U.S. patent application number 10/714212 was filed with the patent office on 2004-04-22 for methods for production of proteins in host cells.
Invention is credited to Donnelly, Mark, Joachimiak, Andrzej.
Application Number | 20040077038 10/714212 |
Document ID | / |
Family ID | 23869226 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077038 |
Kind Code |
A1 |
Donnelly, Mark ; et
al. |
April 22, 2004 |
Methods for production of proteins in host cells
Abstract
The present invention provides methods for the production of
proteins, particularly toxic proteins, in host cells. The invention
provides methods which use a fusion protein comprising a chaperonin
binding domain in host cells induced or regulated to have increased
levels of chaperonin which binds the chaperonin binding domain.
Inventors: |
Donnelly, Mark;
(Warrensville, IL) ; Joachimiak, Andrzej;
(Bolingbrook, PL) |
Correspondence
Address: |
Genencor International, Inc.
925 Page Mill Road
Palo Alto
CA
94034-1013
US
|
Family ID: |
23869226 |
Appl. No.: |
10/714212 |
Filed: |
November 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10714212 |
Nov 13, 2003 |
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09470830 |
Dec 23, 1999 |
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6677139 |
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Current U.S.
Class: |
435/68.1 ;
435/212; 435/252.33; 435/320.1; 435/69.1 |
Current CPC
Class: |
C12N 15/70 20130101 |
Class at
Publication: |
435/068.1 ;
435/069.1; 435/252.33; 435/320.1; 435/212 |
International
Class: |
C12P 021/06; C12N
009/48; C12N 001/21; C12N 015/74 |
Claims
We claim:
1. A method for producing a protein in a host cell, comprising the
step of culturing a host cell comprising a first nucleic acid
encoding an isolated chaperonin binding domain associated with a
second nucleic acid encoding the protein and a third nucleic acid
encoding a chaperonin, under conditions suitable for expression of
said first, said second and said third nucleic acid and wherein
said chaperonin binding domain is capable of binding to said
chaperonin.
2. The method of claim 1 further comprising recovering said protein
from said cell.
3. The method of claim 1 wherein said nucleic acid encoding the
chaperonin is naturally produced by the host cell.
4. The method of claim 3 wherein said cell is grown under
conditions that result in elevation of the levels of the naturally
produced chaperonin.
5. The method of claim 1 wherein said nucleic acid encoding the
chaperonin is heterologous to the host cell.
6. The method of claim 1 wherein said host cell is a bacterial
cell.
7. The method of claim 6 wherein said bacterial cell is a member of
the family Enterobacteriaceae
8. The method of claim 7 wherein said bacterial cell is E.
coli.
9. The method of claim 1 wherein the chaperonin binding domain has
a sequence as shown in SEQ ID NO: 1 through SEQ ID NO: 38.
10. The method of claim 1 wherein said chaperonin binding domain is
obtainable from GroES and said chaperonin is the GroEL
chaperonin.
11. The method of claim 10 wherein the chaperonin binding domain
comprises the amino acid sequence EVETKSAGGIVLTGSAAA or is a
variation thereof capable of binding to GroEL chaperonin with an
affinity of between about 10.sup.-2 and 10.sup.-8 Kd.
12. The method of claim 1 wherein said first and said second
nucleic acid encode a fusion protein.
13. The method of claim 12 wherein said first and said second
nucleic acid encode a fusion protein and are separated by an
enzymatic cleavage site.
14. The method of claim 12 wherein said first and said second
nucleic acid encode a fusion protein and are separated by a
chemical cleavage site.
15. The method of claim 1 wherein said protein is toxic to the host
cell.
16. The method of claim 5 wherein said chaperonin heterologous to
the host cell is under the control of an expression signal capable
of overexpression said chaperonin.
17. An expression vector comprising a first nucleic acid encoding a
chaperonin binding domain and a second nucleic acid encoding a
protein.
