated
beads. After washing unbound material, the N-terminal marker is
directly measured using a fluorescence scanner while the C-terminal
marker can be quantitated using anti-his.times.6 antibodies
conjugated with a fluorescent dye (like rhodamine or Texas Red)
which has optical properties different than BODIPY, thus
facilitating simultaneous dual detection.
[0148] In a different example, the N-terminal marker could be a
biotin or photocleavable biotin incorporated by a misaminoacylated
tRNA, the affinity marker could be a His .times.6 tag and the
C-terminal had C-myc marker. In this case, after the translation,
the reaction mixture is incubated with metal chelating beads or
microtiter plates (for example Talon, ClonTech). After washing the
unbound proteins, the plates or beads can be subjected to detection
reaction using streptavidine conjugated fluorescence dye and C-myc
antibody conjugated with other fluorescent dye. In addition, one
can also use chemiluminescent detection method using antibodies
which are conjugated with peroxidases.
[0149] It will be understood by those skilled in the area of
molecular biology and biochemistry that the N-terminal marker,
affinity marker and C-terminal marker can all consist of epitopes
that can be incorporated into the nascent protein by designing the
message or DNA coding for the nascent protein to have a nucleic
acid sequence corresponding to the particular epitope. This can be
accomplished using known methods such as the design of primers that
incorporate the desired nucleic acid sequence into the DNA coding
for the nascent protein using the polymerase chain reaction (PCR).
It will be understood by those skilled in protein biochemistry that
a wide variety of detection methods are available that can be used
to detect both the N-terminal marker and the C-terminal markers.
Additional examples include the use of chemiluminescence assays
where an enzyme which converts a non-chemiluminescent substrate to
a chemiluminescent product is conjugated to an antibody that is
directed against a particular epitope.
[0150] One example of this approach is based on using a luminometer
to measure luminenscent markers. A biotin detectable marker is
incorporated at the N-terminal using a misaminoacylated tRNA. The
biotin is detected by using a streptavidin which is conjugated to
Renilla luciferase from sea pansy. The C-terminal sequence
comprises an epitope which interacts with a binding agent that has
attached firefly luciferase. After separation of the nascent
protein using a distinct epitope located near the N-terminal end of
the protein, the protein is subjected to a dual luminescent
luciferase assay based on a procedure described by Promega Corp and
known as the Dual-Luciferase.RTM. Reporter Assay. This assay
consists of first adding Luciferase Assay Reagent II available from
Promega Corp. to the isolated nascent protein and then measuring
the level of chemiluminesence. Stop & Glo.RTM. Reagent is then
added which simultaneously quenches the firefly luminescence and
activates the Renilla luminescence. The luciferase assay can be
performed and quantified in seconds. A comparison of the level of
luminescence measured from the firefly and Renilla luciferase
provides an indication of whether a mutation is present or not in
the coding message of the nascent protein.
[0151] In an additional example, the N-terminal marker comprises an
affinity marker which is incorporated at the N-terminal end of the
protein during its translation using a misaminoacylated tRNA. The
affinity marker interacts with a coupling agent which acts to
separate the nascent protein from the translation mixture. The
nascent protein also contains a detectable marker which is located
adjacent or close to the N-terminal of the protein containing the
affinity marker. In addition, it contains a sequence specific
marker at or near the C-terminal end of the protein. The detectable
markers near the N-terminal and C-terminal ends of the nascent
protein are then measured and compared to detect the presence of
chain terminating or out-of-frame mutations.
[0152] There are a variety of additional affinity markers,
N-terminal markers and C-terminal markers available for this
embodiment. The affinity marker could be biotin or photocleavable
biotin, N-terminal marker could be StepTag and C-terminal the C-myc
epitope. In this case, after the translation, the reaction mixture
is incubated with streptavidin coated beads or microtiter plates
coated with streptavidin. After washing the unbound proteins, the
plates or beads can be subjected to detection reaction using
anti-his 6 antibodies conjugated with a fluorescent dye (like
rhodamine or Texas Red) and C-myc antibody conjugated with other
another fluorescent dye such as BODIPY. In addition, one can also
use chemiluminescent detection method using antibodies which are
conjugated with peroxidases. Even in case of peroxidases conjugated
antibodies, one can use fluorescent substrates and use FluorImager
like device to quantitate N-terminal and C-terminal labels.
[0153] For optimal effectiveness, the N-terminal marker and
affinity marker should be placed as close as possible to the
N-terminal end of the protein. For example, if an N-terminal marker
is incorporated using a misaminoacylated initiator, it will be
located at the N-terminal amino acid. In this case, the affinity
marker should be located immediately adjacent to the N-terminal
marker. Thus, if a BODIPY marker which consists of a BODIPY
conjugated to methionine is incorporated by a misaminoacylated
initiator tRNA, it should be followed by an epitope sequence such
as SteptTag (WSHPQFEK) so that the entire N-terminal sequence will
be BODIPY-MWSPQFEK. However, for specific cases it may be
advantageous to add intervening amino acids between the BODIPY-M
and the epitope sequence in order to avoid interaction between the
N-terminal marker and the affinity marker or the coupling agent
which binds the affinity marker. Such interactions will vary
depending on the nature of the N-terminal marker, affinity marker
and coupling agent.
[0154] For optimal effectiveness, the C-terminal marker should be
placed as close as possible to the C-terminal end of protein. For
example, if a His-.times.6 tag is utilized, the protein sequence
would terminate with 6 His. In some cases, an epitope may be
located several residues before the C-terminal end of the protein
in order to optimize the properties of the nascent protein. This
might occur for example, if a specific amino acid sequence is
necessary in order to modify specific properties of the nascent
protein that are desirable such as its solubility or
hydrophobicity.
[0155] In the normal application of this method, the ratio of the
measured level of N-terminal and C-terminal markers for a nascent
protein translated from a normal message can be used to calculate a
standard normalized ratio. In the case of a message which may
contain a mutations, deviations from this standard ratio can then
be used to predict the extent of mutations. For example, where all
messages are defective, the ratio of the C-terminal marker to the
N-terminal marker is expected to be zero. On the other hand, in the
case where all messages are normal, the ratio is expected to be 1.
In the case where only half of the message is defective, for
example for a patient which is heterozygote for a particular
genetic defect which is chain terminating or causes an out-of-frame
reading error, the ratio would be 1/2.
[0156] There are several unique advantages of this method compared
to existing techniques for detecting chain terminating or
out-of-frame mutations. Normally, such mutations are detected by
analyzing the entire sequence of the suspect gene using
conventional DNA sequencing methods. However, such methods are time
consuming, expensive and not suitable for rapid throughput assays
of large number of samples. An alternative method is to utilize gel
electrophoresis, which is able to detect changes from the expected
size of a nascent protein. This approach, sometimes referred to as
the protein truncation test, can be facilitated by using
non-radioactive labeling methods such as the incorporation of
detectable markers with misaminoacylated tRNAs. However, in many
situations, such as high throughput screening, it would be
desirable to avoid the use of gel electrophoresis which is
time-consuming (typically 60-90 minutes). In the present method,
the need for performing gel electrophoresis is eliminated.
Furthermore, since the approach depends on comparison of two
detectable signals from the isolated nascent protein which can be
fluorscent, luminescent or some combination thereof, it is highly
amenable to automation.
[0157] Measuring a sequence dependent marker located near the
C-terminal end of the protein provides information about the
presence of either a frameshift or chain terminating mutation since
the presence of either would result in an incorrect sequence. The
measurement of the N-terminal marker provides an internal control
to which measurement of the C-terminal marker is normalized.
Normalizing the level of the C-terminal marker to the N-terminal
marker eliminates the inherent variabilities such as changes in the
level of protein expression during translation that can undermine
experimental accuracy. Separating the protein from the translation
mixture using an using an affinity marker located at or close to
the N-terminal end of the protein eliminates the occurrence of
false starts which can occur when the protein is initiated during
translation from an internal AUG in the coding region of the
message. A false start can lead to erroneous results since it can
occurs after the chain terminating or out-of-frame mutation. This
is especially true if the internal AUG is in-frame with the
message. In this case, the peptide C-terminal marker will still be
present even if message contains a mutation.
[0158] B. Reporter Groups
[0159] Another embodiment of the invention is directed to a method
for monitoring the synthesis of nascent proteins in a cellular or a
cell-free protein synthesis system without separating the
components of the system. These markers have the property that once
incorporated into the nascent protein they are distinguishable from
markers free in solution or linked to a tRNA. This type of marker,
also called a reporter, provides a means to detect and quantitate
the synthesis of nascent proteins directly in the cellular or
cell-free translation system.
[0160] One type of reporters previously described in U.S. Pat. No.
5,643,722 (hereby incorporated by reference) has the characteristic
that once incorporated into the nascent protein by the protein
synthesizing system, they undergo a change in at least one of their
physical or physio-chemical properties. The resulting nascent
protein can be uniquely detected inside the synthesis system in
real time without the need to separate or partially purify the
protein synthesis system into its component parts. This type of
marker provides a convenient non-radioactive method to monitor the
production of nascent proteins without the necessity of first
separating them from pre-existing proteins in the protein synthesis
system. A reporter marker would also provide a means to detect and
distinguish between different nascent proteins produced at
different times during protein synthesis by addition of markers
whose properties are distinguishable from each other, at different
times during protein expression. This would provide a means of
studying differential gene expression.
[0161] One example of the utilization of reporters is schematically
represented in FIG. 10. A tRNA molecule is misaminoacylated with a
reporter (R) which has lower or no fluorescence at a particular
wavelength for monitoring and excitation. The misaminoacylated tRNA
is then introduced into a cellular or cell-free protein synthesis
system and the nascent proteins containing the reporter analog are
gradually produced. Upon incorporation of the reporter into the
nascent protein (R*), it exhibits an increased fluorescence at
known wavelengths. The gradual production of the nascent protein is
monitored by detecting the increase of fluorescence at that
specific wavelength.
[0162] The chemical synthesis of a reporter can be based on the
linkage of a chemical moiety or a molecular component having
reporter properties with a native amino acid residue. There are
many fluorescent molecules which are sensitive to their environment
and undergo a change in the wavelength of emitted light and yield
of fluorescence. When these chemical moieties, coupled to amino
acids, are incorporated into the synthesized protein, their
environments are altered because of a difference between the bulk
aqueous medium and the interior of a protein which can causes
reduced accessibility to water, exposure to charged ionic groups,
reduced mobility, and altered dielectric constant of the
surrounding medium. Two such examples are shown in FIG. 11A.
[0163] One example of a reporter molecule is based on a fluorescent
acridinium moiety and has the unique property of altering its
emission properties, depending upon polarity or viscosity of the
microenvironment. It has a higher quantum yield of fluorescence
when subjected to hydrophobic environment and/or viscosity. Due to
the hydrophobicity of the reporter itself, it is more likely to be
associated with the hydrophobic core of the nascent protein after
incorporation into the growing nascent polypeptide. An increase in
the fluorescence intensity is a direct measure of protein synthesis
activity of the translation system. Although, the environment of
each reporter residue in the protein will be different, and in some
cases, the reporter may be present on the surface of the protein
and exposed to an aqueous medium, a net change occurs in the
overall spectroscopic properties of the reporters incorporated into
the protein relative to bulk aqueous medium. A change in the
spectroscopic properties of only a subset of reporters in the
protein will be sufficient to detect the synthesis of proteins that
incorporate such reporters.
[0164] An alternative approach is to utilize a reporter which
alters its fluorescent properties upon formation of a peptide bond
and not necessarily in response to changes in its environment.
Changes in the reporter's fluorescence as it partitions between
different environments in the cell-free extract does not produce a
large signal change compared to changes in fluorescence upon
incorporation of the reporter into the nascent protein.
[0165] A second example of a reporter is a marker based on coumarin
such as 6,7-(4', 5'-prolino)coumarin. This compound can be
chemically synthesized by coupling a fluorophore like coumarin with
an amino-acid structural element in such a way that the fluorophore
would alter its emission or absorption properties after forming a
peptide linkage (FIG. 11B). For example, a proline ring containing
secondary amino functions will participate in peptide bond
formation similar to a normal primary amino group. Changes in
fluorescence occur due to the co-planarity of the newly formed
peptide group in relation to the existing fluorophore. This
increases conjugation/delocalization due to the .pi.-electrons of
nitrogen-lone pair and carbonyl-group in the peptide bond.
Synthesis of such compounds is based on on coumarin synthesis using
ethylacetoacetate (FIG. 11C).
[0166] Reporters are not limited to those non-native amino acids
which change their fluorescence properties when incorporated into a
protein. These can also be synthesized from molecules that undergo
a change in other electromagnetic or spectroscopic properties
including changes in specific absorption bands in the UV, visible
and infrared regions of the electromagnetic spectrum, chromophores
which are Raman active and can be enhanced by resonance Raman
spectroscopy, electron spin resonance activity and nuclear magnetic
resonances. In general, a reporter can be formed from molecular
components which undergo a change in their interaction and response
to electromagnetic fields and radiation after incorporation into
the nascent protein.
[0167] In the present invention, reporters may also undergo a
change in at least one of their physical or physio-chemical
properties due to their interaction with other markers or agents
which are incorporated into the same nascent protein or are present
in the reaction chamber in which the protein is expressed. The
interaction of two different markers with each other causes them to
become specifically detectable. One type of interaction would be a
resonant energy transfer which occurs when two markers are within a
distance of between about 1 angstrom (A) to about 50 A, and
preferably less than about 10 A. In this case, excitation of one
marker with electromagnetic radiation causes the second marker to
emit electromagnetic radiation of a different wavelength which is
detectable. A second type of interaction would be based on electron
transfer between the two different markers which can only occur
when the markers are less than about 5 A. A third interaction would
be a photochemical reaction between two markers which produces a
new species that has detectable properties such as fluorescence.
Although these markers may also be present on the misaminoacylated
tRNAs used for their incorporation into nascent proteins, the
interaction of the markers occurs primarily when they are
incorporated into protein due to their close proximity. In certain
cases, the proximity of two markers in the protein can also be
enhanced by choosing tRNA species that will insert markers into
positions that are close to each other in either the primary,
secondary or tertiary structure of the protein. For example, a
tyrosine-tRNA and a tryptophan-tRNA could be used to enhance the
probability for two different markers to be near each other in a
protein sequence which contains the unique neighboring pair
tyrosine-tryptophan.
[0168] In one embodiment of this method, a reporter group is
incorporated into a nascent protein using a misaminoacylated tRNA
so that when it binds to a coupling agent, the reporter group
interacts with a second markers or agents which causes them to
become specifically detectable. Such an interaction can be
optimized by incorporating a specific affinity element into the
nascent protein so that once it interacts with a coupling agent the
interaction between the reporter group and the second marker is
optimized. Such an affinity element might comprise a specific amino
acid sequence which forms an epitope or a nonnative amino acid. In
one example, the reporter group is incorporated at the N-terminal
of the nascent protein by using a misaminoacylated tRNA. The
epitope is incorporated into the nascent protein so that when it
interacts with the coupling agent the reporter comes into close
proximity with a second marker which is conjugated to the coupling
agent.
[0169] One type of interaction between the markers that is
advantageously used causes a fluorescence resonant energy transfer
which occurs when the two markers are within a distance of between
about 1 angstrom (A) to about 50 A, and preferably less than about
10 A. In this case, excitation of one marker with electromagnetic
radiation causes the second marker to emit electromagnetic
radiation of a different wavelength which is detectable. This could
be accomplished, for example, by incorporating a fluorescent marker
at the N-terminal end of the protein using the E. coli initiator
tRNA.sup.fmet. An epitope is then incorporated near the N-terminal
end such as the SteptTag (WSHPQFEK) described by Sigma-Genosys.
Streptavidin is then conjugated using known methods with a second
fluorescent marker which is chosen to efficiently undergo
fluorescent energy transfer with marker 1. The efficiency of this
process can be determined by calculating the a Forster energy
transfer radius which depends on the spectral properties of the two
markers. The marker-streptavidin complex is then introduced into
the translation mixture. Only when nascent protein is produced does
fluorescent energy transfer between the first and second marker
occur due to the specific interaction of the nascent protein
StreptTag epitope with the streptavidin.
[0170] There are a variety of dyes which can be used as marker
pairs in this method that will produce easily detectable signals
when brought into close proximity. Previously, such dye pairs have
been used for example to detect PCR products by hybridizing to
probes labeled with a dye on one probe at the 5'-end and another at
the 3'-end. The production of the PCR product brings a dye pair in
close proximity causing a detectable FRET signal. In one appliation
the dyes, fluoresein and LC 640 were utilized on two different
primers (Roche Molecular Biochemicals-http://www.biochem-
.boehringer-mannheim.com/lightcycler/monito03.htm). When the
fluorescein is excited by green light (around 500 nm) that is
produced by a diode laser, the LC 640 emits red fluorescent light
(around 640 nm) which can be easily detected with an appropriate
filter and detector. In the case of nasent proteins, the pair of
dyes BODIPY FL and LC 640 would function in a similar manner. For
example, incorporation of the BODIPY FL on the N-terminal end of
the protein and the labeling of a binding agent with LC 640 which
is directed against an N terminal epitope would allow detection of
the production of nascent proteins.
[0171] The use of the marker pair BODIPY-FL and coumarin is a
second pair which can be utilized advantageously. In one study,
[Keller, R. C., Silvius, J. R., and De Kruijff, B. (1995) Biochem
Biophys Res Commun 207(2), 508-14] it was found using the spectral
overlap a Forster energy transfer radius (RO) of 50.+-.2 A and
40.+-.2 A for the coumarin-(beta-BODIPY FL ) and the
coumarin-(beta-BODIPY 530/550) couple respectively. Experimentally
this was estimated to be 49.0-51.5 A and 38.5-42.5 A respectively.
It is also possible to use two markers with similar or identical
spectral properties for the marker pair due to the process of
quenching. For example, in one study this process was used in the
case of BODIPY FL in order to study the processing of exogenous
proteins in intact cells [Reis, R. C., Sorgine, M. H., and
Coelho-Sampaio, T. (1998) Eur J Cell Biol 75(2), 192-7] and in a
second case to study the kinetics of intracellular viral assembly
[Da Poian, A. T., Gomes, A. M., and Coelho-Sampaio, T. (1998) J
Virol Methods 70(1), 45-58].
[0172] As stated above, a principal advantage of using reporters is
the ability to monitor the synthesis of proteins in cellular or a
cell-free translation systems directly without further purification
or isolation steps. Reporter markers may also be utilized in
conjunction with cleavable markers that can remove the reporter
property at will. Such techniques are not available using
radioactive amino acids which require an isolation step to
distinguish the incorporated marker from the unincorporated marker.
With in vitro translation systems, this provides a means to
determine the rate of synthesis of proteins and to optimize
synthesis by altering the conditions of the reaction. For example,
an in vitro translation system could be optimized for protein
production by monitoring the rate of production of a specific
calibration protein. It also provides a dependable and accurate
method for studying gene regulation in a cellular or cell-free
systems.