18. The expression vector of claim 17 wherein the chaperonin
binding domain has a sequence as shown in SEQ ID NO: 1 through SEQ
ID NO: 38
19. The expression vector of claim 18 wherein the chaperonin
binding domain is obtainable from GroES.
20. The expression vector of claim 18 wherein the chaperonin
binding domain comprises the amino acid sequence EVETKSAGGIVLTGSAAA
or a variation thereof capable of binding to GroEL chaperonin with
an affinity of between about 10.sup.-2 and 10.sup.-8 Kd.
21. A host cell containing the expression vector of claim 17.
22. The host cell of claim 21 wherein the host cell is a member of
the family Enterobacteriaceae.
23. The host cell of claim 22 wherein the host cell is E. coli.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to methods for the
production of proteins in host cells. Specifically, the present
invention relates to the use of a chaperonin binding domain in
expression systems designed for the production of proteins in host
cells.
BACKGROUND OF THE INVENTION
[0002] One of the simplest and most inexpensive ways to obtain
large quantities of desired polypeptides for commercial or research
uses is through the expression of heterologous genes in bacterial
cells. Often however, net accumulation of the recombinant
polypeptide is low due to degradation, missfoldings or aggregation.
Also, many recombinant polypeptides fail to attain their correct
three-dimensional conformation in E. coli and are found sequestered
within large retractile aggregates, i.e., inclusion bodies.
Processes for recovering active polypeptides from inclusion bodies
can be complex and expensive. Additionally, in some cases, the
large scale production of a protein is limited by the toxicity of
the overexpressed protein toward the host cell or the accumulation
of proteins as inclusion bodies that impede their recovery and
purification.
[0003] In the cell, a class of accessory proteins known as
molecular chaperones function by interacting with nascent
polypeptide chains and aid in the process of correct folding
Georgiou et al. (1996, Current Opinion in Biotechnology,
7:190-197). Molecular chaperones are highly conserved proteins
found in all organisms that control and sometimes catalyse the
ATP-dependent folding of newly synthesized proteins and
polypeptides as they are produced in cells. Chaperones mediate the
stabilization and refolding of proteins under conditions of stress
and are believed to fold crucial portions of proteins, such as
enzymes, independently (Hendrick, J. P., 1993, Ann. Rev. Biochem.
62: 349-384).
[0004] Several E. coli proteins have been shown to exhibit
chaperone activity: the 60 kDa heat shock protein (Hsp60) GroEL, a
chaperonin and the smaller accessory protein GroES (10 kDa); the
DnaK (Hsp70), DnaJ and GrpE complex; and the Clp system. Georgiou
et al. supra. GroEL consists of 14 subunits which are arranged in
two heptameric rings stacked back to back. The central cavity of
the cylinder accepts unfolded substrate polypeptides in the
conformation of a collapsed intermediate. GroEL interacts with
GroES, a single heptameric ring that binds asymetrically to GroEL,
capping one opening of the cylinder. GroES coordinates the ATP
hydrolysis by GroEL with productive folding (Mayhew, M et al.,
Nature vol. 379:420-426.)
[0005] Dale, G. et al. (1994, Protein Engineering 7:925-931) report
that simultaneous overproduction of the GroEL/GroES chaperonins
with dihydrofolate reductase results in an increased solubility of
the enzyme and Amrein, K. et al. (1995, Proc. Natl. Acad. Sci. vol
92: 1048-1052) report on the purification of recombinant human
protein-tyrosine kinase in an E. coli expression system
overproducing the bacterial chaperonins GroES and GroEL.
[0006] Landry, S. et al. (1993, Nature 364:255-258) disclose a
polypeptide loop of the GroES/GroEL complex and Altamirano et al.
(1997, Proc. Natl. Acad. Sci. USA, 94:3576-3578) disclose the use
of immobilized fragments of the GroEL chaperonin in
chromatography.