[0173] C. Affinity Markers
[0174] Another embodiment of the invention is directed to the use
of markers that facilitate the detection or separation of nascent
proteins produced in a cellular or cell-free protein synthesis
system. Such markers are termed affinity markers and have the
property that they selectively interact with molecules and/or
materials containing acceptor groups. The affinity markers are
linked by aminoacylation to tRNA molecules in an identical manner
as other markers of non-native amino acid analogs and derivatives
and reporter-type markers as described. These affinity markers are
incorporated into nascent proteins once the misaminoacylated tRNAs
are introduced into a translation system.
[0175] An affinity marker facilities the separation of nascent
proteins because of its selective interaction with other molecules
which may be biological or non-biological in origin through a
coupling agent. For example, the specific molecule to which the
affinity marker interacts, referred to as the acceptor molecule,
could be a small organic molecule or chemical group such as a
sulfhydryl group (--SH) or a large biomolecule such as an antibody.
The binding is normally chemical in nature and may involve the
formation of covalent or non-covalent bonds or interactions such as
ionic or hydrogen bonding. The binding molecule or moiety might be
free in solution or itself bound to a surface, a polymer matrix, or
a reside on the surface of a substrate. The interaction may also be
triggered by an external agent such as light, temperature, pressure
or the addition of a chemical or biological molecule which acts as
a catalyst.
[0176] The detection and/or separation of the nascent protein and
other preexisting proteins in the reaction mixture occurs because
of the interaction, normally a type of binding, between the
affinity marker and the acceptor molecule. Although, in some cases
some incorporated affinity marker will be buried inside the
interior of the nascent protein, the interaction between the
affinity marker and the acceptor molecule will still occur as long
as some affinity markers are exposed on the surface of the nascent
protein. This is not normally a problem because the affinity marker
is distributed over several locations in the protein sequence.
[0177] Affinity markers include native amino acids, non-native
amino acids, amino acid derivatives or amino acid analogs in which
a coupling agent is attached or incorporated. Attachment of the
coupling agent to, for example, a non-native amino acid may occur
through covalent interactions, although non-covalent interactions
such as hydrophilic or hydrophobic interactions, hydrogen bonds,
electrostatic interactions or a combination of these forces are
also possible. Examples of useful coupling agents include molecules
such as haptens, immunogenic molecules, biotin and biotin
derivatives, and fragments and combinations of these molecules.
Coupling agents enable the selective binding or attachment of newly
formed nascent proteins to facilitate their detection or isolation.
Coupling agents may contain antigenic sites for a specific
antibody, or comprise molecules such as biotin which is known to
have strong binding to acceptor groups such as streptavidin. For
example, biotin may be covalently linked to an amino acid which is
incorporated into a protein chain. The presence of the biotin will
selectively bind only nascent proteins which incorporated such
markers to avidin molecules coated onto a surface. Suitable
surfaces include resins for chromatographic separation, plastics
such as tissue culture surfaces for binding plates, microtiter
dishes and beads, ceramics and glasses, particles including
magnetic particles, polymers and other matrices. The treated
surface is washed with, for example, phosphate buffered saline
(PBS), to remove non-nascent proteins and other translation
reagents and the nascent proteins isolated. In some case these
materials may be part of biomolecular sensing devices such as
optical fibers, chemfets, and plasmon detectors.
[0178] One example of an affinity marker is dansyllysine (FIG. 5).
Antibodies which interact with the dansyl ring are commercially
available (Sigma Chemical; St. Louis, Mo.) or can be prepared using
known protocols such as described in Antibodies: A Laboratory
Manual (E. Harlow and D. Lane, editors, Cold Spring Harbor
Laboratory Press, 1988) which is hereby specifically incorporated
by reference. Many conventional techniques exist which would enable
proteins containing the dansyl moiety to be separated from other
proteins on the basis of a specific antibody-dansyl interaction.
For example, the antibody could be immobilized onto the packing
material of a chromatographic column. This method, known as
affinity column chromatography, accomplishes protein separation by
causing the target protein to be retained on the column due to its
interaction with the immobilized antibody, while other proteins
pass through the column. The target protein is then released by
disrupting the antibody-antigen interaction. Specific
chromatographic column materials such as ion-exchange or affinity
Sepharose, Sephacryl, Sephadex and other chromatography resins are
commercially available (Sigma Chemical; St. Louis, Mo.; Pharmacia
Biotech; Piscataway, N.J.).
[0179] Separation can also be performed through an antibody-dansyl
interaction using other biochemical separation methods such as
immunoprecipitation and immobilization of the antibodies on filters
or other surfaces such as beads, plates or resins. For example,
protein could be isolated by coating magnetic beads with a
protein-specific antibody. Beads are separated from the extract
using magnetic fields. A specific advantage of using dansyllysine
as an affinity marker is that once a protein is separated it can
also be conveniently detected because of its fluorescent
properties.
[0180] In addition to antibodies, other biological molecules exist
which exhibit equally strong interaction with target molecules or
chemical moieties. An example is the interaction of biotin and
avidin. In this case, an affinity analog which contains the biotin
moiety would be incorporated into the protein using the methods
which are part of the present invention. Biotin-lysine amino acid
analogs are commercially available (Molecular Probes; Eugene,
Oreg.).
[0181] Affinity markers also comprise one component of a
multicomponent complex which must be formed prior to detection of
the marker. One particular embodiment of this detection means
involves the use of luminescent metal chelates, in particular
luminescent rare earth metal chelates. It is well known that
certain molecules form very stable complexes with rare earth
metals. It is also well known that introduction of chromophore into
these chelates sensitizes luminescence of these complexes. A
variety of detection schemes based on the use of luminescent rare
earth metal chelates have been described: Hemmila, I. A.,
"Applications of Fluorescence in Immunoassays", (Wiley & Sons
1991).
[0182] In a preferred embodiment, tRNA is misaminoacylated with a
chromophore that also acts as a rare earth shelter. This modified
aminoacyl tRNA is then introduced into cellular or cell-free
protein translation system and the modified amino acid incorporated
into nascent protein. The mixture is then separated using gel
electrophoresis and the gel is incubated with a solution containing
rare earth cation. Under these conditions rare earth cations form
luminescent complexes with amino acids modified with a chelator
present only in nascent proteins. The nascent proteins are then
detected using a mid-range UV transilluminator (350 nm), which
excites the formed lanthanide complex. The image is then recorded
using polaroid camera or CCD array and a filter. In one embodiment,
the derivatives of salicylic acid as one component and terbium ions
as a second component of the binary detection system are used.
[0183] Affinity markers can also comprise cleavable markers
incorporating a coupling agent. This property is important in cases
where removal of the coupled agent is required to preserve the
native structure and function of the protein and to release nascent
protein from acceptor groups. In some cases, cleavage and removal
of the coupling agent results in production of a native amino acid.
One such example is photocleavable biotin coupled to an amino
acid.
[0184] Photocleavable biotin contains a photoreactive moiety which
comprises a phenyl ring derivatized with functionalities
represented in FIG. 12 by X, Y and Z. X allows linkage of PCB to
the bimolecular substrate through the reactive group X'. Examples
of X' include Cl, O--N-hydroxysuccinimidyl, OCH.sub.2CN,
OPhF.sub.5, OPhCl.sub.5, N.sub.3. Y represents a substitution
pattern of a phenyl ring containing one or more substitutions such
as nitro or alkoxyl. The functionality Z represents a group that
allows linkage of the cross-linker moiety to the photoreactive
moiety. The photoreactive moiety has the property that upon
illumination, it undergoes a photoreaction that results in cleavage
of the PCB molecule from the substrate.
[0185] A lysine-tRNA is misaminoacylated with photocleavable
biotin-lysine, or chemically modified to attach a photocleavable
biotin amino acid. The misaminoacylated tRNA is introduced into a
cell-free protein synthesizing system and nascent proteins
produced. The nascent proteins can be separated from other
components of the system by streptavidin-coated magnetic beads
using conventional methods which rely on the interaction of beads
with a magnetic field. Nascent proteins are released then from
beads by irradiation with UV light of approximately 280 nm
wavelength.
[0186] Many devices designed to detect proteins are based on the
interaction of a target protein with specific immobilized acceptor
molecule. Such devices can also be used to detect nascent proteins
once they contain affinity markers such as biodetectors based on
sensing changes in surface plasmons, light scattering and
electronic properties of materials that are altered due to the
interaction of the target molecule with the immobilized acceptor
group.
[0187] Nascent proteins, including those which do not contain
affinity-type markers, may be isolated by more conventional
isolation techniques. Some of the more useful isolation techniques
which can be applied or combined to isolate and purify nascent
proteins include chemical extraction, such as phenol or chloroform
extract, dialysis, precipitation such as ammonium sulfate cuts,
electrophoresis, and chromatographic techniques. Chemical isolation
techniques generally do not provide specific isolation of
individual proteins, but are useful for removal of bulk quantities
of non-proteinaceous material. Electrophoretic separation involves
placing the translation mixture containing nascent proteins into
wells of a gel which may be a denaturing or non-denaturing
polyacrylamide or agarose gel. Direct or pulsed current is applied
to the gel and the various components of the system separate
according to molecular size, configuration, charge or a combination
of their physical properties. Once distinguished on the gel, the
portion containing the isolated proteins removed and the nascent
proteins purified from the gel. Methods for the purification of
protein from acrylamide and agarose gels are known and commercially
available.
[0188] Chromatographic techniques which are useful for the
isolation and purification of proteins include gel filtration,
fast-pressure or high-pressure liquid chromatography, reverse-phase
chromatography, affinity chromatography and ion exchange
chromatography. These techniques are very useful for isolation and
purification of proteins species containing selected markers.
[0189] Another embodiment of the invention is directed to the
incorporation of non-native amino acids or amino acid derivatives
with marker or affinity properties at the amino-terminal residue of
a nascent protein (FIG. 13). This can be accomplished by using the
side chain of an amino acid or by derivatizing the terminal amino
group of an amino acid. In either case the resulting molecule is
termed an amino acid derivative. The amino-terminal residue of a
protein is free and its derivatization would not interfere with
formation of the nascent polypeptide. The non-native amino acid or
amino acid derivative is then used to misaminoacylate an initiator
tRNA which only recognizes the first AUG codon signaling the
initiation of protein synthesis. After introduction of this
misaminoacylated initiator tRNA into a protein synthesis system,
marker is incorporated only at the amino terminal of the nascent
protein. The ability to incorporate at the N-terminal residue can
be important as these nascent molecules are most likely to fold
into native conformation. This can be useful in studies where
detection or isolation of functional nascent proteins is
desired.
[0190] Not all markers incorporated with misaminoacylated initiator
tRNAs at the amino-terminal residue of the nascent protein show the
same acceptance by the protein translational machinery.
Furthermore, the range of incorporation of different markers can be
more restrictive compared to the use of non-initiator tRNAs such as
lysyl-tRNA. Although the factors influencing this descrimination
between markers for incorporation by a misaminoacylated initiator
tRNA are not fully elucidated, one possibility is that the
initiation factor (IF2) which is used for carrying
formylmethionine-tRNA.sup.fmet to ribosomes plays a role. A second
possibility is that the interaction between the marker structure
and the ribosomes plays a role. For example, the marker, BODIPY-FL
is accepted by the protein translational machinery to a greater
extent than smaller fluorescent markers such as NBD. For this
reason, BODIPY-FL is a superior marker for use in the detection of
nascent protein when incorporated through initiator tRNAs.
[0191] A marker group can also be incorporated at the N terminal by
using a mutant tRNA which does not recognize the normal AUG start
codon. In some cases this can lead to a higher extent of specific
incorporation of the marker. For example, the mutant of initiator
tRNA, where the anticodon has been changed from CAU.fwdarw.CUA
(resulting in the change of initiator methionine codon to amber
stop codon) has shown to act as initiator suppressor tRNA (Varshney
U, RajBhandary U L, Proc Natl Acad Sci U S A 1990
February;87(4):1586-90; Initiation of protein synthesis from a
termination codon). This tRNA initiates the protein synthesis of a
particular gene when the normal initiation codon, AUG is replaced
by the amber codon UAG. Furthermore, initiation of protein
synthesis with UAG and tRNA(fMet.sup.CUA) was found to occur with
glutamine and not methionine. In order to use this tRNA to
introduce a marker at the N terminal of a nascent protein, this
mutant tRNA can be enzymatically aminoacylated with glutamine and
then modified with suitable marker. Alternatively, this tRNA could
be chemically aminoacylated using modified amino acid (for example
methionine-BODIPY). Since protein translation can only be initiated
by this protein on messages containing UAG, all proteins will
contain the marker at the N-terminal end of the protein.
[0192] D. Mass Spectrometry
[0193] Mass spectrometry measures the mass of a molecule. The use
of mass spectrometry in biology is continuing to advance rapidly,
finding applications in diverse areas including the analysis of
carbohydrates, proteins, nucleic acids and biomolecular complexes.
For example, the development of matrix assisted laser desorption
ionization (MALDI) mass spectrometry (MS) has provided an important
tool for the analysis of biomolecules, including proteins,
oligonucleotides, and oligosacharrides [Karas, 1987 #6180;
Hillenkamp, 1993 #6175]. This technique's success derives from its
ability to determine the molecular weight of large biomolecules and
non-covalent complexes (>500,000 Da) with high accuracy (0.01%)
and sensitivity (subfemtomole quantities). Thus far, it has been
found applicable in diverse areas of biology and medicine including
the rapid sequencing of DNA, screening for bioactive peptides and
analysis of membrane proteins.
[0194] Markers incorporated by misaminoacylated tRNAs into nascent
proteins, especially at a specific position such at the N-terminal
can be used for the detection of nascent proteins by mass
spectrometry. Without such a marker, it can be very difficult to
detect a band due to a nascent protein synthesized in the presence
of a cellular or cell-free extract due the presence of many other
molecules of similar mass in the extract. For example, in some
cases less than 0.01% of the total protein mass of the extract may
comprise the nascent protein(s). Furthermore, molecules with
similar molecular weight as the nascent protein may be present in
the mixture. Such molecules will overlap with peaks due to the
nascent protein. This problem is particularly severe if the nascent
protein is a transcription or translation factor already present in
the cell-free or cellular protein synthesis. The synthesis of
additional amounts of this protein in the protein synthesis system
would be difficult to detect using known methods in mass
spectrometry since peak intensities are not correlated in a linear
manner with protein concentration.
[0195] Detection by mass spectrometry of a nascent protein produced
in a translation system is also very difficult if the mass of the
nascent protein produced is not known. This might situation might
occur for example if the nascent protein is translated from DNA
where the exact sequence is not known. One such example is the
translation of DNA from individuals which may have specific
mutations in particular genes or gene fragments. In this case, the
mutation can cause a change in the protein sequence and even result
in chain truncation if the mutation results in a stop codon. The
mass of nascent proteins produced in a translation system might
also not be known if DNA is derived from unknown sources such as as
colonies of bacteria which can contain different members of a gene
library or fragments thereof.
[0196] In one embodiment of the invention, the incorporation of
markers of a specific mass (mass markers) into nascent proteins can
be used to eliminate all of the above mentioned problems associated
with the conventional mass spectrometric approach. First, a tRNA
misaminoacylated with a marker of a known mass is added to the
protein synthesis system. The synthesis system is then incubated to
produce the nascent proteins. The mass spectrum of the protein
synthesis system is then measured. The presence of the nascent
protein can be directly detected by identifying peaks in the mass
spectrum of the protein synthesis system which correspond to the
mass of the unmodified protein and a second band at a higher mass
which corresponds to the mass of the nascent protein plus the
modified amino acid containing the mass of the marker.
[0197] There are several steps that can be taken to optimize the
efficient detection of nascent proteins using this method. The mass
of the marker should exceed the resolution of the mass
spectrometer, so that the increased in mass of the nascent protein
can be resolved from the unmodified mass. For example, a marker
with a mass exceeding 100 daltons can be readily detected in
proteins with total mass up to 100,000 using both matrix assisted
laser desorption (MALDI) or electrospray ionization (ESI)
techniques. The amount of misaminoacylated tRNA should be adjusted
so that the incorporation of the mass marker occurs in
approximately 50% of the total nascent protein produced. An
initiator tRNA is preferable for incorporation of the mass marker
since it will only be incorporated at the N-terminal of the nascent
protein, thus avoiding the possibility that the nascent protein
will contain multiple copies of the mass marker.
[0198] One example of this method is the incorporation of the
marker BODIPY-FL, which has a mass of 282, into a nascent protein
using a misaminoacylated initiator tRNA. Incorporation of this
marker into a nascent protein using a misaminoacylated initiator
tRNA causes a band to appear at approximately 282 daltons above the
normal band which appears for the nascent protein. Since the
incorporation of the marker is less than one per protein due to
competition of non-misaminoacylated E. coli tRNA.sup.fmet, a peak
corresponding to the unmodified protein also appears.
Identification of these two bands separated by the mass of the
marker allows initial identification of the band due to the nascent
protein. Further verification of the band due to the nascent
protein can be made by adjusting the level of the misaminoacylated
initiator tRNA in the translation mixture. For example, if the
misaminoacylated initiator tRNA is left out, than only a peak
corresponding to the unmodified protein appears in the mass
spectrum of the protein synthesis system. By comparing the mass
spectrum from the protein synthesis system containing and not
containing the misaminacylated tRNA with the BODIPY-FL, the
presence of the nascent protein can be uniquely identified, even
when a protein with similar or identical mass is already present in
the protein synthesis system.
[0199] For the purpose of mass spectrometric identification of
nascent proteins, it is sometimes advantageous to utilize a
photocleavable marker. In this case, peaks due to nascent proteins
in the mass spectrum can be easily identified by measuring and
comparing spectra from samples of the protein synthesis system that
have been exposed and not exposed to irradiation which photocleaves
the marker. Those samples which are not exposed to irradiation will
exhibit bands corresponding the mass of the nascent protein which
has the incorporated mass marker, whereas those samples which are
exposed to irradiation will exhibit bands corresponding to the mass
of the nascent proteins after removal of the mass marker. This
shift of specific bands in the mass spectrum due to irradiation
provides a unique identifier of bands which are due to the nascent
proteins in the protein synthesis system.
[0200] One example of this method involves the use of the
photocleavable marker, photocleavable biotin. When photocleavable
biotin is incorporated into the test protein .alpha.-hemolysin, a
toxin produced by staphyloccus, by using the misaminoacylated E.
coli initiator tRNA.sup.met, the mass spectrum exhibits two peaks
corresponding to the mass of the nascent protein 35,904 Da, and a
second peak at 36,465 Da corresponding to the mass of the nascent
protein plus the mass of photocleavable biotin. After photocleavage
of the marker by exposing the cell-free or cellular extract to UV
light with a wavelength of approximately 365 nm for approximately
10 minutes, the two bands undergo changes in intensity due to
cleavage of the marker from the nascent protein. For example, in
the case of a form of photocleavable biotin containing a single
spacer the change in the mass will be 561.57. These characteristic
changes are then used to uniquely identify the peaks corresponding
the nascent protein. In the case of MALDI mass spectrometry, the
probe laser pulses when adjusted to sufficient intensity can be
used to accomplish photocleavage of photocleavable biotin. In this
case, changes can be conveniently measured during the course of the
measurements, thereby facilitating detection of peaks associated
with the nascent protein. A similar approach can also be used to
identify more than one nascent protein of unknown mass in a cell or
cell-free translation system.