[0007] In spite of advances in understanding chaperonins and the
production of proteins in host cells, there remains a need to
develop expression vectors and systems which allow for production
of proteins in host cells.
SUMMARY OF THE INVENTION
[0008] The present invention generally relates to chaperonin
protein binding domains and to the use of an isolated chaperonin
protein binding domain in the production of heterologous proteins,
peptides or polypeptides in a host cell. The present invention is
based, in part, upon the finding that a toxic gene product could be
recombinantly produced by a host cell when expressed as a fusion
protein associated with an isolated chaperonin binding domain.
[0009] Accordingly, the present invention provides a method for
producing a protein in a host cell, comprising the step of
culturing a host cell comprising a first nucleic acid encoding an
isolated chaperonin binding domain associated with a second nucleic
acid encoding the protein and a third nucleic acid encoding a
chaperonin under conditions suitable for expression of said first,
said second and said third nucleic acid and wherein said chaperonin
binding domain is capable of binding to said chaperonin. In a
further embodiment, the chaperonin binding domain and the
chaperonin are capable of binding with an affinity of between about
10.sup.-2 and 10.sup.-8 Kd. The method may further comprise
recovering said protein from said cell. In one aspect, the protein
is one toxic to the host cell. A protein may be toxic to a host
cell due to its intrinsic nature or toxic due to the presence of
elevated levels in the host cell.
[0010] In another embodiment of the present invention, the first
and second nucleic acid encode a fusion protein. The first and
second nucleic acid may be directly linked or indirectly linked by
nucleic acid encoding an enzymatic cleavage site, a chemical
cleavage site, or another protein or peptide.
[0011] In one aspect of the invention, nucleic acid encoding the
chaperonin is naturally produced by the host cell and the cell is
grown under conditions that result in elevated levels of the
chaperonin. In another aspect, nucleic acid encoding the chaperonin
is heterologous to the host cell and the heterologous chaperonin is
under the control of at least one expression signal capable of
overexpressing the chaperonin in the host cell. The present
invention encompasses any host cell that is capable of expression
of recombinant proteins. In one embodiment, the host cell is a
bacterium. In another embodiment, the host cell is a eubacterium.
In yet further embodiments, the host cell is a gram-positive or a
gram-negative bacterium. In a further embodiment, the bacterial
cell is a member of the family Entero.bacteriaceae. In an
additional embodiment, the bacterial cell is an Escherichia
species, in particular E. coli.
[0012] There are several well characterized chaperonin systems
known in the art having two or more interacting partners, for
example, Hsp60 and Hsp10 (GroEL/GroES); Hsp70 and Hsp40 and GrpE
(DnaK/DNAJ/GrpE); ClipA/X and ClipP; Hsp90 and Hsp70 and other
factors; TriC and other factors. The present invention encompasses
chaperonin binding domains obtainable from these systems as long as
the chaperonin binding domain is capable of binding to a chaperonin
with an affinity of between about 10.sup.-2 and 10.sup.-8 Kd. In
one embodiment, the chaperonin binding domain has the sequence as
shown in SEQ ID NO: 1 through SEQ ID NO: 38. In yet another
embodiment, the chaperonin binding domain is obtainable from the
GroES co-chaperonin and said chaperonin is the GroEL chaperonin. In
another embodiment, the binding domain comprises the amino acid
sequence EVETKSAGGIVLTGSAAA. In a further embodiment, the binding
domain comprises a variation of the sequence EVETKSAGGIVLTGSAAA,
said variant being capable of binding to GroEL chaperonin with an
affinity of 10.sup.-2 to 10.sup.-8 Kd. The present invention also
provides expression vectors and host cells comprising a chaperonin
protein binding domain.
[0013] Examples of heterologous proteins include therapeutically
significant proteins, such as growth factors, cytokines, ligands,
receptors and inhibitors, as well as vaccines and antibodies;
enzymes such as hydrolases including proteases, carbohydrases, and
lipases; isomerases such as racemases, epimerases, tautomerases, or
mutases; transferases, kinases and phophatases; and commercially
important industrial proteins or polypeptides, such as proteases,
carbohydrases such as amylases and glucoamylases, cellulases,
oxidases and lipases. The nucleic acid encoding the heterologous
protein may be naturally occurring, a variation of a naturally
occurring protein or synthetic.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the nucleic acid (SEQ ID NO:1) and amino acid
(SEQ ID NO:2) for the region of the chaperonin containing the
GroEL/GroES binding domain.
[0015] FIG. 2 shows the growth of JM105 in the presence of
increasing amount of ethanol: diamonds, no ethanol; squares, 1%;
triangles, 2%; X, 3%; *, 4% ethanol in LB media.
[0016] FIG. 3 shows the growth rate of BAX transformants with
different leader sequences in the presence and absence of 2%
ethanol.
[0017] FIG. 4 shows and analysis of proteins present upon induction
of the host cells with IPTG and growth in the presence of ethanol.
Lanes 1 & 5--MW markers; lanes 2-4--pATP011 (chaperonin binding
domain); 6-8--pWS213.(OmpA leader). Bar between lanes 3&4 and
7&8 indicates position of BAX protein on the gel.
[0018] FIG. 5 shows the design of a linker for attaching the
GroEL-binding loop of GroES to proteins. Oligonucleotides matching
the two sequences shown above were synthesized chemically, annealed
to generate the duplex DNA fragment, and cloned into appropriate
vectors. Linkage to a gene via the EcoRI overhang generates protein
20 amino acids (1905 Daltons) longer.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0019] Definitions
[0020] The in vivo cellular processes of protein folding and
assembly are controlled by molecular mechanisms associated with
molecular chaperones such as chaperonins. As used herein, the term
"chaperonin" refers to those molecules including heat shock
proteins Hsp60 and like proteins that are expressed in any organism
which are associated with protein folding. The present invention
encompasses any chaperonin from any microbial source, virus or
bacteriophage including the chaperonin systems, Hsp60 and Hsp10
(GroEL/GroES); Hsp70 and Hsp40 and GrpE (DnaK/DNAJ/GrpE); ClipA/X
and ClipP; Hsp90 and Hsp70; and TriC. In a preferred embodiment,
the chaperonin binding domain and the chaperonin are obtainable
from the heat shock protein 60 (HSP60) class of proteins. Other
chaperonins include mammalian or yeast HSP68, HSP70, HSP72, HSP73,
clathrin uncoating ATPase, IpG heavy chain binding protein (BiP),
glucose-regulated proteins 75, 78, and 80 (GRP75, GRP78, and
GRP80), HSC70, and yeast KAR2, BiP, SSA1-4, SSB1, SSD1 and the
like. Chaperone proteins which can increase protein secretion also
include enzymes which catalyze covalent modification of proteins,
such as mammalian or yeast protein disulfide isomerase (PDI),
prolyl-4-hydroxylase B-subunit, ER p59, glycosylation site binding
protein (GSBP) and thyroid hormone binding protein (T3BP).
[0021] Chaperonins are known to be associated with protein folding
and anti-aggregation activities (Craig, et al., 1994, Cell vol. 78,
365-372; Hendrick, et al., 1993, Annu. Rev. Biochem. Vol. 62,
349-84; HartI, 1994, TIBS vol.19:20-25). Often, multiple sub-units
are associated with one chaperonin complex. The present invention
encompasses each chaperonin sub-unit used individually or in
combination with other subunits providing that the individual
subunit or combination of subunits is able to function by binding
to a chaperonin protein binding domain. In the present invention,
preferred chaperonins are those present in members of the family
Enterobacteriaceae and in particular from Eschericia species. In
the present invention, a preferred chaperonin is the GroEL
chaperonin which is associated with the co-chaperonin GroES. A
chaperonin of the present invention may be naturally occurring in
the host cell or heterologous to the host cell and may be
introduced by recombinant means.