[0201] Markers with affinity properties which are incorporated by
misaminoacylated tRNAs into nascent proteins can also be very
useful for the detection of such proteins by mass spectrometry.
Such markers can be used to isolate nascent proteins from the rest
of the cell-free or cellular translation system. In this case, the
isolation of the nascent proteins from the rest of the cell-free
mixture removes interference from bands due to other molecules in
the protein translation system. An example of this approach is the
incorporation of photocleavable biotin into the N-terminal end of a
nascent proteins using misaminoacylated tRNA. When this marker is
incorporated onto the N-terminal end of a nascent protein using an
E. coli tRNA.sup.met, it provides a convenient affinity label which
can be bound using streptavidin affinity media such as streptavidin
agarose. Once the nascent protein is separated by this method from
the rest of the protein synthesis system, it can be released by
UV-light and analyzed by mass spectrometry. In the case of MALDI
mass spectrometry, release of the nascent protein can most
conveniently be accomplished by using the UV-laser excitation
pulses of the MALDI system. Alternatively, the sample can be
irradiated prior to mass spectrometric analysis in the case of
MALDI or ESI mass spectrometry.
[0202] E. Electrophoresis
[0203] Another embodiment of the invention is directed to methods
for detecting by electrophoresis the interaction of molecules or
agents with nascent proteins which are translated in a translation
system. This method allows a large number of compounds or agents to
be rapidly screened for possible interaction with the expressed
protein of specific genes, even when the protein has not been
isolated or its function identified. It also allows a library of
proteins expressed by a pool of genes to be rapidly screened for
interaction with compounds or agents without the necessity of
isolating these proteins or agents. The agents might be part of a
combinatorial library of compounds or present in a complex
biological mixture such as a natural sample. The agents might
interact with the nascent proteins by binding to them or to cause a
change in the structure of the nascent protein by chemical or
enzymatic modification.
[0204] In addition to gel electrophoresis, which measures the
electrophoretic mobility of proteins in gels such as
polyacyralimide gel, this method can be performed using capillary
electrophoresis. CE measures the electrophoretic migration time of
a protein which is proportional to the charge-to-mass ratio of the
molecule. One form of CE, sometimes termed affinity capillary
electrophoresis, has been found to be highly sensitive to
interaction of proteins with other molecules including small
ligands as long as the binding produces a change in the
charge-to-mass ratio of the protein after the binding event. The
highest sensitivity can be obtained if the protein is conjugated to
a marker with a specifically detectable electromagnetic spectral
property such as a fluorescent dye. Detection of a peak in the
electrophoresis chromatogram is accomplished by laser induced
emission of mainly visible wavelengths. Examples of fluorescent
dyes include fluoroscein, rhodamine, Texas Red and BODIPY.
[0205] It is very difficult to detect a nascent protein synthesized
in a cellular or cell-free extract by CE without subsequent
isolation and labeling steps due the need for high sensitivity
detection and the presence of many other molecules of similar
mass/charge ratio in the extract. For example, in typical cases
less than 0.01% of the total protein mass of the extract may
comprise the nascent protein(s). Other molecules with similar
electrophoretic migration times as the nascent protein may be
present in the mixture. Such molecules will overlap with peaks due
to the nascent protein.
[0206] It is also very difficult using conventional methods of CE
to detect the interaction of molecules with nascent proteins
produced in a cell free or cellular synthesis system. Affinity
capillary electrophoresis has been found to be sensitive to
interaction of proteins with other molecules including small
ligands as long as the binding produces a change in the
charge-to-mass ratio of the protein after the binding event.
However, the selective addition of a marker such as a fluorescent
dye to a nascent protein is not possible using conventional means
because most markers reagents will nonspecifically label other
molecules in the protein synthesis system besides the nascent
proteins. In some cases, it may be possible utilize a specific
substrate or ligand which binds only to the nascent protein.
However this approach requires a detailed knowledge of the binding
properties of the nascent protein and special design of a ligand
with marker properties. The nascent protein may also be isolated
from the protein synthesis system and then selectively labeled with
a detectable marker. However, this also requires the development of
a procedure for isolation of the nascent protein which can be time
consuming and requires extensive knowledge of properties of the
protein or protein engineering to incorporate an affinity epitope.
Even after a nascent protein has been isolated, it is often
difficult to uniformly label the protein with a marker so that the
charge/mass ratio of each labeled protein remains the same. In the
most advantageous form of labeling, a highly fluorescent marker is
incorporated at only one specific position in the protein thus
avoiding a set of proteins with different electrophoretic
mobilities.
[0207] The method of the invention also overcomes major problems
associated with the rapid screening of samples for new therapeutic
compounds using capillary electrophoresis (CE) such as described in
U.S. Pat. No. 5,783,397 (hereby incorporated by reference) when the
target protein is a nascent protein expressed in a translation
system. This includes the need to uniformly label expressed target
proteins in a translation system with markers for high sensitivity
analysis by CE which normally requires lengthy isolation steps and
special techniques for uniform labeling.
[0208] The method can also be used in conjunction with expression
cloning method for isolating novel cDNA clones such as described in
U.S. Pat. No. 565,451, which is specifically incorporated by
reference. This patent describes novel methods to identify cDNA
clones by a) collecting pools of about 100 individual bacterial
colonies; and b) expressing proteins encoded by the cDNAs in the
pools in vitro. Proteins which can be identified in this manner
include but are not limited to nucleic acid binding protein,
cytoskeletal protein, growth factor, differentiation factor,
post-translationally modified protein, phosphorylated protein,
proteolytically cleaved protein, glycosylated protein, subunit or a
multiple component of a protein complex, enzyme, isoform of a known
protein, mutant form of known protein. Importantly, the method
includes as a crucial step identifying a desired protein from
protein translation system. Two such methods described for
identifying the protein involve radioactive labeling and chemical
labeling. However, these steps can be extremely time-consuming and
are not conducive to rapidly screening an extract for the desired
protein.
[0209] The present invention avoids all of the problems discussed
above. In one embodiment of the invention a tRNA misaminoacylated
with a detectable marker is added to the protein synthesis system.
The system is incubated to incorporate the detectable marker into
the nascent proteins. One or more molecules (agents) are then
combined with the nascent proteins (either before or after
isolation) to allow agents to interact with nascent proteins.
Aliquots of the mixture are then subjected to electrophoresis.
Nascent proteins which have interacted with the agents are
identified by detecting changes in the electrophoretic mobility of
nascent proteins with incorporated markers. In the case where the
agents have interacted with the nascent proteins, the proteins can
be isolated and subsequently subjected to further analysis. In
cases where the agents have bound to the nascent proteins, the
bound agents can be identified by isolating the nascent
proteins.
[0210] In one example of this method, the fluorescent marker
BODIPY-FL is used to misaminoacylate an E. coli initiator
tRNA.sup.fmet as previously described. The misaminoacylated tRNA is
then added to a protein synthesis system and the system incubated
to produce nascent protein containing the BODIPY-FL at the
N-terminal. A specific compound which may bind to the nascent
protein is then added to the protein synthesis system at a specific
concentration. An aliquot from the mixture is then injected into an
apparatus for capillary electrophoresis. Nascent proteins in the
mixture are identified by detection of the fluorescence from the
BODIPY-FL using exciting light from an Argon laser tuned to 488 nm.
Interaction of the specific compound is determined by comparing the
electrophoretic mobility measured of the nascent protein exposed to
the specific compound with a similar measurement of the nascent
protein that has not been exposed. The binding strength of the
compound can then be ascertained by altering the concentration of
the specific compounds added to the protein synthesis system and
measuring the change in the relative intensity of bands assigned to
the uncomplexed and complexed nascent protein.
[0211] The method is not limited to studying the interaction of one
agent with one nascent protein translated in a protein translation
system. For example, a library of compounds can be screened to
identify those which serve are ligands for specific target protein.
In addition to interactions which involve the binding of one or
more agents to the nascent proteins interactions which result in a
modification of the nascent protein including but not limited to
phosphorylation, proteolysis, glycosylation, formation of a complex
with other biological molecules can be detected using the marker
incorporated in the nascent proteins when combined with
electrophoresis. For example, the interaction of an antibody with
the nascent proteins can be detected due to a change in the
effective electrophoretic mobility of the complex formed. A similar
approach could be used to identify the presence of one or more
compounds in a complex mixture which bind to the nascent protein.
Such a mixture might constitute a library of compounds produced by
combinatorial chemistry or compounds which might be present in a
complex biological mixture such as natural samples which may
contain therapeutic compounds.
[0212] F. Microscale Methods
[0213] While the present invention contemplates capillary
electrophoresis (see above), other methods are also contemplated.
In particular, microscale methods can be employed in conjunction
with the novel markers (e.g. BODIPY) and methods of the present
invention. The methods are "microscale" in that the dimensions of
the channels on the device (and the corresponding fluid volumes)
are very small (typically in the picometer range). For example,
channels are typically between approximately 0.10 and 0.50 .mu.m in
depth and between approximately 5 and 500 .mu.m in width.
[0214] Although there are many formats, materials, and size scales
for constructing integrated fluidic systems, the present invention
contemplates microfabricated devices as a solution to the many
inefficiencies of larger scale screening. Devices can be
microfabricated from a number of materials. Silicon is the material
used for the construction of computing microprocessors and its
fabrication technologies have developed at an unprecedented pace
over the past 30 years. The principal modern method for fabricating
semiconductor integrated circuits is the so-called planar process.
The planar process relies on the unique characteristics of silicon
and comprises a complex sequence of manufacturing steps involving
deposition, oxidation, photolithography, diffusion and/or ion
implantation, and metallization, to fabricate a "layered"
integrated circuit device in a silicon substrate. See e.g., W.
Miller, U.S. Pat. No. 5,091,328, hereby incorporated by reference.
While this technology was initially applied to making
microelectronic devices, the same techniques are currently being
used for micromechanical systems.
[0215] Continuous flow liquid transport has been described using a
microfluidic device developed with silicon. See J. Pfahler et al.,
Sensors and Actuators, A21-A23 (1990), pp. 431-434. Pumps have also
been described, using external forces to create flow, based on
micromachining of silicon. See H. T. G. Van Lintel et al., Sensors
and Actuators 15:153-167 (1988). SDS capillary gel electrophoresis
of proteins in microfabricated channels has also been described.
See Yao S et al., "SDS capillary gel electrophoresis of proteins in
microfabricated channels," PNAS 96:5372 (1999). Compared to more
conventional two-dimensional denaturing gel electorphoresis (which
is generally time consuming and requires considerable amounts of
sample), this microchannel-based separation technique was shown to
be quick and offer high resolution.
[0216] As a mechanical building material, silicon has well-known
fabrication characteristics. The economic attraction of silicon
devices is that their associated micromachining technologies are,
essentially, photographic reproduction techniques. In these
processes, transparent templates or masks containing opaque designs
are used to photodefine objects on the surface of the silicon
substrate. The patterns on the templates are generated with
computer-aided design programs and can delineate structures with
line-widths of less than one micron. Once a template is generated,
it can be used almost indefinitely to produce identical replicate
structures. Consequently, even extremely complex micromachines can
be reproduced in mass quantities and at low incremental unit
cost--provided that all of the components are compatible with the
silicon micromachining process. While the present invention
contemplates other substrates, such as glass or quartz, for use in
photolithographic methods to construct microfabricated analysis
devices, silicon is preferred because of the added advantage of
allowing a large variety of electronic components to be fabricated
within the same structure.
[0217] In one embodiment, the present invention contemplates
silicon micromachined components in an integrated analysis system.
Sample (e.g. a test compound) and one or more reagents (e.g. a
BODIPY labelled nascent protein) are injected either continuously
or in pulses into the device through entry ports and they are
transported through channels to a reaction chamber, such as a
thermally controlled reactor where mixing and reactions take place.
The biochemical products can be then moved down a new channel (or
by an electrophoresis module, if desired) where migration data is
collected by a detector and transmitted to a recording instrument.
If desired, a polymer can be used in the channels to provide
resolution by molecular sieving. The biochemical products can be
isolated by diverting the flow to an external port for subsequent
additional analysis. Importantly, the fluidic and electronic
components are designed to be fully compatible in function and
construction with the biological reactions and reagents. In this
embodiment, potential test compounds can be rapidly screened for
interaction with a labeled nascent protein or multiple nascent
proteins that are co-expressed in a translation reaction system. In
this manner the system can be used to screen for interaction so as
to identify useful drugs.
[0218] In another embodiment, one or more components of the protein
synthesis system are introduced into the device through entry ports
and they are transported through channels to a reaction chamber,
such as a thermally controlled reactor, where the expression of the
nascent protein which contains the marker such as BODIPY occurs.
The labeled nascent protein can than be mixed with one or more
reagents (e.g. a test compound) that are introduced into the device
through entry ports. After the reaction takes placed, the
biochemical products can be then moved down a new channel (or by an
electrophoresis module, if desired) where migration data is
collected by a detector and transmitted to a recording instrument.
It is to be understood that components of the protein synthesis
system which can be introduced into the device can include
misaminoacylated tRNAs, DNA, mRNA, amino acids and nucleotides. The
components can be introduced either continuously or in discrete
pulses. The DNA may also be produced within the micromachined
device by enzymatic reactions such as the polymerase chain reaction
as has been described. See Kopp et al., "Chemical Amplification:
Continuous Flow PCR on a Chip," Science 280:1046 (1998).
[0219] In silicon micromachining, a simple technique to form closed
channels involves etching an open trough on the surface of a
substrate and then bonding a second, unetched substrate over the
open channel. There are a wide variety of isotropic and anisotropic
etch reagents, either liquid or gaseous, that can produce channels
with well-defined side walls and uniform etch depths. Since the
paths of the channels are defined by the photo-process mask, the
complexity of channel patterns on the device is virtually
unlimited. Controlled etching can also produce sample entry holes
that pass completely through the substrate, resulting in entry
ports on the outside surface of the device connected to channel
structures.
[0220] G. Multiple Misaminoacylated tRNAs
[0221] It may often be advantageous to incorporate more than one
marker into a single species of protein. This can be accomplished
by using a single tRNA species such as a lysine tRNA
misaminoacylated with both a marker such as dansyllysine and a
coupling agent such as biotin-lysine. Alternatively, different
tRNAs which are each misaminoacylated with different markers can
also be utilized. For example, the coumarin derivative could be
used to misaminoacylate a tryptophan tRNA and a dansyl-lysine used
to misaminoacylate a lysine tRNA.
[0222] One use of multiple misaminoacylated tRNAs is to study the
expression of proteins under the control of different genetic
elements such as repressors or activators, or promoters or
operators. For example, the synthesis of proteins at two different
times in response to an internal or external agent could be
distinguished by introducing misaminoacylated tRNAs at different
times into the cellular or cell-free protein synthesis system. A
tRNA.sup.tyr might be charged with marker A and a tRNA.sup.lys
charged with marker B, yielding A-tRNA.sup.tyr and B-tRNA.sup.lys,
respectively. In this case, protein one under the control of one
promoter can be labeled by adding the A-tRNA.sup.tys to the
reaction system. If a second misaminoacylated tRNA, B-tRNA.sup.lys
is then added and a second promoter for protein two activated, the
nascent protein produced will contain both label A and B.
Additional markers could also be added using additional tRNA
molecules to further study the expression of additional proteins.
The detection and analysis of multiply labeled nascent proteins can
be facilitated by using the multi-colored electrophoresis pattern
reading system, described in U.S. Pat. No. 5,190,632, which is
specifically incorporated by reference, or other multi-label
reading systems such as those described in U.S. Pat. Nos. 5,069,769
and 5,137,609, which are both hereby specifically incorporated by
reference.
[0223] A second use of multiple misaminoacylated tRNAs is in the
combined isolation and detection of nascent proteins. For example,
biotin-lysine marker could be used to misaminoacylate one tRNA and
a coumarin marker used to misaminoacylate a different tRNA.
Magnetic particles coated with streptavidin which binds the
incorporated lysine-biotin would be used to isolate nascent
proteins from the reaction mixture and the coumarin marker used for
detection and quantitation.
[0224] A schematic diagram of the basics of the above methods is
shown in FIG. 14. In a first step, the marker selected (M), which
may have reporter (R) or affinity (A) properties, is chemically or
enzymatically misaminoacylated to a single tRNA species or a
mixture of different tRNAs. Prior to protein synthesis, a
predetermined amount of the misaminoacylated tRNA, charged with the
fluorescent marker is mixed with the cell-free protein synthesis
reaction system at concentrations sufficient to allow the
misaminoacylated tRNA to compete effectively with the corresponding
tRNA. After an incubation of about 1-3 hours, the reaction mixture
is analyzed using conventional polyacrylamide or agarose gel
electrophoresis. After electrophoresis, the gel is illuminated by
UV radiation. Bands due to the nascent protein exhibit distinct
fluorescence and can be easily and rapidly distinguished, either
visually or photographically, from non-fluorescent bands of
preexisting proteins. Nascent proteins can be isolated by excising
the fluorescent band and electroeluting the protein from the
extracted gel pieces. The quantities and molecular weights of the
nascent proteins can be determined by comparison of its
fluorescence with the fluorescence produced by a set of proteins
with known molecular weights and known quantities. The results of
the assay can be recorded and stored for further analysis using
standard electronic imaging and photographic or spectroscopic
methods.
[0225] H. Resulting Compositions
[0226] Another embodiment of the invention is directed to a
composition comprising nascent proteins isolated or purified by
conventional methods after translation in the presence of markers.
Compositions can be utilized in manufacturing for the preparation
of reagents such as coatings for tissue culture products and in the
pharmaceutical industry.
[0227] Incorporation of markers into nascent proteins utilized in
manufacturing facilitates analysis of the final manufactured
product or process by detection of marker. For example, nascent
proteins produced may be used as coatings for tissue culture
products. The reproducibility of a particular coating process could
be accurately determined by detecting variations of marker
emissions over the surface of the coated product. In addition,
non-toxic markers incorporated into proteins encompassed within a
pharmaceutical preparation such as a hormone, steroid, immune
product or cytokine can be utilized to facilitate safe and
economical isolation of that protein preparation. Such products
could be used directly without the need for removal of marker. When
very low concentrations of marker are preferred, limiting amounts
of marked proteins could be used to follow a protein through a
purification procedure. Such proteins can be efficiently purified
and the purity of the resulting composition accurately determined.
In addition, the presence of markers may facilitate study and
analysis of pharmaceutical compositions in testing. For example,
markers can be utilized to determine serum half-life, optimum serum
levels and the presence or absence of break-down products of the
composition in a patient.