[0022] As used herein, the phrase "isolated binding domain" of a
chaperonin or "chaperonin protein binding domain" or "chaperonin
binding domain" refers to a region of a protein or polypeptide that
is able to bind with an affinity of between 10.sup.-2 and 10.sup.-8
Kd to a chaperonin or portion or fragment thereof of said
chaperonin. In one embodiment of the present invention, the
chaperonin binding domain has the sequence as shown in any of SEQ
ID NO: 1 through SEQ ID NO:38. In another embodiment of the present
invention, the chaperonin binding domain is obtainable from the
GroES co-chaperonin. As used herein, the chaperonin protein binding
domain obtainable from GroES refers to the residues shown in FIG. 1
comprising the sequence EVETKSAGGIVLTGSAAA. In another embodiment,
the binding domain comprises amino acid variations of
EVETKSAGGIVLTGSAAA capable of binding to a GroEL chaperonin with an
affinity of between 10.sup.-2 and 10.sup.-8 Kd. A chaperonin
protein binding domain is associated with a second nucleic acid
encoding a heterologous protein when the first and second nucleic
acids are directed linked, such as in a fusion protein, or are
indirectly linked such as having an enzymatic cleavage site,
chemical cleavage site or other nucleic acid inserted between the
first and the second nucleic acid.
[0023] As used herein, "nucleic acid" refers to a nucleotide or
polynucleotide sequence, and fragments or portions thereof, and to
DNA or RNA of genomic or synthetic origin which may be
double-stranded or single-stranded, whether representing the sense
or antisense strand. As used herein "amino acid" refers to peptide
or protein sequences or portions thereof.
[0024] The terms "isolated" or "purified" as used herein refer to a
nucleic acid or amino acid that is removed from at least one
component with which it is naturally associated.
[0025] As used herein, the term "heterologous protein" refers to a
protein or polypeptide that is encoded by nucleic acid introduced
into a host cell. Examples of heterologous proteins include enzymes
such as hydrolases including proteases, carbohydrases, and lipases;
isomerases such as racemases, epimerases, tautomerases, or mutases;
transferases, kinases and phophatases. The heterologous gene may
encode therapeutically significant proteins or peptides, such as
growth factors, cytokines, ligands, receptors and inhibitors, as
well as vaccines and antibodies. The gene may encode commercially
important industrial proteins or peptides, such as proteases,
carbohydrases such as amylases and glucoamylases, cellulases,
oxidases and lipases. The gene of interest may be a naturally
occurring gene, a mutated gene or a synthetic gene. The term
"homologous protein" refers to a protein or polypeptide that
naturally occurs in the host cell. The present invention
encompasses homologous proteins that are introduced into the host
cell via recombinant means. The term "toxic" as used herein refers
to any protein that inhibits the growth of a bacterial cell. A
protein may be toxic to a host cell due to an intrinsic harmful
nature or due to expression levels in the host bacterial cell. An
illustrative example of a toxic protein disclosed herein is the
mouse apoptosis modulator protein, Bax. Examples of proteins
considered to be toxic due to their intrinsic nature include
nucleoses, proteoses and phospholiposes.
[0026] As used herein, the term "overexpressing" when referring to
the production of a protein in a host cell means that the protein
is produced in greater amounts than it is produced in its naturally
occurring environment.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] The present invention provides a means for the production of
proteins or polypeptides in a host cell, especially proteins or
polypeptides that are toxic to the cell due to the protein's
intrinsic nature or due to expression levels of the protein
produced recombinantly in the cell.
[0028] The present invention provides methods for producing a
protein in a host cell wherein the cell comprises nucleic acid
encoding a fusion protein comprising a chaperonin binding domain
and the protein and wherein the cell naturally produces a
chaperonin that binds to the chaperonin binding domain. In this
embodiment, the host cell is grown under conditions suitable for
inducing or enhancing the levels of the naturally occurring
chaperonin. The present invention encompasses methods for producing
a protein in a host cell wherein the cell comprises nucleic acid
encoding a fusion protein comprising a chaperonin binding domain
and protein and said host cell further comprises nucleic acid
encoding a chaperonin that has been recombinantly introduced into
said host cell. In this embodiment, the chaperonin may be
homologous or heterologous to said host cell and is associated with
expression signals capable of overexpressing the chaperonin.
[0029] In an illustrative example disclosed herein, the mammalian
gene bax, a member of the bcl-2 family of apoptosis modulators
(Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Bcl-2
Heterodimerizes in vivo with a conserved Homolog, Bax, that
accelerates Programmed cell death, Cell 74, 609-619) was used.
Although homologous to bcl-2 and bcl-XL, which inhibit apoptosis,
bax has the opposite function and is an effector of cell death
(McDonnell, T. J., et al., 1996, Importance of the Bcl-2 family in
cell death regulation, Experientia 52, 1008-1017). These three
genes were expressed in E. coli as fusions with the OmpA leader
sequence. Bcl-2 and Bcl-X.sub.L proteins were produced in the
periplasm of E. coli, but only trace amounts of Bax was produced by
this approach. Expression of Bax appeared to be highly toxic to the
host cell. No expression of the native form of Bax was observed
from any clones when we placed the bax cDNA sequence adjacent to
the lac promoter.
[0030] When the bax gene was linked to a chaperonin binding domain
obtainable from GroES and produced as a fusion protein in E. coli
simultaneous with overproduction of the GroEL chaperonin,
overexpression of the Bax protein was observed, suggesting that the
toxic effect of Bax on the host cell had been reduced.
[0031] I. Chaperonin Nucleic Acid and Amino Acid Sequences
[0032] The present invention encompasses chaperonin proteins that
are associated with increased protein secretion and those that are
associated with the folding and unfolding of polypeptides,
including but not limited to, the heat shock 60 family of proteins
(Hsp60).
[0033] In one embodiment, the chaperonin is obtained from an
organism listed in Table I and has the respective chaperonin
binding domain as given in Table I. In a preferred embodiment
herein, the chaperonin is GroEL the nucleic acid and amino acid
sequence of which is disclosed in Hemmingsen, et al., 1988, Nature
vol. 333, pages 330-334. A method for isolation of GroEL is
described, for example, in the reference Hendrix, R. W. 1979, J.
Mol. Biol. 129:375-392.
[0034] The chaperonin may be naturally occurring in the host cell
in which case the host cell comprising the chaperonin is subjected
to conditions that result in an increase in the production of the
chaperonin. This provides elevated levels of chaperonin to which
the chaperonin binding domain attaches. Methods for inducing the
natural levels of chaperonin in a host cell include heat shock
(Welch W. J., 1993, Philos Trans R. soc Lond B Biol Sci vol. 339,
pages 327-333); chemical shock, such as by the addition of ethanol,
methanol, glucose, and drugs such as those described in Volker et
al., 1994, Microbiology, vol 140: pages 741-752; Barbosa et al.,
Gene, 1994, vol. 148, pages 51-57; and Hartke, 1997, Curr
Microbiol, vol 34, pages 23-26.
[0035] The chaperonin may be heterologous to the cell and
introduced into the cell via recombinant means. The heterologous
chaperonin, or portions or fragments thereof capable of binding to
the chaperonin binding domain, may be introduced via a replicating
plasmid or integrated into the host genome by means known to those
of skill in the art. The present invention also encompasses host
cells having additional copies of homologous chaperonins, or
portions or fragments thereof capable of binding to a chaperonin
binding domain, introduced into the cell.
[0036] In one illustrative example disclosed herein, the host cell
used was E. coli which naturally produced the GroEL chaperonin as
well as the GroES co-chaperonin, and further comprised nucleic acid
encoding a fusion of the chaperonin binding domain having the amino
acid sequence EVETKSAGGIVLTGSAAA with the mouse apoptosis modulator
protein, Bax. The recombinant E. coli was subjected to growth
conditions that stimulated overproduction of the naturally
occurring GroEL chaperonin and expression of the fusion protein was
observed.