[0228] Alternatively, nascent proteins may contain specific markers
which serve as therapeutically useful compounds such as toxic
substances. These proteins are administered to a patient and the
therapeutic moiety released after proteins have identified and
possibly bound to their respective targets. Release may be
electrical stimulation, photochemical cleavage or other means
whereby the moiety is specifically deposited in the area targeted
by the nascent proteins. In addition, moieties such as modified
toxins may be utilized which become toxic only after release from
nascent proteins. Nascent protein may also serve as a
pharmaceutical carrier which bestows the incorporated marker with
active therapeutic function or prevents marker from breaking down
in the body prior to its therapeutic or imaging action.
[0229] The incorporation of cleavable markers in nascent proteins
further provides a means for removal of the non-native portion of
the marker to facilitate isolation of the protein in a completely
native form. For example, a cleavable affinity marker such as
photocleavable biotin introduced into a nascent protein facilitates
economical isolation of the protein and allows for the removal of
the marker for further use as a pharmaceutical composition.
[0230] Pharmaceutical compositions of proteins prepared by
translation in the presence of markers may further comprise a
pharmaceutically acceptable carrier such as, for example, water,
oils, lipids, polysaccharides, glycerols, collagens or combinations
of these carriers. Useful immunological compositions include
immunologically active compositions, such as a vaccine, and
pharmaceutically active compositions, such as a therapeutic or
prophylactic drug which can be used for the treatment of a disease
or disorder in a human.
[0231] I. Kits
[0232] Another embodiment of the invention is directed to
diagnostic kits or aids containing, preferably, a cell-free
translation containing specific misaminoacylated tRNAs which
incorporate markers into nascent proteins coded for by mRNA or
genes, requiring coupled transcription-translation systems, and are
only detectably present in diseased biological samples. Such kits
may be useful as a rapid means to screen humans or other animals
for the presence of certain diseases or disorders. Diseases which
may be detected include infections, neoplasias and genetic
disorders. Biological samples most easily tested include samples of
blood, serum, tissue, urine or stool, prenatal samples, fetal
cells, nasal cells or spinal fluid. In one example, misaminoacylate
fmet-tRNAs could be used as a means to detect the presence of
bacteria in biological samples containing prokaryotic cells. Kits
would contain translation reagents necessary to synthesize protein
plus tRNA molecules charged with detectable non-radioactive
markers. The addition of a biological sample containing the
bacteria-specific genes would supply the nucleic acid needed for
translation. Bacteria from these samples would be selectively lysed
using a bacteria directed toxin such as Colicin E1 or some other
bacteria-specific permeabilizing agent. Specific genes from
bacterial DNA could also be amplified using specific
oligonucleotide primers in conjunction with polymerase chain
reaction (PCR), as described in U.S. Pat. No. 4,683,195, which is
hereby specifically incorporated by reference. Nascent proteins
containing marker would necessarily have been produced from
bacteria. Utilizing additional markers or additional types of
detection kits, the specific bacterial infection may be
identified.
[0233] The present invention also contemplates kits which permit
the GFTT described above. For example, the present invention
contemplates kits to detect specific diseases such as familial
adenomatous polyposis. In about 30 to 60% of cases of familial
adenomatous polyposis, the diseased tissues also contain chain
terminated or truncated transcripts of the APC gene (S. M. Powell
et al., N. Engl. J. Med. 329:1982-87, 1993). Chain termination
occurs when frameshift cause a stop codon such as UAG, UAA or UGA
to appear in the reading frame which terminates translation. Using
misaminoacylated tRNAs which code for suppressor tRNAs, such
transcripts can be rapidly and directly detected in inexpensive
kits. These kits would contain a translation system, charged
suppressor tRNAs containing detectable markers, for example
photocleavable coumarin-biotin, and appropriate buffers and
reagents. Such a kit might also contain primers or "pre-primers,"
the former comprising a promoter, RBS, start codon, a region coding
an affinity tag and a region complementary to the template, the
latter comprising a promoter, RBS, start codon, and region coding
an affinity tag--but lacking a region complementary to the
template. The pre-primer permits ligation of the region
complementary to the template (allowing for customization for the
specific template used). A biological sample, such as diseased
cells, tissue or isolated DNA or mRNA or PCR products of the DNA,
is added to the system, the system is incubated and the products
analyzed. Analysis and, if desired, isolation is facilitated by a
marker such as coumarin or biotin which can be specifically
detected by its fluoresence using streptavidin coupled to HRP. Such
kits provide a rapid, sensitive and selective non-radioactive
diagnostic assay for the presence or absence of the disease.
[0234] Experimental
[0235] The following examples illustrate embodiments of the
invention, but should not be viewed as limiting the scope of the
invention. In some of the examples below, particular reagents and
methods were employed as follows:
[0236] Reagents: tRNA.sup.fmet, aminoacyl-tRNA synthetase, amino
acids, buffer salts, and RNase free water were purchased from Sigma
(St. Louis, Mo.). Many of the fluorescent dyes were obtained from
Molecular Probes (Eugene, Oreg.). The translation supplies
including routine kits were purchased from Promega (Madison, Wis.).
Sephadex G-25 was from Amersham-Pharmacia Biotech (Piscataway,
N.J.). The in vitro translation kits and plasmid DNAs coding for
CAT (PinPoint.TM.) and Luciferase (pBESTluc.TM.) were from Promega
(Wisconsin-Madison, Wis.) while DHFR plasmid DNA (pQE16-DHFR) was
obtained from Qiagen (Valencia, Calif.). The plasmid DNA for
.alpha.-hemolysin, pT7-WT-H6-.alpha.HL was kindly supplied by Prof.
Hagan Bayley (Texas A & M University) and large scale
preparation of .alpha.-HL DNA was carried out using Qiagen plasmid
isolation kit. The bacterioopsin plasmid DNA (pKKbop) was from the
laboratory stock.
[0237] Preparation of FluoroTag tRNAs: The purified tRNA.sup.fmet
was first aminoacylated with the methionine. In typical reaction,
1500 picomoles (.about.1.0 OD.sub.260) of tRNA was incubated for 45
min at 37.degree. C. in aminoacylation mix using excess of
aminoacyl tRNA-synthetases. After incubation, the mixture was
neutralized by adding 0.1 volume of 3 M sodium acetate, pH 5.0 and
subjected to chloroform:acid phenol extraction (1:1). Ethanol (2.5
volumes) was added to the aqueous phase and the tRNA pellet
obtained was dissolved in the water (25 .mu.l). The coupling of
NHS-derivatives of fluorescent molecules to the .alpha.-amino group
of methionine was carried out in 50 mM sodium carbonate, pH 8.5 by
incubating the aminoacylated tRNAf.sup.met (25 .mu.l) with
fluorescent reagent (final concentration=2 mM) for 10 min at
0.degree. C. and the reaction was quenched by the addition of
lysine (final concentration=100 mM). The modified tRNA was
precipitated with ethanol and passed through Sephadex G-25 gel
filtration column (0.5.times.5 cm) to remove any free fluorescent
reagent, if present. The modified tRNA was stored frozen
(-70.degree. C.) in small aliquots in order to avoid free-thaws.
The modification extent of the aminoacylated-tRNA was assessed by
acid-urea gel electrophoresis. This tRNA was found to stable at
least for 6 month if stored properly.
[0238] Cell free synthesis of proteins and their detection: The in
vitro translation reactions were typcially carried out using E.
coli T7 transcription-translation system (Promega) with optimized
premix. The typical translation reaction mixture (10 .mu.l)
contained 3 .mu.l of extract, 4 .mu.l of premix, 1 .mu.l of
complete amino acid mix, 30 picomoles of fluorescent-methionyl-tRNA
and 0.5 .mu.g of appropriate plasmid DNA. The optimized premix
(1.times.) contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate,
210 mM potassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mM ATP,
0.8 mM GTP, 0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenol pyruvate, 0.6
mM cAMP and 16 mM magnesium acetate. The translation reaction was
allowed to proceed for 45 min at 37.degree. C. For SDS-PAGE, 4-10
.mu.l aliquot of the reaction mix was precipitated with 5-volume
acetone and the precipitated proteins were collected by
centrifugation. The pellet was dissolved in 1.times. loading buffer
and subjected to SDS-PAGE after boiling for 5 min. SDS-PAGE was
carried out according to Laemmli and the gel was scan using
Molecular Dynamics FluorImager 595 using Argon laser as excitation
source. Alternatively, the nascent proteins in polyacrylamide gels
were also detected using an UV-transilluminator and the photographs
were carried out using Polaroid camera fitted with green filter
(Tiffen green #58, Polaroid DS34 camera filter kit).
[0239] For visualization of BODIPY-FL labeled protein, 488 nm as
excitation source was used along with a 530.+-.30 narrow band
excitation filter. The gel was scanned using PMT voltage 1000 volts
and either 100 or 200 micron pixel size.
[0240] Enzyme/Protein activities: Biological activity of
.alpha.-hemolysin was carried out as follows. Briefly, various
aliquots (0.5-2 .mu.l) of in vitro translation reaction mixture
were added to 500 .mu.l of TBSA (Tris-buffered saline containing 1
mg/ml BSA, pH 7.5). To this, 25 .mu.l of 10% solution of rabbit red
blood cells (rRBCs) was added and incubated at room temperature for
20 min. After incubation, the assay mix was centrifuged for 1 min
and the absorbance of supernatant was measured at 415 nm (release
of hemoglobin). The equal amount of rRBCs incubated in 500 .mu.l of
TBSA is taken as control while rRBCs incubated with 500 .mu.l of
water as taken 100% lysis. The DHFR activity was measured
spectrophotometrically. Luciferase activity was determined using
luciferase assay system (Promega) and luminescence was measures
using Packard Lumi-96 luminometer.
[0241] Purification of .alpha.-HL and measurement BODIPY-FL
incorporation into .alpha.-HL: The translation of plasmid coding
for .alpha.-HL (His.sub.6) was carried out at 100 .mu.l scale and
the .alpha.-HL produced was purified using Talon-Sepharose
(ClonTech) according manufacturer instructions. The fluorescence
incorporated into .alpha.-HL was then measured on Molecular
Dynamics FluorImager along with the several known concentration of
free BODIPY-FL (used as standard). The amount of protein in the
same sample was measured using a standard Bradford assay using
Pierce Protein Assay kit (Pierce, Rockford, Ill.).
EXAMPLE 1
[0242] Preparation of Markers
[0243] Synthesis of Coumarin Amino Acid: 4-(Bromomethyl)-7-methoxy
coumarin (FIG. 15, compound 1; 6.18 mmole) and
diethylacetamidomalonate (FIG. 15, compound 2; 6.18 mmole) were
added to a solution of sodium ethoxide in absolute ethanol and the
mixture refluxed for 4 hours. The intermediate obtained (FIG. 15,
compound 3) after neutralization of the reaction mixture and
chloroform extraction was further purified by crystallization from
methanolic solution. This intermediate was dissolved in a mixture
of acetone and HCl (1:1) and refluxed for one hour. The reaction
mixture was evaporated to dryness, and the amino acid hydrochloride
precipitated using acetone. This hydrochloride was converted to
free amino acid (FIG. 15, compound 4) by dissolving in 50% ethanol
and adding pyridine to pH 4-5. The proton (.sup.1H) NMR spectrum of
the free amino acid was as follows: (m.p. 274-276.degree. C.,
decomp.) --OCH.sub.3 (.delta.3.85 s, 3H), --CH.sub.2-(.delta.3.5 d,
2H), .alpha.-CH-- (.delta.2.9 t, 1H), CH--CO (.delta.6.25 s, 1H),
ring H (.delta.7.05 s, 1H), (6 7.8 d, 2H).
[0244] Synthesis of Fmoc derivative of coumarin: Coumarin amino
acid (1.14 mmol) was reacted with Fluorenylmethyloxycarbonyl
N-hydroxysuccininmidyl ester (Fmoc-NHS ester) 1.08 mmol) in the
presence of 1.14 mmol of triethylamine for 30 minutes at room
temperature. The reaction mixture was acidified and the precipitate
washed with 1 N HCl and dried. The NMR spectrum of the free amino
acid was as follows:
[0245] (MP 223-225.degree. C.) --OCH.sub.3 (.delta.3.85 s, 3H),
--CH.sub.2Br (.delta.3.5 broad singlet, 2H), .alpha.-CH-(.delta.3.0
t, 1H), CH--CO (.delta.6.22 m, 1H), ring H (.delta.7.05 s, 1H),
(.delta.7.8 d, 2H), fluorene H CH.sub.2--CH (.delta.4.2 m, 2H),
CH.sub.2--CH (.delta.4.25 m, 1H), aromatic regions showed
characteristic multiplets.
[0246] Synthesis of PCB: Photocleavable biotin was synthesized as
described below. 2-bromo, 2'-nitroacetophenone (FIG. 15, compound
5) was converted first into its hexamethyltetraamommonium salt
which was decomposed to obtain 2-amino, 2'-nitroacetophenone (FIG.
15, compound 6). Biotin N-hydroxysuccinimidyl ester (FIG. 15,
compound 7; Sigma Chemical; St. Louis, Mo.) was reacted with a
6-aminocaproic acid (FIG. 15, compound 8) to obtain the
corresponding acid (FIG. 15, compound 9). This acid was coupled
with the 2-amino, 2'nitroacetophenone using DCC to obtain the
ketone (FIG. 15, compound 10). The ketone was reduced using sodium
borohydride to obtain the alcohol (FIG. 15, compound 11) which was
further converted into its chloroformate derivative (FIG. 15,
compound 12). The proton NMR spectrum of the derivative (compound
12) was as follows: (.delta.1.3 m, 3H), (.delta.1.4 m, 2H),
(.delta.1.5 m, 5H) (.delta.1.62 m, 1H), (.delta.2.1 t, 2H)
(.delta.2.4 t, 2H), (.delta.2.6 d, 1H), (.delta.2.8 m, 1H),
(.delta.3.0 t, 1H), (.delta.3.1 m 1H), (.delta.4.15 qt, 1H)
(.delta.4.42 qt, 1H), (.delta.5.8 t, 1H), (.delta.6.25 s, 1H),
(.delta.6.45 s, 1H), (.delta.7.5 t, 1H), (.delta.7.75 m, 4H),
(.delta.7.9 d, 1H).
EXAMPLE 2
[0247] Misaminoacylation of tRNA
[0248] The general strategy used for generating misaminoacylated
tRNA is shown in FIG. 16 and involved truncation of tRNA molecules,
dinucleotide synthesis (FIG. 17), aminoacylation of the
dinucleotide (FIG. 18) and ligase mediated coupling.
[0249] a) Truncated tRNA molecules were generated by periodate
degradation in the presence of lysine and alkaline phosphatase
basically as described by Neu and Heppel (J. Biol. Chem.
239:2927-34, 1964). Briefly, 4 mmoles of uncharged E. coli
tRNA.sup.Lys molecules (Sigma Chemical; St. Louis, Mo.) were
truncated with two successive treatments of 50 mM sodium
metaperiodate and 0.5 M lysine, pH 9.0, at 60.degree. C. for 30
minutes in a total volume of 50 .mu.l. Reaction conditions were
always above 50.degree. C. and utilized a 10-fold excess of
metaperiodate. Excess periodate was destroyed treatment with 5
.mu.l of 1M glycerol. The pH of the solution was adjusted to 8.5 by
adding 15 .mu.l of Tris-HCl to a final concentration of 0.1 M. The
reaction volume was increased to 150 .mu.l by adding 100 .mu.l of
water. Alkaline phosphatase (15 .mu.l, 30 units) was added and the
reaction mixture incubated again at 60.degree. C. for two hours.
Incubation was followed by ethanol precipitation of total tRNA,
ethanol washing, drying the pellet and dissolving the pellet in 20
.mu.l water. This process was repeated twice to obtain the
truncated tRNA.
[0250] b) Dinucleotide synthesis was carried out basically as
performed by Hudson (J. Org. Chem. 53:617-24, 1988), and can be
described as a three step process, deoxycytidine protection,
adenosine protection and dinucleotide synthesis.
[0251] Deoxycytidine protection: All reaction were conducted at
room temperature unless otherwise indicated. First, the 5' and 3'
hydroxyl groups of deoxycytidine were protected by reacting with
4.1 equivalents of trimethylsilyl chloride for 2 hours with
constant stirring. Exocyclic amine function was protected by
reacting it with 1.1 equivalents of Fmoc-Cl for 3 hours.
Deprotection of the 5' and 3' hydroxyl was accomplished by the
addition of 0.05 equivalents of KF and incubation for 30 minutes.
The resulting product (FIG. 17, compound 19) was produced at an 87%
yield. Phosphate groups were added by incubating this compound with
1 equivalent of bis-(2-chlorophenyl)phosphorochloridate and
incubating the mixture for 2 hours at 0.degree. C. The yield in
this case was 25-30%.
[0252] Adenosine protection: Trimethylsilyl chloride (4.1
equivalents) was added to adenosine residue and incubated for 2
hours, after which, 1.1 equivalents of Fmoc-Cl introduced and
incubation continued for 3 hours. The TMS groups were deprotected
with 0.5 equivalents of fluoride ions as described above. The Fmoc
protected adenosine (compound 22) was obtained in a 56% yield. To
further protect the 2'-hydroxyl, compound 22 was reacted with 1.1
equivalents of tetraisopropyl disiloxyl chloride (TIPDSCl.sub.2)
for 3 hours which produces compound 23 at a 68-70% yield. The
compound was converted to compound 24 by incubation with 20
equivalents of dihydropyran and 0.33 equivalents of
p-toluenesulfonic acid in dioxane for about 4-5 hours. This
compound was directly converted without isolation into compound 25
(FIG. 17) by the addition of 8 equivalents of tetrabutyl ammonium
fluoride in a mixture of tetrahydro-furan, pyridine and water.
[0253] Dinucleotide synthesis: The protected deoxycytidine,
compound 20, and the protected adenosine, compound 25 (FIG. 17),
were coupled by the addition of 1.1 equivalents of 2-chlorophenyl
bis-(1-hydroxy benzotriazolyl) phosphate in tetrahydrofuran with
constant stirring for 30 minutes. This was followed by the addition
of 1.3 equivalents of protected adenosine, compound 25, in the
presence of N-methylimidazole for 30 minutes. The coupling yield
was about 70% and the proton NMR spectrum of the coupled product,
compound 26 (FIG. 17), was as follows: (.delta.8.76 m, 2H),
(.delta.8.0 m, 3H), (.delta.7.8 m, 3H) (.delta.7.6 m, 4H),
(.delta.7.5 m, 3H), (.delta.7.4 m, 18H), (.delta.7.0 m, 2H),
(.delta.4.85 m, 14H), (.delta.4.25 m, 1H); (.delta.3.6 m, 2H),
(.delta.3.2 m, 2H), (.delta.2.9 m, 3H), (.delta.2.6 m, 1H),
(.delta.2.0-1.2 m, 7H).
[0254] c) Aminoacylation of the dinucleotide was accomplished by
linking the protected marker amino acid, Fmoc-coumarin, to the
dinucleotide with an ester linkage. First, the protected amino acid
was activated with 6 equivalents of benzotriazol-1-yl-oxy
tris-(dimethylamino) phosphonium hexafluoro phosphate and 60
equivalents of 1-hydroxybenzotriazole in tetrahydrofuran. The
mixture was incubated for 20 minutes with continuous stirring. This
was followed with the addition of 1 equivalent of dinucleotide in 3
equivalents N-methylimidazole, and the reaction continued at room
temperature for 2 hours. Deprotection was carried out by the
addition of tetramethyl guanidine and 4-nitrobenzaldoxime, and
continuous stirring for another 3 hours. The reaction was completed
by the addition of acetic acid and incubation, again with
continuous stirring for 30 minutes at 0.degree. C. which produced
the aminoacylated dinucleotide (FIG. 18).
[0255] d) Ligation of the tRNA to the aminoacylated dinucleotide
was performed basically as described by T. G. Heckler et al.
(Tetrahedron 40: 87-94, 1984). Briefly, truncated tRNA molecules
(8.6 O.D..sub.260 units/ml) and aminoacylated dinucleotides (4.6
O.D..sub.260 units/ml), were incubated with 340 units/ml T4 RNA
ligase for 16 hours at 4.degree. C. The reaction buffer included 55
mM Na-Hepes, pH 7.5, 15 mM MgCl.sub.2, 250 .mu.M ATP, 20 .mu.g/ml
BSA and 10% DMSO. After incubation, the reaction mixture was
diluted to a final concentration of 50 mM NaOAc, pH 4.5, containing
10 mM MgCl.sub.2. The resulting mixture was applied to a
DEAE-cellulose column (1 ml), equilibrated with 50 mM NaOAc, pH
4.5, 10 mM MgCl.sub.2, at 4.degree. C. The column was washed with
0.25 mM NaCl to remove RNA ligase and other non-tRNA components.
The tRNA-containing factions were pooled and loaded onto a
BD-cellulose column at 4.degree. C., that had been equilibrated
with 50 mM NaOAc, pH 4.5, 10 mM MgCl.sub.2, and 1.0 M NaCl.
Unreacted tRNA was removed by washes with 10 ml of the same buffer.
Pure misaminoacylated tRNA was obtained by eluting the column with
buffer containing 25% ethanol.
EXAMPLE 3
[0256] Preparation of Extract and Template
[0257] Preparation of extract: Wheat germ embryo extract was
prepared by floatation of wheat germs to enrich for embryos using a
mixture of cyclohexane and carbon tetrachloride (1:6), followed by
drying overnight (about 14 hours). Floated wheat germ embryos (5 g)
were ground in a mortar with 5 grams of powdered glass to obtain a
fine powder. Extraction medium (Buffer I: 10 mM trisacetate buffer,
pH 7.6, 1 nM magnesium acetate, 90 mM potassium acetate, and 1 mM
DTT) was added to small portions until a smooth paste was obtained.
The homogenate containing disrupted embryos and 25 ml of extraction
medium was centrifuged twice at 23,000.times.g. The extract was
applied to a Sephadex G-25 fine column and eluted in Buffer II (10
mM trisacetate buffer, pH 7.6, 3 mM magnesium acetate, 50 mM
potassium acetate, and 1 mM DTT). A bright yellow band migrating in
void volume and was collected (S-23) as one ml fractions which were
frozen in liquid nitrogen.
[0258] Preparation of template: Template DNA was prepared by
linearizing plasmid pSP72-bop with EcoRI. Restricted linear
template DNA was purified by phenol extraction and DNA
precipitation. Large scale mRNA synthesis was carried out by in
vitro transcription using the SP6-ribomax system (Promega; Madison,
Wis.). Purified mRNA was denatured at 67.degree. C. for 10 minutes
immediately prior to use.
EXAMPLE 4
[0259] Cell-Free Translation Reactions
[0260] The incorporation mixture (100 .mu.l) contained 50 .mu.l of
S-23 extract, 5 mM magnesium acetate, 5 mM Tris-acetate, pH 7.6, 20
mM Hepes-KOH buffer, pH 7.5; 100 mM potassium acetate, 0.5 mM DTT,
0.375 mM GTP, 2.5 mM ATP, 10 mM creatine phosphate, 60 .mu.g/ml
creatine kinase, and 100 .mu.g/ml mRNA containing the genetic
sequence which codes for bacterioopsin. Misaminoacylated PCB-lysine
or coumarin amino acid-tRNA.sup.lys molecules were added at 170
.mu.g/ml and concentrations of magnesium ions and ATP were
optimized. The mixture was incubated at 25.degree. C. for one
hour.
EXAMPLE 5
[0261] Isolation of Nascent Proteins Containing PCB-Lysine
[0262] Streptavidin coated magnetic Dynabeads M-280 (Dynal; Oslo,
Norway), having a binding capacity of 10 .mu.g of biotinylated
protein per mg of bead. Beads at concentrations of 2 mg/ml, were
washed at least 3 times to remove stabilizing BSA. The translation
mixture containing PCB-lysine incorporated into nascent protein was
mixed with streptavidin coated beads and incubated at room
temperature for 30 minutes. A magnetic field was applied using a
magnetic particle concentrator (MPC) (Dynal; Oslo, Norway) for
0.5-1.0 minute and the supernatant removed with pipettes. The
reaction mixture was washed 3 times and the magnetic beads
suspended in 50 .mu.l of water.
[0263] Photolysis was carried out in a quartz cuvette using a
Black-Ray longwave UV lamp, Model B-100 (UV Products, Inc.; San
Gabriel, Calif.). The emission peak intensity was approximately
1100 .mu.W/cm.sup.2 at 365 nm. Magnetic capture was repeated to
remove the beads. Nascent proteins obtained were quantitated and
yields estimated at 70-95%.
EXAMPLE 6
[0264] The Lower Limit of Detection using Fluorescence
[0265] Bovine serum albumin (BSA), suspended at 0.25 mg/ml in
borate buffer, pH 8.0, was combined with a 25 fold molar excess
fluorescamine (Sigma Chemical; St. Louis, Mo.) at 50 mg/ml to
produce a modified, fluorescent BSA. Various amounts of modified
protein (1 ng, 5 ng, 10 ng, 25 ng, 50 ng, 75 ng, 100 ng, 150 ng,
200 ng) were suspended in loading buffer (bromophenol blue,
glycerol, 2-mercaptoethanol, Tris-HCl, pH 6.8, SDS), and added to
individual wells of a 1.5 mm thick, 12% polyacrylamide gel with a
3% stacker. The water cooled gel was electrophoresed for 4 hours at
50 volts. After electrophoresis, the gel was removed from the
electrophoresis apparatus, placed on a UV transilluminator and
photographed with polaroid Type 667 film using an exposure time of
10 seconds. The lowest limit of detection observed under theses
conditions was 10 ng. These results indicate that using equipment
found in a typical molecular biology lab, fluorescently labeled
proteins can be detected at ng quantities. Using even more
sophisticated detection procedures and devices the level of
detection can be increased even further.
EXAMPLE 7
[0266] Nascent Proteins Containing Coumarin-Amino Acid
[0267] Cell-free translation is performed as described using
charged tRNA.sup.lys molecules misaminoacylated with lysine coupled
to a benzopyrene fluorophore moiety and human .gamma.-interferon
mRNA which contains 21 codons for lysine. Samples of the mixture
are supplemented with buffer containing bromophenol blue, glycerol,
2-mercaptoethanol, Tris-HCl, pH 6.8, and SDS, and directly applied
to a 12% poly-acrylamide gel (3% stacker) along with a set of
molecular weight markers. Electrophoresis is performed for 3 hours
at 50 volts. The gel is removed from the electrophoresis apparatus
and photographed under UV light. Bands of fluorescently labeled
interferon protein are specifically detected at a molecular weight
of about 25 KDa. No other significant fluorescent activity is
observed on the gel. Free misaminoacylated tRNA molecules may be
electrophoresed off of the gel and not specifically detected.
EXAMPLE 8
[0268] In Vivo Half-life of a Pharmaceutical Composition
[0269] Cell-free translation reactions are performed by mixing 10
.mu.l of PCB-coumarin amino acid-tRNA.sup.leu, prepared by chemical
misaminoacylation as described above and suspended in TE at 1.7
mg/ml), 50 .mu.l of S-23 extract, 10 .mu.l water and 10 .mu.l of a
solution of 50 mM magnesium acetate, 50 mM Tris-acetate, pH 7.6,
200 mM Hepes-KOH buffer, pH 7.5; 1 M potassium acetate, 5 mM DTT,
3.75 mM GTP, 25 mM ATP, 100 mM creatine phosphate and 600 .mu.g/ml
creatine kinase. This mixture is kept on ice until the addition of
20 .mu.l of 500 .mu.g/ml human IL-2 mRNA (containing 26 leucine
codons) transcribed and isolated from recombinant IL-2 cDNA. The
mixture is incubated at 25.degree. C. for one hour and placed on
ice. 100 .mu.l of streptavidin coated magnetic Dynabeads (2 mg/ml)
are added to the mixture which is placed at room temperature for 30
minutes. After incubation, the mixture is centrifuged for 5 minutes
in a microfuge at 3,000.times.g or, a magnetic field is applied to
the solution using a MPC. Supernatant is removed and the procedure
repeated three times with TE. The final washed pellet is
resuspended in 50 .mu.l of 50 mM Tris-HCl, pH 7.5 and transferred
to a quartz cuvette. UV light from a Black-Ray longwave UV lamp is
applied to the suspension for approximately 1 second. A magnetic
field is applied to the solution with a MPC for 1.0 minute and the
supernatant removed with a pipette. The supernatant is sterile
filtered and mixed with equal volumes of sterile buffer containing
50% glycerol, 1.8% NaCl and 25 mM sodium bicarbonate. Protein
concentration is determined by measuring the O.D..sub.260.
[0270] 0.25 ml of the resulting composition is injected i.v. into
the tail vein of 2 Balb/c mice at concentrations of 1 mg/ml. Two
control mice are also injected with a comparable volume of buffer.
At various time points (0, 5 minutes, 15 minutes, 30 minutes, 60
minutes, 2 hours and 6 hours), 100 .mu.l serum samples are obtained
from foot pads and added to 400 .mu.l of 0.9% saline. Serum sample
are added to a solution of dynabeads (2 mg/ml) coated with
anti-coumarin antibody and incubated at room temperature for 30
minutes. A magnetic field is applied to the solution with a MPC for
1 minute and the supernatant removed with a pipette. Fluorescence
at 470 mn is measured and the samples treated with monoclonal
antibody specific for rat IL-2 protein. IL-2 protein content is
quantitated for each sample and equated with the amount of
fluorescence detected. From the results obtained, in vivo IL-2
half-life is accurately determined.
EXAMPLE 9
[0271] Incorporation of Various Fluorophores into
.alpha.-Hemolysin
[0272] E. coli tRNA.sup.fmet was first quantitatively aminoacylated
with methionine and the .alpha.-amino group was specifically
modified using NHS-derivatives of several fluorophores. The list of
fluorescent reporter molecules (fluorophores) tested and their
properties are given in Table 2. Under the modification conditions,
the modified Met-tRNA.sup.fmet is found to be stable as assessed by
acid-urea gel. Since all the fluorescent molecules tested have
different optical properties (excitation and emission), we have
determined their relative fluorescence intensity under the
condition which were used for the quantitation of gels containing
nascent protein.
[0273] Fluorescent detection of nascent protein was first evaluated
using .alpha.-hemolysin (.alpha.-HL) as a model protein (with
C-terminal His.sub.6-tag). .alpha.-HL is relatively small protein
(32 kDa) and could be produced efficiently in in vitro translation.
In addition, its activity can be measured directly in the protein
translation mixture using a rabbit red blood cell hemolysis assay.
In vitro translation of .alpha.-HL was carried out using an E. coli
T7 S30 transcription/translation extract (Promega Corp., Madison,
Wis.) in the presence of several different modified
methionyl-tRNA.sup.fmet as described above. After the reaction, an
aliquot (3-5 .mu.l ) was subjected to SDS-PAGE analysis and the
fluorescent bands were detected and quantitated using a FluorImager
F595 (Molecular Dynamics, Sunnyvale, Calif.).
[0274] The data is presented in FIG. 20. Lane 1 is a no DNA
control. Lane 2 shows the results with BODIPY-FL-SSE. Lane 3 shows
the results with BODIPY-FL-SE. Lane 4 shows the results with NBD
(see Table 2 for the structure). Lane 5 shows the results with
Bodipy-TMR. Lane 6 shows the results with BODIPY R6G. Lanes 7, 8, 9
and 10 show the results achieved with FAM, SFX, PYMPO and TAMRA,
respectively (see Table 2 for structures).
[0275] The results clearly indicate the .alpha.-HL produced in
presence of BODIPY-FL-methionyl-tRNA.sup.fmet (lanes 2 and 3)
exhibited the highest fluorescence (all the data is normalized to
the BODIPY-FL-SSE. The two different BODIPY-FL reagents (BODIPY-FL
sulfosuccinimidyl ester (SSE) and BODIPY-FL succinimidyl ester
(SE)), differ only with respect to solubility. The next best
fluorophore evaluated, 6-(tetramethylrhodamine--
5-(and-6)-carboxamido)hexanoic acid, succinimidyl ester (TAMRA-X,
SE), exhibited 35% of the fluorescence (corrected for relative
fluorescence) of BODIPY-FL-SSE. Two other forms of BODIPY,
BODIPY-TMR, SE
(6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-i-
ndacene-2-propionyl)amino)hexanoic acid, succinimidyl ester) and
BODIPY-R6G, SE
(4,4-difluoro-5-phenyl-bora-3a,4a-diaza-s-indacene-3-propi- onic
acid, succinimidyl ester) exhibited less than 3% of the
fluorescence of BODIPY-FL, SSE . Succinimidyl
6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ami- nohexanoate (NBD-X-SE), a
fluorescent molecule which has previously been incorporated into
the neuorkinin-2 receptor. exhibited only 6% of the BODIPY-FL-SSE.
The two fluorescein analogs 5-(and-6)-carboxyfluorescein,
succinimidyl-ester (FAM, SE) and 6-(fluorescein-5-(and-6)
carboxamido)hexanoic acid, succinimidyl ester (SFX, SE) also showed
very low fluorescence (8.4% and 4.6%, respectively relative to
BODIPY-FL).
EXAMPLE 10
[0276] Optimizing Incorporation
[0277] In order to optimize the amount of BODIPY-FL incorporated
into nascent proteins, the translation reaction for .alpha.-HL was
carried out in presence of increasing amounts of
BODIPY-FL-methionyl-tRNA.sup.fmet ranging from 3-60 picomoles per
reaction. All reactions yielded similar amount of .alpha.-HL as
determined by hemolysis activity of rabbit red blood cells
indicating that the exogenously added BODIPY-FL-methionyl-tRN-
A.sup.fmet in this range did not inhibit protein synthesis. In
contrast, the intensity of the fluorescent band corresponding to
.alpha.-HL continued to increase up to 30 picomoles
BODIPY-FL-methionyl-tRNA per 10 .mu.l reaction (data not shown).
Increases above this level produced no further increase in
fluorescence, thus subsequent reactions were performed using this
level of BODIPY-FL-methionyl-tRNA.
[0278] A second step used to optimize BODIPY-FL incorporation was
based on eliminating N-formyl-tetrahydrofolate (fTHF) from the
reaction mixture. In prokaryotes, N-formyl-tetrahydrofolate (fTHF)
acts as a cofactor for the enzyme methionyl-tRNA transformylase,
which formylates the initiator tRNA after its aminoacylation with
methionine. Protein synthesis is then initiated using this modified
tRNA (formyl-methionine-tRNA). Without limiting the invention to
any particular mechanism, it is believed that eliminating fTHF from
the reaction mixture reduces the competition for initiation of
protein synthesis between this endogenous initiator tRNA and
exogenously added modified-initiator RNA by preventing the
formylation of endogenous initiator tRNA. This was confirmed by
measuring fluorescence directly from SDS-PAGE for reactions for
which fTHF was present and absent from the reaction mixture. In the
later case, a 2-3 fold increase in fluorescence was found (data not
shown).
EXAMPLE 11
[0279] Incorporation Into Other Proteins
[0280] In order to explore the general applicability of this
approach, transcription/translation reactions with
BODIPY-FL-methionyl-tRNA.sup.fme- t were carried out using various
plasmid DNAs coding for dihydrofolate reductase (DHFR), luciferase,
chloramphenicol acetyl-transferase (CAT) and bacteriorhodopsin
(BR). BR was included because it represents membrane proteins,
which are typically very hydrophobic. An optimized coupled
transcription/translation system was used along with free BODIPY-FL
and BODIPY-FL-methionyl-tRNA.sup.fmet using the Talon metal chelate
resin (ClonTech, Palo Alto, Calif.) in order to examine
incorporation into other proteins. The results are shown in FIGS.
21A (visualization using laser based Molecular Dynamics FluorImager
595) and 21B (visualization using a UV-transilluminator). Lane 1 is
a no DNA control. Lanes 2, 3, 4, 5 and 6 are hemolysin, DHFR,
Luciferase, CAT and bacteriohodopsin, respectively.
[0281] Fluorescent bands are observed using a fluorescence scanner
for each of the proteins at positions corresponding to their
relative molecular. In the case of luciferase, bands are observed
which correspond to the expected products of false initiation at
internal methionines (Promega Technical Bulletin TB219). Bands
corresponding to all of the proteins could also be observed
visually and recorded photographically using a UV transilluminator
(UVP TMW-20) combined with an emission filter that allows light
with .lambda.>450 nm (FIG. 21B). Excitation in this case is
likely occur in the UV absorbing band of BODIPY-FL which extends
from 300-400 nm.
[0282] The amount of BODIPY incorporation was then determined by
measuring the amount of incorporated BODIPY-FL and protein present
in the purified sample by comparison with solutions of different
concentrations of BODIPY-FL and using a Bradford protein assay,
respectively. The average of three such measurements yielded a
molar ratio of 0.29.+-.0.03%. However, the incorporation yield is
likely to be higher since fluorescence quenching of BODIPY-FL with
protein residues such as tryptophan and tyrosine may lower the
fluorescence quantum yield compared to BODIPY-FL in aqueous
solution.
[0283] The effects of the fluorescence labeling procedure on the
activity of the nascent proteins synthesized was also evaluated.
This is important in cases where it is desirable to perform
downstream functional analysis such as in the case of in vitro
expression cloning and other proteomic applications of in vitro
technology. Although it is possible for the N-terminal fluorescent
label to alter the function of a protein, the low molar
incorporation level (.about.0.3%) should not significantly alter
the overall activity of the extract. This is confirmed by various
enzyme assays and no significant difference is found for the
activity measured for DHFR, .alpha.-HL and luciferase synthesized
in the presence and absence of the
BODIPY-FL-methionyl-tRNA.sup.fmet (see Table 3).
EXAMPLE 12
[0284] Measuring the Sensitivity
[0285] In order to estimate the sensitivity of the method, various
dilutions of the translation extract corresponding to 0.003-0.5
.mu.l of the original reaction mixture were analyzed by SDS-PAGE.