[0037] II. Chaperonin Binding Domain
[0038] The present invention encompasses chaperonin binding domains
that are capable of binding to a chaperonin with an affinity of
between 10.sup.-2 and 10.sup.-8 Kd. Examples of chaperonin binding
domains are provided in Table I. Table I provides the sequence of
chaperonin binding domains and a list of the respective
microorganism from which the binding domain is obtained (Hunt et
al., 1996, Nature vol. 379, pages 37-45).
1 TABLE I Organism Chaperonin binding domain ch10_ecoli
EVETKSAGGIVLTGSAAAK ch10_acype EVESKSAGGIVLTGSAAGK ch10_haedu
EVETCSAGGIVLTGSATVK ch10_pseae EEETKTAGGIVLPGSAAEK ch10_chrvi
EEERLSAGGIVIPDSATEK cg10_coxbu EEERTSAGGIVIPDSAAEK ch10_legmi
EEERTTAGGIVIPDSATEK ch13_rhime ESEEKTKGGIIIPDTAKEK ch10_legpn
EEERTTAGGIVIPDSATEK ch10_bruab ESEAKTAGGIIIPDTAKEK ch12_braja
DAEEKTAGGIIIPDTVKEK ch10_agrtu ESEAKTKGGIIIPDTAKEK ch10_cloab
EAEETTKSGIVLPSSAKEK ch10_amops EEERTTAGWIVIPDSATEK ch11_rhime
ESEEKTKGGIIIPDTAKEK ch10_lacla EEEEKSMGGIVLTSASQEK ch10_stral
DAEQTTASGLVTPDTAKEK ch10_thep3 ETEEKTASGIVLPDTAKEK ch10_bacsu
ESEEKTASGIVLPDSAKEK ch10_bacst ETEEKTASGLVLPDTAKEK ch10_myctu
EAETTTASGLVIPDTAKEK ch13_braja DAEEKTAGGIIIPDTAKEK ch10_staau
EQEQTTKSGIVLTDSAKEK ch10_mycbo EAETTTASGLVIPDTAKEK ch10_mycle
EAETMTPSGLVIPENAKEK ch10_clope EAEETTKSGIIVTGTAKER ch10_synp7
EAEEKTAGGIILPDNAKEK ch10_synp6 EAEEKTAGGIILPDNAKEK ch10_syny3
PAEEKTAGGILLPDNAKEK ch10_chlpn EEEATARGGIILPDTAKKK ch10_lepin
QEAEEKIGSIFVPDTAKEK ch10_chips EEDSTARGGIILPDTAKKK ch10_chltr
EEASTARGGIILPDTAKKK ch10_rat AAETVTKGGIMLPEKSQGK ch10_bovin
AAETVTKGGIMLPEKSQGK ch10_ricts QNDE.AHGKILIPDTAKEK ch10_spiol
EVENKTSGGLLLAESSKEK ch10_arath IQPAKTESGILLP.EKSSK
[0039] In a preferred embodiment, the chaperonin binding domain is
the sequence EVETKSAGGIVLTGSAAA or portions or variations thereof
which bind to the GroEL chaperonin with an affinity of between
about 10.sup.-2 to about 10.sup.-8 Kd.
[0040] For construction of a fusion protein, the chaperonin binding
domain may be directly linked to the desired protein, peptide or
polypeptide, or indirectly linked, ie comprising additional nucleic
acid between the nucleic acid encoding the chaperonin binding
domain and the protein or peptide or polypeptide. Such additional
nucleic acid may encode enzymatic cleavage sites or chemical
cleavage sites. Nucleic acid encoding the chaperonin may be 5' or
3' to the nucleic acid encoding the protein, peptide or
polypeptide.