As a control, extract from a reaction performed without DNA was
analyzed. As seen in FIG. 22B, fluorescence from .alpha.-HL bands
corresponding to as small as 0.007 .mu.l of the original reaction
mixture were detectable. Based on the our estimation of total
nascent protein produced in the in vitro system, which ranged from
50-80 .mu.g/ml, this corresponds to 0.35-0.5 nanograms of
.alpha.-hemolysin. This compares favorably with the sensitivity
obtainable using radioisotope labeling of nascent proteins where
such a low expression of nascent protein may required longer
exposure to X-ray film which might result in serious background
problem. It also exceeds the sensitivity of measuring proteins on
gels currently with commercially available dyes such as coomasie
blue (8-100 nanograms). Further improvements in sensitivity are
expected by increasing the level of BODIPY-FL incorporation and by
reducing background fluorescence, which appears to be due to
fluorescent impurities in the gel material, extract and modified
tRNA added.
EXAMPLE 13
[0286] Synthesis As A Function Of Time
[0287] The ability of the fluorescent labeling approach to monitor
the nascent protein synthesis as a function of time was also
evaluated. For this purpose, small aliquots of the .alpha.-HL
transcription/translation mixture (4 .mu.l) were withdrawn at
various times during the reaction and analyzed by SDS-PAGE. As seen
in FIG. 22A, bands due to .alpha.-HL can clearly be detected as
early as 5 minutes after initiation of the incubation. Synthesis of
fluorescently labeled a-HL appears to saturate after 15 minutes of
translation.
EXAMPLE 14
[0288] The Modifying Reagent
[0289] In the case of post-aminoacylation modifications used to
form a misaminoacylated tRNA, an important factor is the modifying
reagent used to add the modification to the natural amino acid. For
example, in the case of the fluorophore BODIPY FL, there are two
different commercially available BODIPY FL NHS reagents known as
BODIPY-FL-SE and BODIPY-FL-SSE (Molecular Probes). Both reagents
are based on N-hydroxysucinimide (NHS) as the leaving group.
However, the two forms differ in aqueous solubility due to the
presence in one form (SSE) of a sulonate (sulfo) group (see Table 2
for structures). In this example, optimized reactions based on
standard biochemical procedures were performed aimed at adding the
BODIPY FL fluorophore to a purified tRNA.sup.fmet which is
aminoacylated with methionine using these two different reagents.
For this purpose, first the tRNA.sup.fmet was aminoacylated with
the methionine. In typical reaction, 1500 picomoles (.about.1.0
OD.sub.260) of tRNA was incubated for 45 min at 37(C. in
aminoacylation mix using excess of aminoacyl tRNA-synthetases. The
aminoacylation mix consisted of 20 mM imidazole-HCl buffer, pH 7.5,
150 mM NaCl, 10 mM MgCl.sub.2, 2 mM ATP and 1600 units of aminoacyl
tRNA-synthetase. The extent of aminoacylation was determined by
acid-urea gel as well as using .sup.35S-methionine. After
incubation, the mixture was neutralized by adding 0.1 volume of 3 M
sodium acetate, pH 5.0 and subjected to chloroform:acid phenol (pH
5.0) extraction (1:1). Ethanol (2.5 volumes) was added to the
aqueous phase and the tRNA pellet obtained was dissolved in water
(37.5 (I) and used for modification.
[0290] A. Modification of Aminoacylated tRNA With BODIPY-FL-SSE
[0291] To the above aminoacylated-tRNA solution, 2.5 (1 of 1N
NaHCO.sub.3 was added (final conc. 50 nM, pH=8.5) followed by 10 (1
of 10 mM solution of BODIPY-FL-SSE (Molecular Probes) in water. The
mixture was incubated for 10 min at 0.degree. C. and the reaction
was quenched by the addition of lysine (final concentration=100
mM). To the resulting solution 0.1 volume of 3 M NaOAc, pH=5.0 was
added and the modified tRNA was precipitated with 3 volumes of
ethanol. Precipitate was dissolved in 50 ml microliters of water
and purified on Sephadex G-25 gel filtration column (0.5.times.5
cm) to remove any free fluorescent reagent, if present. The
modified tRNA was stored frozen (-70.degree. C.) in small aliquots
in order to avoid free-thaws.
[0292] B. Modification of Aminoacylated tRNA With BODIPY-FL-SE
[0293] To the above aminoacylated-tRNA solution, 2.5 (1 of 1N
NaHCO.sub.3 (final conc. 50 mM, pH=8.5) and 20 (1 of DMSO was added
followed by 10 (1 of 10 mM solution of BODIPY-FL-SE (Molecular
Probes) in DMSO. The mixture was incubated for 10 min at 0.degree.
C. and the reaction was quenched by the addition of lysine (final
concentration=100 mM). To the resulting solution 0.1 volume of 3 M
NaOAc, pH=5.0 was added and the modified tRNA was precipitated with
3 volumes of ethanol. Precipitate was dissolved in 50 ml of water
and purified on Sephadex G-25 gel filtration column (0.5.times.5
cm) to remove any free fluorescent reagent, if present. The
modified tRNA was stored frozen (-70.degree. C.) in small aliquots
in order to avoid free-thaws.
[0294] C. Analysis p It was found empirically using HPLC that the
extent of modification of the (alpha-amino group of methionine is
substantially greater using the sulfonated form of NHS BODIPY FL
compared to the non-sulfonated form of NHS-BODIPY FL reagent. In
addition the misaminoacylated tRNA.sup.fmet formed using the
sulfonated form was found to exhibit superior properties. When used
in an optimized S30 E. coli translation systems to incorporate
BIDOPY FL into the protein (hemolysin using a plasmid containing
the HL gene under control of a T7 promoter), the band on an
SDS-PAGE gel corresponding to the expressed HL exhibited an
approximately 2 times higher level of fluorescence when detected
using a argon laser based fluoroimager compared to a similar system
using the misaminoacylated formed using the non-sulfonated
form.
[0295] D. Coumarin
[0296] A similar result to that described above was obtained by
comparing the non-sulfonated and sulfonated NHS derivitives of
coumarin, which are also commercially available and referred to
respectively as succinimidyl 7-amino-methyl-amino-coumarin acetate
(AMCA-NHS; Molecular Probes) and sulfosuccinimidyl
7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS; Pierce
Chemicals). In this case, optimized reactions were performed using
these two different reagents based on standard biochemical
procedures in order to add the coumarin fluorophore to a purified
tRNA.sup.fmet which is aminoacylated with methionine.
[0297] To the aminoacylated-tRNA solution described above, 2.5 (1
of 1N NaHCO.sub.3 was added (final conc. 50 mM, pH=8.5) followed by
10 (1 of 10 mM solution of sulfosuccinimidyl
7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS; Pierce
Chemicals) in water. The mixture was incubated for 10 min at 0(C.
and the reaction was quenched by the addition of lysine (final
concentration=100 mM). To the resulting solution 0.1 volume of 3 M
NaOAc, pH=5.0 was added and the modified tRNA was precipitated with
3 volumes of ethanol. Precipitate was dissolved in 50 microliters
of water and purified on Sephadex G-25 gel filtration column
(0.5.times.5 cm) to remove any free fluorescent reagent, if
present. The modified tRNA was stored frozen (-70.degree. C.) in
small aliquots in order to avoid free-thaws.
[0298] To the above aminoacylated-tRNA solution, 2.5 (1 of 1N
NaHCO.sub.3 (final conc. 50 mM, pH=8.5) and 20 (1 of DMSO was added
followed by 10 (1 of 10 mM solution of succinimidyl
7-amino-methyl-amino-coumarin acetate (AMCA-NHS; Molecular Probes)
in DMSO. The mixture was incubated for 10 min at 0.degree. C. and
the reaction was quenched by the addition of lysine (final
concentration=100 mM). To the resulting solution 0.1 volume of 3 M
NaOAc, pH=5.0 was added and the modified tRNA was precipitated with
3 volumes of ethanol. Precipitate was dissolved in 50 microliter of
water and purified on Sephadex G-25 gel filtration column
(0.5.times.5 cm) to remove any free fluorescent reagent, if
present. The modified tRNA was stored frozen (-70.degree. C.) in
small aliquots in order to avoid free-thaws.
[0299] In this case, the coumarin-methionine-tRNA.sup.fmet formed
using the non-sulfonated form of coumarin-NHS (AMCA-NHS) when used
in standard E. coli S30 translation mixtures generated very low
levels of detectable fluorescent bands when detected using UV light
from a standard UV transilluminator. In contrast, the sulfonated
form (AMCA-sulfo-NHS) when added using the same procedures led to
easily detectable bands using the UV-transilluminator.
[0300] Attempts to incorporate coumarin using an initiator tRNA by
modifying the .alpha.-amino group of methionine have been reported
in the literature but failed. Coumarin attachment to a initiator
tRNA subsequently required more extensive and complicated chemical
attachment using a chemical cross linker. This was achieved by
first aminoacylating the tRNA with methionine followed by reaction
of aminoacylated tRNA with DTDG monosuccinimidyl ester (DTDG is
Dithiodiglycolic acid). The reaction product was then reduced using
DTT and subsequently reacted with CPM
(3-(4'-Maleimidophenyl)-4-methyl-diethylamino coumarin. (Odom, O.
W, Kudlicki, W and Hardestry, B. 1998. In vitro engineering using
acyl-derivatized tRNA, In Protein synthesis: Methods and Protocols,
PP.93-103, Humana press, Totowa, N.J.). Due to the need for special
procedures designed for each marker, such an approach is not
practical for general attachment of a wide variety of markers to
tRNAs through post-chemical aminoacylation procedures.
[0301] One likely factor that makes sulfonated NHS reagents used
for postchemical aminoacylation of tRNAs is its solubility in
aqueous buffer. In contrast, non-sulfonated reagents such as the
BIDOPY FL NHS reagent require organic buffer such as DMSO for
postchemical modification. While it is still not clear why use of
organic buffers lowers the overall marker incorporation, one
possibility is that hydrolysis of the aminoacyl bond formed between
the amino acid and tRNA reduces the overall level of
modification.
EXAMPLE 15
[0302] Imparting Water Solubility
[0303] In general, the property of water solubility can be imparted
to chemical reagents in several ways. Some of these are summarized
below:
[0304] Introduction of polar functional group into leaving group
(such as sulfonated-NHS).
[0305] Introduction of the polar functional group into a spacer
arm.
[0306] Introduction of the polar functional group into the reagent
moiety itself.
[0307] While the introduction of the --SO.sub.3.sup.-Na.sup.+
(sulfo-) group is peferred, other polar ionizable groups (such as
DSP) can also be used where DSP is shown below: 1
[0308] Final water soubility can be engineered into a spacer arm
for eample by using a polyether spacer (e.g. one based on
tetraethylene glycol). In general, any moiety that has a free
carboxyl group can be converted into its sulfo-NHS active ester.
This reaction involves N-hydroxy-slfosuccinimide (monosodium salt),
the marker and a coupling agent such as DCC
(dicyclohexylcarbodiimide): 2
[0309] In a typical reaction, marker 1, 55 mmol, is dissolved in 10
ml DMF (dimethylformamide) and (2) (5 mmol) is added, followed by
(3) (1.1 equivalents). The mixture is stirred overnight at room
temperature, precipitate filtered off, and the filtrate evaporated
under reduced pressure at room temperature. The product is purified
if necessary using column chromatography or is recrystallized.
[0310] One preferred embodiment of this invention involves the
post-chemical modification of tRNAs to form a misaminoacylated tRNA
by using markers that contain a sulfonated NHS reagent. While such
reagents are not generally available commercially, such reagents
can be routinely produced out of a variety of useful markers. For
example, fluoroescein, which has a high fluorescent quantum yield
for both UV and visible excitation could be prepared in a form
which contains a sulfonated NHS ester.
EXAMPLE 16
[0311] Triple Marker System
[0312] In this example, a three marker system is employed to detect
nascent proteins, i.e. an N-terminus marker, a C-terminus marker,
and an affinity marker (the latter being an endogenous affinity
marker). The experiment involves 1) preparation of a tRNA with a
marker, so that a marker can be introduced (during translation) at
the N-terminus of the protein; 2) translation of hemolysin with
nucleic acid coding for wild type and mutant hemolysin; and 4)
quantitation of the markers.
[0313] 1. Preparation of biotin-methionyl-tRNA.sup.fmet
[0314] The purified tRNA.sup.fmet (Sigma Chemicals, St. Louis, Mo.)
was first aminoacylated with methionine. The typical aminoacylation
reaction contained 1500 picomoles (-1.0 OD.sub.260) of tRNA, 20 mM
imidazole-HCl buffer, pH 7.5, 10 mM MgCl.sub.2, 1 mM methionine, 2
mM ATP, 150 mM NaCl and excess of aminoacyl tRNA-synth-etases
(Sigma). The reaction mixture was incubated for 45 min at
37.degree. C. After incubation, the reaction mixture was
neutralized by adding 0.1 volume of 3 M sodium acetate, pH 5.0 and
subjected to chloroform:acid phenol extraction (1:1). Ethanol (2.5
volumes) was added to the aqueous phase and the tRNA pellet
obtained was dissolved in the water (25 .mu.l). The coupling of
NHS-biotin to the .alpha.-amino group of methionine was carried out
in 50 mM sodium bicarbonate buffer, pH 8.0 by incubating the
aminoacylated tRNA.sup.fmet (25 .mu.l) with NHS-biotin (final
concentration=2 mM) for 10 min at 0.degree. C. and the reaction was
quenched by the addition of free lysine (final concentration=100
mM). The modified tRNA was precipitated with ethanol and passed
through Sephadex G-25 gel filtration column (0.5.times.5 cm) to
remove any free reagent, if present.
[0315] 2. In vitro Translation of .alpha.-HL DNA
[0316] A WT and Amber (at position 135) mutant plasmid DNA was
using coding for .alpha.-hemolysin (.alpha.-HL), a 32 kDa protein
bearing amino acid sequence His-His-His-His-His-His (His-6) at its
C-terminal. In vitro translation of WT and amber mutant .alpha.-HL
gene (Amb 135) was carried out using E. Coli T7 circular
transcription/translation system (Promega Corp., Wisconsin, Wis.)
in presence of Biotin-methionyl-tRNA.sup.fmet (AmberGen, Inc.).The
translation reaction of 100 .mu.l contained 30 .mu.l E. Coli
extract (Promega Corp., Wisconsin, Wis.), 40 .mu.l premix without
amino acids, 10 .mu.l amino acid mixture (1 mM), 5 .mu.g of plasmid
DNA coding for WT and mutant .alpha.-HL, 150 picomoles of
biotin-methionyl-tRNA.sup.fmet and RNase-free water. The premix
(1.times.) contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate,
210 mM potassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mM ATP,
0.8 mM GTP, 0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenol pyruvate, 0.6
mM cAMP and 6 mM magnesium acetate. From the translation reaction
premix, n-formyl-tetrahydrofolate (fTHF) was omitted. The
translation was carried out at 37.degree. C. for 1 hour. The
translation reaction mixture incubated without DNA is taken as
control. After the translation reaction mixture was diluted with
equal volume of TBS (Tris-buffered saline, pH 7.5). Each sample was
divided into two aliquots and processed individually as described
below.
[0317] 3. Preparation of Anti-.alpha.-HL Antibody Microtiter
Plate
[0318] Anti-rabbit-IgG coated microtiter plate (Pierce Chemicals,
Rockford, Ill.) was washed with Superblock buffer solution (Pierce)
and incubated with 100 .mu.g/ml of anti-.alpha.-HL polyclonal
antibody solution (Sigma Chemicals, St. Louis, Mo.) prepared in
Superblock buffer on microtiter plate shaker for I hour at room
temperature. The plate was then washed (3 times.times.200 .mu.l)
with Superblock buffer and stored at 4.degree. C. till further
use.
[0319] 4. Quantitation of N-terminal (Biotin) Marker
[0320] The translation reaction mixture (50 .mu.l) for the control,
WT and amber .alpha.-HL DNA were incubated in different wells of
anti-.alpha.-HL microtiter plate for 30 minutes on the shaker at
room temperature. After incubation, the wells were washed 5 times
(5-10 min each) with 200 .mu.l Superblock buffer and the
supernatant were discarded. To these wells, 100 .mu.l of 1:1000
diluted streptavidin-horse radish peroxidase (Streptavidin-HRP;
0.25 mg/ml; Promega) was added and the plate was incubated at room
temperature for 20 min under shaking conditions. After the
incubation, excess streptavidin-HRP was removed by extensive
washing with Superblock buffer (5 times .times.5 min each).
Finally, 200 .mu.l of substrate for HRP (OPD in HRP buffer; Pierce)
was added and the HRP activity was determined using
spectrophotometer by measuring absorbance at 441 nm.
[0321] 5. Quantitation of C-terminal (His-6-taq) Marker
[0322] Translation reaction mixture (50 .mu.l ) from example 2 for
control, WT and Amber .alpha.-HL DNA were incubated in different
wells of anti-.alpha.-HL microtiter plate for 30 min on the shaker
at room temperature. After incubation, the wells were washed 5
times (5-10 min each) with 200 .mu.l Superblock buffer and the
supernatant were discarded. To these wells, 100 .mu.l of 1:1000
diluted anti-His-6 antibody (ClonTech, Palo Alto, Calif.) was added
to the well and incubated at room temperature for 20 min under
shaking conditions. After the incubation, excess antibodies were
removed with extensive washing with Superblock buffer (5
times.times.5 min each). Subsequently, the wells were incubated
with secondary antibody (anti-mouse IgG-HRP, Roche-BM,
Indianapolis, Ind.) for 20 min at room temperature. After washing
excess 2.sup.nd antibodies, HRP activity was determined as
described above.
[0323] 6. Gel-Free Quantitation of N- and C-Terminal Markers
[0324] The results of the above-described quantitation are shown in
FIG. 23A (quantitation of N-terminal, Biotin marker) and FIG. 23B
(quantitation of C-terminal, His-6 marker). In case of in vitro
transcription/translation of WT .alpha.-HL DNA in presence of
biotin-methionyl-tRNA, the protein synthesized will have translated
His-6 tag at the C-terminal of the protein and some of the
.alpha.-HL molecules will also carry biotin at their N-terminus
which has been incorporated using biotinylated-methionine-tRNA.
When the total translation reaction mixture containing .alpha.-HL
was incubated on anti-.alpha.-HL antibody plate, selectively all
the .alpha.-HL will bind to the plate via interaction of the
antibody with the endogenous affinity marker. The unbound proteins
can be washed away and the N- and C-terminal of the bound protein
can be quantitated using Streptavidin-HRP and anti-His-6
antibodies, respectively. In case of WT .alpha.-HL, the protein
will carry both the N-terminal (biotin) and C-terminal (His-6) tags
and hence it will produce HRP signal in both the cases where
streptavidin-HRP and secondary antibody-HRP conjugates against
His-6 antibody used (HL, FIG. 23A). On the other hand, in case of
amber mutant .alpha.-HL, only N-terminal fragment of .alpha.-HL
(first 134 amino acids) will be produced and will have only
N-terminal marker, biotin, but will not have His-6 marker due to
amber mutation at codon number 135. As a result of this mutation,
the protein produced using amber .alpha.-HL DNA will bind to the
antibody plate but will only produce a signal in the case of
strepavidin-HRP (HL-AMB, FIG. 23A) and not for anti-His.times.6
antibodies (HL-AMB, FIG. 23B).
EXAMPLE 17
[0325] Electrophoretic Mobility Shift Assay
[0326] To demonstrate the changes in the electrophoretic mobility
of fluorescently labeled nascent protein on the SDS-gels either due
to proteolysis or oligomerization in presence of membranes, we have
use plasmid DNA of .alpha.-hemolysin (.alpha.-HL) which codes 32
kDa protein bearing a sequence His-His-His-His-His-His (His-6) at
its C-terminal. In vitro translation of .alpha.-HL gene was carried
out using E. coli T7 circular transcription/translation system
(Promega Corp., Wisconsin-Madison, Wis.) in presence of
BODIPY-FL-methionyl-tRNA.sup.fmet (AmberGen, Inc.) This experiment
involved 1) preparation of the tRNA-marker for introduction of the
N-terminus marker during translation, 2) translation, 3)
purification, 4) protease treatment or 5) oligomerization.
[0327] 1. Preparation of BODIPY-FL-methionyl-tRNA:
[0328] BODIPYL-FL-methionyl-tRNA was prepared by first
aminoacylating pure tRNA.sup.fmet (Sigma Chemicals, St. Louis, Mo.)
using methionine and subsequently modifying cc-amino group of
methionine using BODIPY-FL-SSE
(4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene propionic
acid, sulfosuccinimidyl ester; Molecular Probes, Eugene, Oreg.).
The typical aminoacylation reaction (100 .mu.l ) contained 1500
picomoles (-1.0 OD.sub.260) of tRNA, 20 mM imidazole-HCl buffer, pH
7.5, 10 mM MgCl.sub.2, 1 mM methionine, 2 mM ATP, 150 mM NaCl and
excess of aminoacyl tRNA-synthetases (Sigma). The reaction mixture
was incubated for 45 min at 37.degree. C. After incubation, the
reaction mixture was neutralized by adding 0.1 volume of 3 M sodium
acetate, pH 5.0 and subjected to chloroform:acid phenol extraction
(1:1). Ethanol (2.5 volumes) was added to the aqueous phase and the
tRNA pellet obtained was dissolved in the water (25 .mu.l). The
coupling of BODIPY-FL-SSE to the .alpha.-amino group of methionine
was carried out in 50 .mu.l reaction volume using 50 mM sodium
bicarbonate buffer, pH 8.0 by incubating 25 .mu.l aminoacylated
tRNA.sup.fmet (1.5 nanomoles) with 10 .mu.l of BODIPY-FL-SSE (10
mM) for 10 min at 0.degree. C. and the reaction was quenched by the
addition of free lysine (final concentration=100 mM). The modified
tRNA was precipitated with ethanol, and the pellet was dissolved in
RNase-free water and passed through Sephadex G-25 gel filtration
column (0.5.times.5 cm) to remove any free fluorescent reagent, if
present.
[0329] 2. In vitro Translation of .alpha.-hemolysin DNA
[0330] The translation reaction of 100 .mu.L contained 30 .mu.l E.
coli extract (Promega Corp., Wisconsin, Wis.), 40 .mu.l premix
without amino acids, 10 .mu.l amino acid mixture (1 mM), 5 .mu.g of
plasmid DNA coding for .alpha.-HL, 150 picomoles of
BODIPY-FL-methionyl-tRNA.sup.fmet and RNase free water. The premix
(1.times.) contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate,
210 mM potassium glutamate, 1.7 mM DTT, 4% PEG 8000. 1.25 mM ATP,
0.8 mM GTP, 0.8 mM UTP, 60 mM phosphoenol pyruvate, 0.6 mM cAMP and
6 mM magnesium acetate. From the translation reaction premix,
n-formyl-tetrahydrofolate (fTHF) was omitted. The translation was
carried out at 37.degree. C. for 1 hour. The translation reaction
mixture incubated without DNA is taken as control.
[0331] 3. Purification of Hls-6-.alpha.-HL
[0332] Fifty microliters of the translation reaction mixture (from
above) was subjected to Talon-Sepharose (ClonTech, Palo Alto,
Calif.) chromatography for the purification of Hls-6-.alpha.-HL.
This was carried out by loading the crude extract onto the
Talon-Sepharose column which was pre-equillbrated with 50 mM
Tris-HCl, pH 8.0 containing 150 mM NaCl and washing the column to
remove unbound proteins. The bound protein was then eluted by
adding 100 mM imidazole in the above buffer. The eluted .alpha.-HL
was dialyzed against 50 mM Tris-HCl buffer, pH 7.5.
[0333] 4. EMSA for Protease Detection
[0334] The purified fluorescently labeled .alpha.-HL (-5 .mu.g)
(example 3) was incubated with 0.0.5 .mu.g of pure trypsin (Sigma
Chemicals, St. Louis, Mo.) in 50 nM acetate buffer, pH 5.0 (100:1;
protein:protease ratio) for 5 min at 37.degree. C. The proteolysis
reaction was arrested by the addition of 1.times. SDS-gel loading
buffer and boiling the samples for 5 min. The SDS-PAGE was carried
out as described by Laemmli (Laemmli, U. K. 1970, Nature 227,
680-685) using 4-20% gradient gel (ready-gel, Bio-Rad, Richmond,
Calif.). After the gel electrophoresis, the gel was visualized
using FluorImager F595 (Molecular Dynamics, Sunnyvale, Calif.).
[0335] Trypsin was used under very limited conditions (single-hit
kinetics) to obtain very defined cleavage of .alpha.-HL (50 mM
acetate buffer, pH 5.0, 100:1: protein:protease ratio, 5 min at
37.degree. C.). Under these conditions, the glycine rich loop in
.alpha.-HL is most susceptible to cleavage and as a result
proteolytic fragment of 17 kDa was observed (Vecesey-Semjen, B.,
Knapp, S., Mollby, R., Goot, F. G. 1999, Biochemistry, 38
4296-4302). When the fluorescently labeled .alpha.-HL was subjected
to very mild trypsin treatment, it resulted a cleavage of
.alpha.-HL yielding the N-terminal fragment of approximately 17-18
kDa mass as evidence by change in the mobility of fluorescent band
on SDS-PAGE (FIG. 24: Lane 1 shows untreated protein and Lane 2
shows protease treated protein). This result indicates that such
assay could be used to screen for proteases or any other enzymatic
activities like kinase, transferase etc. that could potentially
result in the electrophoretic mobility shift of the nascent
protein. Though we have used pure nascent protein for this
particular assay, there is no reason why one can not use a nascent
protein without any purification (total translation reaction
mixture).
[0336] 5. EMSA for Oligomerization of Nascent Protein on
Membranes
[0337] The total translation reaction mixture (10 .mu.l ) (see
above) was incubated in absence and in presence of rabbit red blood
cells (rRBCs, Charles River Farm, Conn.) for 30 min. at 0.degree.
C. After the incubation, rRBCs were washed free of excess unbound
C.alpha.-HL and the rRBCs were incubated in Tris buffer saline
(TBS) containing 1 mg/ml BSA (TBSA) at 37.degree. C. for 20 min
during which lysis of rRBCs occurred. The rRBC membranes were
isolated after centrifugation, dissolved in 1.times. SDS-gel
loading buffer and subjected to SDS-PAGE (4-20% gradient gel)
without heating the sample. After the gel electrophoresis, the gel
was visualized using FluorImager F595.
[0338] .alpha.-HL is expressed a soluble monomeric protein and in
presence of various membranes it can oligomerize to form heptameric
pore (Walker, B., Krishnasastry, M., Zom, L., Kasianowicz, J. and
Bayley, H., 1992, J. Biol. Chem. 267 10902-10909). In addition,
some intermediate forms of the oligomers were also observed. In
this experiment, in order to see the applicability of EMSA to
detect the shift in mobility of .alpha.-HL due to oligomerization
in presence lipid membranes, the total translation reaction mixture
(with out any purification) was used. When the total translation
extract containing nascent .alpha.-HL was incubated with rRBCs, it
resulted in the oligomerization of .alpha.-HL on the rRBC membranes
yielding a distinct fluorescent bands corresponding to various
molecular masses that were SDS-resistant (FIG. 25: Lane 1 shows
untreated protein only and Lane 2 shows protein treated with
rRBCs).
[0339] This result demonstrates that such assay could be used to
study proteins, interact with variety of natural and artificial
membranes and as a result the mobility of the protein in
shifted.
EXAMPLE 18
[0340] Incorporation Using Lysyl-tRNA.sup.lys
[0341] This example describes the incorporation of fluorescent
labels into nascent protein using lysyl-tRNA.sup.lys. More
specifically, a variety of fluorescent molecules were incorporated
into 1) hemolysin during translation in an E. coli translation
system, and 2) luciferase during translation in a wheat germ
system, using lysyl tRNA.sup.lys. The experiment involved 1)
preparation of the tRNA-marker compounds, 2) translation, and 3)
detection on gels.
[0342] 1. Preparation of Fluorescent Labeled Misaminoacylated
tRNA.sup.lys
[0343] The purified tRNA.sup.lys (Sigma Chemicals, St. Louis, Mo.)
was first amino-acylated with lysine. The typical aminoacylation
reaction (100 .mu.l) contained 1500 picomoles (-1.0 OD.sub.260) of
tRNA, 20 mM imidazole-HCl buffer, pH 7.5, 10 mM MgCl.sub.2, 1 mM
lysine, 2 mM ATP, 150 mM NaCl and excess of aminoacyl
tRNA-synthetases (Sigma). The reaction mixture was incubated for 45
min at 37.degree. C. After incubation, the reaction mixture was
neutralized by adding 0.1 volume of 3 M sodium acetate, pH 5.0 and
subjected to chloroform:acid phenol extraction (1:1). Ethanol (2.5
volumes) was added to the aqueous phase and the tRNA pellet
obtained was dissolved in the water (25.mu.l). The coupling of
NHS-derivatives of various fluorescent molecules (see Table 2) to
the .alpha.-amino group of lysine was carried out in 50 mM CAPS
buffer, pH 10.5 by incubating the aminoacylated tRNA.sup.lys (25
.mu.l) with fluorescent reagent (final concentration=2 mM) for 10
min at 0.degree. C. and the reaction was quenched by the addition
of free lysine (final concentration=100 mM). The modified tRNA was
precipitated with ethanol, dissolved in 50 .mu.l of RNase-free
water and passed through Sephadex G-25 gel filtration column
(0.5.times.5 cm) to remove any free fluorescent reagent, if
present. The modified tRNA was stored frozen (-70.degree. C.) in
small aliquots in order to avoid free-thaws. The modification
extent of the aminoacylated-tRNA was assessed by acid-urea gel
electrophoresis (Varshney, U., Lee, C. P. & RajBhandary, U. L.,
1991 J. Biol. Chem. 266, 24712-24718).
[0344] 2. Cell Free Synthesis of Proteins in Prokaryotic (E. coli)
Translation Extracts
[0345] The typical translation reaction mixture (10 .mu.l)
contained 3 .mu.l of E. coli extract (Promega Corp.,
Wisconsin-Madison, Wis.), 4 .mu.l of premix, 1 .mu.l of amino acid
mix (1 mM), 30 picomoles of fluorescent-lysyl-tRNA and 0.5 .mu.g of
.alpha. hemolysin (.alpha.HL) plasmid DNA. The premix (1.times.)
contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate, 210 mM
potassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mM ATP, 0.8 mM
GTP, 0.8 mM CTP, 60 mM phosphoenal pyruvate, 0.6 mM cAMP and 6 mM
magnesium acetate. The translation reaction was allowed to proceed
for 45 min at 37.degree. C. For SDS-PAGE, 4-10 .mu.l aliquot of the
reaction mix was precipitated with 5-volume acetone and the
precipitated proteins were collected by centrifugation. The pellet
was dissolved in 1.times. loading buffer and subjected to SDS-PAGE
after boiling for 5 min. SDS-PAGE was carried out according to
Laemmmli (Lammli, U.K. 1970, Nature, 227, 680-685).
[0346] 3. Cell-Free Synthesis in Eukaryotic (TnT Wheat Germ)
Translation Extracts.
[0347] The typical translation reaction mixture (10 .mu.l)
contained 5 .mu.l of TnT wheat germ extract (Promega Corp.,
Wisconsin-Madison, Wis.), 0.4 .mu.l of TnT reaction buffer, 1 .mu.l
of amino acid mix (1 mM), 0.2 .mu.l of T7 RNA polymerase, 30
picomoles of fluorescent-lysyl-tRNA and 0.5 .mu.g of luciferase RNA
(Promega) and RNase-free water. The translation reaction ws allowed
to proceed for 45 min at 30.degree. C. and reaction mixture was
centrifuged for 5 min to remove insoluble material. The clarified
extract was then precipitated with 5-volume acetone and the
precipitated proteins were collected by centrifugation. The pellet
was dissolved in 1.times. loading buffer and subjectd to SDS-PAGE
after boiling for 5 min. SDS-PAGE was carried out according to
Laemmli (Lammli, U.K. 1970), Nature, 227, 680-685).
[0348] 4. Detection of Nascent Protein
[0349] The gel containing nascent proteins was scanned using
FluorImager F595 (Molecular Dynamics, Sunnyvale, Calif. using Argon
laser (488 nm) as excitation source, in addition, the nascent
proteins in polyacrylamide gels were also detected using an
UV-transilluminator and the photographs were carried out using
Polaroid camera fitted with Tiffen green filter (Polaroid,
Cambridge, Mass.). FIGS. 26A and 26B show the results of in vitro
translation of .alpha.-HL produced in presence of various
fluorescent tRNA.sup.lys. It is clear from the results one can
incorporate a variety of fluorescent molecules into nascent protein
using misaminoacylated tRNA (fluorophore-modified
lysyl-tRNA.sup.lys) including dyes like NBD, fluorescein
derivatives etc. (Lane 1: No DNA control; lane 2: BODIPY-FL-SSE
(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene propionic
acid, sulfosuccinimidyl ester); lane 3: BODIPY-FL-SE
(4,4-difluoro-5,7-dimethyl-4bora-3a, 4a-diaza-s-indacene proionic
acid, succinimidyl ester); lane 4 : NBD-X-SE (Succinimidyl
6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoate); lane 5;
BODIPY-TMR-SE
((6-994,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-- 3a,
4a-diaza-s-indacene-2-propionyl)amino)hexanoic acid, succinimidyl
ester); lane 6: BODIPY-R6G-SE
((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-- s-indacene-3propionic
acid, succinimidyl ester); lane 7; FAM-SE
(5-(6-)-carboxyfluorescein, succinimidyl ester); lane 8:SFX-SE
(6-fluorescein-5-(and 6-)carboxyamido)hexonoic acid, succinimidyl
ester); lane 9:
PyMPO-SE(1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphe-
nyl)oxazol-2-yl)pyridininium bromide (PyMPO)) and lane 10: TAMRA-SE
(6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid,
succinimidyl ester).
EXAMPLE 19
[0350] NHS-derivatives of Coumarin
[0351] Given the above noted results for the two BODIPY molecules
(i.e. BODIPY-FL-SSE and BODIPY-FL-SE), attempts were made to
derivatize other markers to make them suitable for incorporation.
The present example involves 1) the preparation of the labeled
tRNA, 2) translation, and 3) detection of the nascent protein
containing the label (or marker).
[0352] 1. Preparation of Fluorescent Labeled Misaminoacylated
tRNA.sup.fmet
[0353] The purified tRNA.sup.fmet (Sigma Chemicals, St. Louis, Mo.)
was first aminoacylated with methionine. The typical aminoacylation
reaction (100 .mu.l) contained 1500 picomoles (approximately 1.0
OD.sub.260) of tRNA, 20 mM imidazole-HCl buffer, pH 7.5, 10 mM
MgCl.sub.2, 1 mM lysine, 2 mM ATP, 150 mM NaCl and excess of
aminoacyl tRNA-synthetases (Sigma). The reaction mixture was
incubated for 45 min at 37.degree. C. After incubation, the
reaction mixture was neutralized by adding 0.1 volume of 3 M sodium
acetate, pH 5.0 and subjected to chloroform:acid phenol extraction
(1:1). Ethanol (2.5 volumes) was added to the aqueous phase and the
tRNA pellet obtained was dissolved in the water (25 .mu.l). The
coupling of NHS-derivatives of coumarin [namely sulfosuccinimidyl
7-amino-4-methylcoumarin-3-acetate [1] (AMCA-sulfo-NHS; Pierce
Chemicals), Alexa 350-N-hydroxy-succinimide ester (Molecular
Probes) and succinimidyl 7-amethyl-amino-coumarin acetate
(AMCA-NHS: Molecular Probes)] to the .alpha.-amino group of
methionine was carried out in 50 mM sodium bicarbonate buffer, pH
8.5 by incubating the aminoacylated tRNA.sup.fmet (25 .mu.l) with
fluorescent reagent (final concentration=2 mM) for 10 min at
0.degree. C. and the reaction was quenched by the addition of free
lysine (final concentration=100 mM). In case of AMCA-NHS, reagent
was dissolved in DMSO and the coupling reaction was carried out in
presence of 40% DMSO. The modified tRNA was precipitated with
ethanol, dissolved in 50 .mu.l of RNase-free water and passed
through Sephadex G-25 gel filtration column (0.5.times.5 cm) to
remove any free fluorescent reagent, if present. The modified tRNA
was stored frozen (-70.degree. C.) in small aliquots in order to
avoid free-thaws. The modification extent of the aminoacylated-tRNA
was assessed by acid-urea gel electrophoresis (Varshney, U., Lee,
C. P. & RajBhandary, U. L., 1991, J. Biol. Chem. 266,
24712-24718).
[0354] 2. Cell Free Systhesis of Proteins in Prokaryotic (E. coli)
Translation Extracts:
[0355] The typical translation reaction mixture (10 .mu.l)
contained 3 .mu.l of E. coli S-30 extract (Promega Corp.,
Wisconsin-Madison, Wis.), 4 .mu.l of premix, 1 .mu.l of amino acid
mix (1 mM), 30 picomoles of fluorescent-methionyl-tRNA and 0.5
.mu.g of .alpha.-hemolysin (.alpha.-HL) plasmid DNA. The premix
(1.times.) contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate,
210 mM potassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mM ATP,
0.8 mM GTP, 0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenol pyruvate, 0.6
mM cAMP and 6 mM magnesium acetate. The translation reaction was
allowed to proceed for 45 min at 37.degree. C. For SDS-PAGE, 4-10
.mu.l aliquot of the reaction mix was precipitated with 5-volume
acetone and the precipitated proteins were collected by
centrifugation. The pellet was dissolved in 1.times. loading buffer
and subjected to SDS-PAGE after boiling for 5 min. SDS-PAGE was
carried out according to Laemmli (Laemmli, U. K. 1970, Nature, 277,
680-685).
[0356] 3. Detection of Nascent Protein
[0357] The gel containing nascent proteins was visualized using an
UV-transilluminator equipped with long wavelength UV bulb (>300
nm) and the photographs were carried out using Polaroid camera
fitted with Tiffen green filter (Polaroid, Cambridge, Mass.). FIG.
27 indicates that the result in vitro translation of .alpha.-HL
produced in presence of various fluorescent tRNAs (Lane 1 shows the
results for the no DNA control; Lane 2 shows the results with
Met-tRNA.sup.fmet modified with AMCA-NHS; Lane 3 shows the results
with Met-tRNA.sup.fmet modified with AMCA-sulfo-NHS; and Lane 4
shows the results with Met-tRNA.sup.fmet modified with Alexa-NHS).
Clearly, one can incorporate the coumarin derivative molecules into
nascent protein using misaminoacylated tRNA which are modified with
the water soluble NHS-esters of fluorescent molecules (lane 3).
Moreover, a dye with negative charge (Alexa, lane 4) seems to not
incorporate as well as its neutral counterpart (AMCA.; lane 3).
[0358] From the above results and general teachings of the present
specification, one skilled in the art can select other markers and
render them (e.g. chemically render them) suitable for
incorporation in accordance with the methods of the present
invention.
EXAMPLE 20
[0359] Capillary Electrophoresis
[0360] The example describes the use of capillary electrophoresis
(CE) for detection of in vitro synthesized fluorescent proteins by
mobility shift. The example describes 1) the preparation of the
tRNA comprising a BODIPY marker, 2) in vitro translation, 3)
purification, 4) protease digestion and 5) detection by mobility
shift assay.
[0361] 1. Preparation of BODIPY-FL-methionyl-tRNA.sup.fmet
[0362] The tRNA.sup.fmet was aminoacylated with the methionine. In
typical reaction, 1500 picomoles (.about.1.0 OD.sub.260) of tRNA
was incubated for 45 min at 37.degree. C. in aminoacylation mix
using an excess of aminoacyl tRNA-synthetases. The aminoacylation
mix comprised 20 mM imidazole-HCl buffer, pH 7.5, 150 mM NaCl, 10
mM MgCl.sub.2, 2 mM ATP and 1600 units of aminoacyl
tRNA-synthetase. The extent of aminoacylation was determined by
acid-urea gel as well as by using .sup.35S-methionine. After
incubation, the mixture was neutralized by adding 0.1 volume of 3 M
sodium acetate, pH 5.0 and subjected to chloroform:acid phenol (pH
5.0) extraction (1:1). Ethanol (2.5 volumes) was added to the
aqueous phase and the tRNA pellet obtained was dissolved in water
(37.5 ul) and used for modification. To the above
aminoacylated-tRNA solution, 2.5 ul of 1N NaHCO.sub.3 was added
(final conc. 50 mM, pH=8.5) followed by 10 ul of 10 mM solution of
BODIPY-FL-SSE (Molecular Probes) in water. The mixture was
incubated for 10 min at 0.degree. C. and the reaction was quenched
by the addition of lysine (final concentration=100 mM). To the
resulting solution 0.1 volume of 3 M NaOAc, pH=5.0 was added and
the modified tRNA was precipitated with 3 volumes of ethanol.
Precipitate was dissolved in 50 ul of water and purified on
Sephadex G-25 gel filtration column (0.5.times.5 cm) to remove any
free fluorescent reagent, if present. The modified tRNA was stored
frozen (-70.degree. C.) in small aliquots in order to avoid
free-thaws.
[0363] 2. In vitro Translation of .alpha.-hemolysin DNA
[0364] The translation reaction of 500 ul contained 150 ul E. coli
extract (Promega Corp., Wisconsin, Wis.), 200 ul premix without
amino acids, 50 ul amino acid mixture (1 mM), 25 ug of plasmid DNA
coding for .alpha.-HL, 1000 picomoles of
BODIPY-FL-methionyl-tRNA.sup.fmet and RNase free water. The premix
(1.times.) contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate,
210 mM potassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mM ATP,
0.8 mM GTP, 0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenol pyruvate, 0.6
mM cAMP and 16 mM magnesium acetate. From the translation reaction
premix, n-formyl-tetrahydrofolate (fTHF) was omitted. The
translation was carried out at 37.degree. C. for 1 hour. The
translation reaction mixture incubated without DNA is taken as
control.
[0365] 3. Purification of His-6-.alpha.-HL
[0366] Five hundred microliters of the translation reaction mixture
(see step 2 above) was subjected to Talon-Sepharose (ClonTech, Polo
Alto, Calif.) chromatography for the purification of
His-6-.alpha.-HL. This was carried out by loading the,crude extract
onto the Talon-Sepharose column which was pre-equilibrated with 50
mM Tris-HCl, pH 8.0 containing 150 mM NaCl and washing the column
to remove unbound proteins. The bound protein was then eluted by
adding 100 mM imidazole in the above buffer. The eluted .alpha.-HL
was dialyzed against 50 mM Tris-HCl buffer, pH 7.5. The
fluorescence of purified and dialyzed .alpha.-HL was checked on
Molecular Dynamics FluorImager F595.
[0367] 4. Protease Digestion
[0368] The purified fluorescently labeled .alpha.-HL (.about.5 ug)
(see step 3 above) was incubated with 0.1 ug of pure trypsin (Sigma
Chemicals, St. Louis, Mo.) in 50 mM Tris-HCl buffer, pH 7.5 (50:1;
protein:protease ratio) for 10 min at room temperature. The
proteolysis reaction was arrested by the addition of 1.times.
CE-SDS-gel loading buffer and boiling the samples for 10 min.
[0369] 5. Mobility Shift Assay
[0370] The SDS-capillary gel electrophoresis (SDS-CGE) was
performed on a Bio-Rad BioFocus 3000CE system. The capillary was
fused-silica with a 75 urn ID, 24 cm total length and 19.5 cm to
the detector. Fifty microliters of fluorescently labeled protein
sample (.alpha.-HL) was mixed with 50 ul of SDS-CGE sample loading
buffer and incubated at 95.degree. C. for 10 min. The capillary was
rinsed with 0.1 M NaOH, 0.1 M HCl and SDS-run buffer for 60, 60 and
120 sec respectively, prior to each injection. Sample were injected
using electrophoretic injection (20 sec at 10 kV). Separation was
performed at 15 kV (constant voltage) for 25 min. Capillary and
sample was maintained at 20.degree. C. The detection of sample was
carried out using 488 nm Argon laser and 520 nm emission
filter.
[0371] The results of SDS-CGE are shown in the FIG. 28. As seen in
the Figure, fluorescently label .alpha.-HL migrates as a major
species eluting around 24 min under the electrophoresis conditions
(Top panel). In addition, the electrophoregram also show the
presence of minor impurities present in the sample, which are
eluting around 17 and 20.5 min. When the fluorescently
labeled-.alpha.-HL sample was treated with trypsin and analyzed
using SDS-CGE, it showed peaks eluting earlier (13, 14 and 15min)
and major peak at 21 min (Bottom panel). This result indicated that
the .alpha.-HL was proteolysed by the trypsin and various
proteolytic fragments have N-terminal fluorescently labeled are
seen in the electrophoregram.
EXAMPLE 21
[0372] Incorporation of Three Markers
[0373] This is an example wherein a protein is generated in vitro
under conditions where N- and C-terminal markers are incorporated
along with a marker incorporated using a misaminoacylated tRNA. The
Example involves 1) PCR with primers harboring N-terminal and
C-terminal detectable markers, 2) preparation of the tRNA, 3) in
vitro translation, 4) detection of nascent protein.
[0374] 1. PCR of .alpha.-Hemolysin DNA
[0375] Plasmid DNA for .alpha.-hemolysin, pT7-WT-H6-.alpha.HL, was
amplified by PCR using following primers. The forward primer (HL-5)
was:
5'-GAATTC-TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGGAACAAAAATTAATC-
TCGGAAGAGGATTTGGCAGATTCTGATATTAATATTAAAACC-3' and the reverse
primer (HL-3) was: 5'-AGCTTCATTAATGATGGTGATGG-TGGTGAC 3'. The
underlined sequence in forward primer is T7 promoter, the region in
bold corresponds to ribosome binding site (Shine-Dalgarno's
sequence), the bold and underlined sequences involve the C-myc
epitope and nucleotides shown in italics are the complimentary
region of .alpha.-hemolysin sequence. In the reverse primer, the
underlined sequence corresponds to that of His.times.6 epitope. The
PCR reaction mixture of 100 ul contained 100 ng template DNA, 0.5
uM each primer, 1 mM MgCl.sub.2, 50 ul of PCR master mix (Qiagen,
Calif.) and nuclease free water (Sigma Chemicals, St. Louis, Mo.)
water. The PCR was carried out using Hybaid Omni-E thermocyler
(Hybaid, Franklin, Mass.) fitted with hot-lid using following
conditions: 95.degree. C. for 2 min, followed by 35 cycles
consisted of 95.degree. C. for 1 min, 61.degree. C. for 1 min and
72.degree. C. for 2 min and the final extension at 72.degree. C.
for 7 min. The PCR product was then purified using Qiagen PCR
clean-up kit (Qiagen, Calif.). The purified PCR DNA was used in the
translation reaction.
[0376] 2. Preparation of BODIPY-FL-lysyl-tRNA.sup.lys
[0377] The purified tRNA.sup.lys (Sigma Chemicals, St. Louis, Mo.)
was first aminoacylated with lysine. The typical aminoacylation
reaction contained 1500 picomoles (.about.1.0 OD.sub.260) of tRNA,
20 mM imidazole-HCl buffer, pH 7.5, 10 mM MgCl.sub.2, 1 mM lysine,
2 mM ATP, 150 mM NaCl and excess of aminoacyl tRNA-synthetases
(Sigma Chemicals, St. Louis, Mo.). The reaction mixture was
incubated for 45 min at 37.degree. C. After incubation, the
reaction mixture was neutralized by adding 0.1 volume of 3 M sodium
acetate, pH 5.0 and subjected to chloroform:acid phenol extraction
(1:1). Ethanol (2.5 volumes) was added to the aqueous phase and the
tRNA pellet obtained was dissolved in water (35 ul). To this
solution 5 ul of 0.5 M CAPS buffer, pH 10.5 was added (50 mM final
conc.) followed by 10 ul of 10 mM solution of BODIPY-FL-SSE. The
mixture was incubated for 10 min at 0.degree. C. and the reaction
was quenched by the addition of lysine (final concentration=100
mM). To the resulting solution 0.1 volume of 3 M NaOAc, pH=5.0 was
added and the modified tRNA was precipitated with 3 volumes of
ethanol. Precipitate was dissolved in 50 ul of water and purified
on Sephadex G-25 gel filtration column (0.5.times.5 cm) to remove
any free fluorescent reagent, if present. The modified tRNA was
stored frozen (-70.degree. C.) in small aliquots in order to avoid
free-thaws. The modification extent of the aminoacylated-tRNA was
assessed by acid-urea gel electrophoresis. Varshney et al., J.
Biol. Chem. 266:24712-24718 (1991).
[0378] 3. Cell-Free Synthesis of Proteins in Eukaryotic (Wheat
Germ) Translation Extracts.
[0379] The typical translation reaction mixture (20 ul) contained
10 ul of TnT wheat germ extract (Promega Corp., Wisconsin-Madison,
Wis.), 0.8 ul of TnT reaction buffer, 2 ul of amino acid mix (1
mM), 0.4 ul of T7 RNA polymerase, 30 picomoles of
BODIPY-FL-lysyl-tRNA.sup.lys, 1-2 ug plasmid or PCR DNA (Example 1)
and RNase-free water. The translation reaction was allowed to
proceed for 60 min at 30.degree. C. and reaction mixture was
centrifuged for 5 min to remove insoluble material. The clarified
extract was then precipitated with 5-volumes of acetone and the
precipitated proteins were collected by centrifugation. The pellet
was dissolved in 1.times. loading buffer and subjected to SDS-PAGE
after boiling for 5 min. SDS-PAGE was carried out according to
Laemmli, Nature, 227:680-685.
[0380] 4. Detection of Nascent Protein
[0381] After the electrophoresis, gel was scanned using FluorImager
595 (Molecular Dymanics, Sunnyvale, Calif.) equipped with argon
laser as excitation source. For visualization of BODIPY-FL labeled
nascent protein, we have used 488 nm as the excitation source as it
is the closest to its excitation maximum and for emission, we have
used 530.+-.30 filter. The gel was scanned using PMT voltage 1000
volts and either 100 or 200 micron pixel size.
[0382] The results are shown in FIG. 29. It can be seen from the
Figure that one can in vitro produce the protein from the PCR DNA
containing desired marker(s) present. In the present case, the
protein (.alpha.-hemolysin) has a C-myc epitope at N-terminal and
His.times.6 epitope at C-terminal. In addition, BODIPY-FL, a
fluorescent reporter molecule is incorporated into the protein.
Lane 1: .alpha.-Hemolysin plasmid DNA control; lane 2: no DNA
control; lane 3: PCR .alpha.-hemolysin DNA and lane 4: hemolysin
amber 135 DNA. The top (T) and bottom (B) bands in all the lane are
from the non-specific binding of fluorescent tRNA to some proteins
in wheat germ extract and free fluorescent-tRNA present in the
translation reaction, respectively.
EXAMPLE 22
[0383] Primer Design
[0384] It is not intended that the presen invention be limited to
particular primers. A variety of primers are contemplated for use
in the present invention to ultimately incorporate markers in the
the nascent protein (as explained above). The Example involves 1)
PCR with primers harboring markers, 2) in vitro translation, and 3)
detection of nascent protein.
[0385] For PCR the following primers were used: forward primer:
5'GGATCCTAATACGACTCACTATAGGGAGACCACCATGGAACAAAAATTAATATCGGAAGAGGATTTGAATG-
TTTCTCCATACAGGTCACGGGGA-3' Reverse Primer:
5'-TTATTAATGATGGTGATGGTGGTG-TTC- TGTAGGAATGGTATCTCGTTTTTC-3' The
underlined sequence in the forward primer is T7 promoter, the bold
and underlined sequences involve the C-myc epitope and nucleotides
shown in italics are the complimentary region of .alpha.-hemolysin
sequence. In the reverse primer, the underlined sequence
corresponds to that of His.times.6 epitope. A PCR reaction mixture
of 100 ul can be used containing 100 ng template DNA, 0.5 uM each
primer, 1 mM MgCl.sub.2, 50 ul of PCR master mix (Qiagen, Calif.)
and nuclease free water (Sigma Chemicals, St. Louis, Mo.) water.
The PCR can be carried out using Hybaid Omni-E thermocyler (Hybaid,
Franklin, Mass.) fitted with hot-lid using following conditions:
95.degree. C. for 2 min, followed by 35 cycles consisted of
95.degree. C. for 1 min, 61.degree. C. for 1 min and 72.degree. C.
for 2 min and the final extension at 72.degree. C. for 7 min. The
PCR product can then be purified using Qiagen PCR clean-up kit
(Qiagen, Calif.). The purified PCR DNA can then be used in a
variety of translation reactions. Detection can be done as
described above.
[0386] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. It is intended that
the specification and examples be considered exemplary only, with
the scope of particular embodiments of the invention indicated by
the following claims.
2TABLE 2 Name and Molecular Fluorescence weight Formula Properties
BODIPY-FL, SSE M. WT. 491 3 Excitation = 502 nm Emmision = 510 nm
Extinction = 75,000 NBD M. WT. 391 4 Excitation = 466 nm Emmision =
535 nm Extinction = 22,000 Bodipy-TMR-X, SE M. WT. 608 5 Excitation
= 544 nm Emmision = 570 nm Extinction = 56,000 Bodipy-R6G M. WT.
437 6 Excitation = 528 nm Emmision = 547 nm Extinction = 70,000
Fluorescein (FAM) M. WT. 473 7 Excitation = 495 nm Emmision = 520
nm Extinction = 74,000 Fluorescein (SFX) M. WT. 587 8 Excitation =
494 nm Emmision = 520 nm Extinction = 73,000 PyMPO M. WT. 582 9
Excitation = 415 nm Emmision = 570 nm Extinction = 26,000 5/6-TAMRA
M. WT. 528 10 Excitation = 546 nm Emmision = 576 nm Extinction =
95,000
[0387]
3TABLE 3 -FluoroTag .TM. tRNA +FluoroTag .TM. tRNA Enzyme/Protein
Translation reaction Translation reaction .alpha.-Hemolysin 0.085
0.083 OD.sub.415nm/.mu.l Luciferase 79052 78842 RLU/.mu.l DHFR
0.050 0.064 .DELTA.OD.sub.339nm/.mu.l
[0388]
Sequence CWU 1
1
18 1 11 DNA Artificial Sequence Synthetic 1 gccagccatg g 11 2 9 DNA
Artificial Sequence Synthetic 2 uaaggaggu 9 3 22 DNA Artificial
Sequence Synthetic 3 uaaggaggun nnnnnnnnna ug 22 4 17 PRT
Artificial Sequence Synthetic 4 Trp Glu Ala Ala Ala Arg Glu Ala Cys
Cys Arg Glu Cys Cys Ala Arg 1 5 10 15 Ala 5 6 PRT Artificial
Sequence Synthetic 5 His His His His His His 1 5 6 10 PRT
Artificial Sequence Synthetic 6 Glu Gln Lys Leu Ile Ser Glu Glu Asp
Leu 1 5 10 7 8 PRT Artificial Sequence Synthetic 7 Asp Tyr Lys Asp
Asp Asp Asp Lys 1 5 8 8 PRT Artificial Sequence Synthetic 8 Trp Ser
His Pro Gln Phe Glu Lys 1 5 9 9 PRT Artificial Sequence Synthetic 9
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 10 8 PRT Artificial
Sequence Synthetic 10 Met Trp Ser Pro Gln Phe Glu Lys 1 5 11 111
DNA Artificial Sequence Synthetic 11 gaattctaat acgactcact
atagggttaa ctttaagaag gagatataca tatggaacaa 60 aaattaatct
cggaagagga tttggcagat tctgatatta atattaaaac c 111 12 30 DNA
Artificial Sequence Synthetic 12 agcttcatta atgatggtga tggtggtgac
30 13 94 DNA Artificial Sequence Synthetic 13 ggatcctaat acgactcact
atagggagac caccatggaa caaaaattaa tatcggaaga 60 ggatttgaat
gtttctccat acaggtcacg ggga 94 14 51 DNA Artificial Sequence
Synthetic 14 ttattaatga tggtgatggt ggtgttctgt aggaatggta tctcgttttt
c 51 15 5 PRT Artificial Sequence Synthetic 15 Ala Val Tyr Lys Trp
1 5 16 33 DNA Artificial Sequence Synthetic 16 auguacacua
aacaugauga uauccgaaaa uga 33 17 10 PRT Artificial Sequence
Synthetic 17 Met Tyr Thr Lys Asp His Asp Ile Arg Lys 1 5 10 18 10
PRT Artificial Sequence Synthetic 18 Lys Arg Ile Asp Asp His Lys
Thr Tyr Met 1 5 10
* * * * *
References