[0041] III. Expression Systems
[0042] The present invention encompasses expression vectors and
host cells comprising a chaperonin binding domain for the
production of proteins, peptides or polypeptides in host cells.
Nucleic acid encoding a chaperonin binding domain can be isolated
from a naturally occurring source or chemically synthesized as can
nucleic acid encoding a desired protein, peptide or polypeptide.
Once nucleic acid encoding a binding domain of the present
invention, or a protein, peptide or polypeptide, is obtained,
fusion proteins comprising the chaperonin binding domain and the
protein, peptide or polypeptide and recombinant host cells
comprising the fusion proteins may be constructed using techniques
well known in the art. Molecular biology techniques are disclosed
in Sambrook et al., Molecular Biology Cloning: A Laboratory Manual,
Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1989). Nucleic acid encoding a chaperonin
binding domain and/or protein is obtained and transformed into a
host cell using appropriate vectors. A variety of vectors and
transformation and expression cassettes suitable for the cloning,
transformation and expression in host cells are known by those of
skill in the art.
[0043] Typically, the vector or cassette contains sequences
directing transcription and translation of the nucleic acid, a
selectable marker, and sequences allowing autonomous replication or
chromosomal integration. Suitable vectors comprise a region 5' of
the gene which harbors transcriptional initiation controls and a
region 3' of the DNA fragment which controls transcriptional
termination. These control regions may be derived from genes
homologous or heterologous to the host as long as the control
region selected is able to function in the host cell.
[0044] Initiation control regions or promoters, which are useful to
drive expression of the chaperonin binding domain, or fusion
protein comprising the chaperonin binding domain, in a host cell
are known to those skilled in the art. Virtually any promoter
capable of driving expression is suitable for the present
invention. Nucleic acid encoding the chaperonin binding domain is
linked operably through initiation codons to selected expression
control regions for effective expression of the chaperonin binding
domain.
[0045] Once suitable cassettes are constructed they are used to
transform the host cell. General transformation procedures are
taught in Current Protocols In Molecular Biology (vol. 1, edited by
Ausubel et al., John Wiley & Sons, Inc. 1987, Chapter 9) and
include calcium phosphate methods, transformation using PEG and
electroporation.
[0046] A host cell which contains the coding sequence for a
chaperonin or chaperonin binding domain of the present invention
and expresses the protein may be identified by a variety of
procedures known to those of skill in the art. These procedures
include, but are not limited to, DNA-DNA or DNA-RNA hybridization
and protein bioassay or immunoassay techniques which include
membrane-based, solution-based, or chip-based technologies for the
detection and/or quantification of the nucleic acid or protein.
[0047] A host cell comprising a fusion protein comprising a
chaperonin binding domain is used to express proteins, peptides or
polypeptides which are normally toxic to the host cell. A toxic
protein may affect the growth of the cell due to its intrinsic
qualities or due to the affects on the cell due to
overexpression.
[0048] The manner and method of carrying out the present invention
may be more fully understood by those of skill in the art by
reference to the following examples, which examples are not
intended in any manner to limit the scope of the present invention
or of the claims directed thereto.
EXAMPLES
[0049] Materials and Methods
[0050] Genes, Strains, Media and Growth Conditions.
[0051] E. coli strain JM83 was obtained from Dr. Deborah Hanson,
Argonne National Laboratory and strain JM105 was from Pharmacia.
For cloning, strains were cultured on LB or 2xYT medium with 100
.mu.g/ml ampicillin where appropriate, and for physiological
studies, strains were grown on LB medium supplemented with
appropriate carbon sources and electron acceptors. The plasmid
pASK40 was from Skerra et al., 1991, Bio/Technology 9:273-278,
pJF118EH was from Dr. Michael Bagdasarian (Furste et al., 1986,
Gene 48, 119-31), pTRC99a and pUC19 were from Pharmacia. Reagents
used in polymerase chain reactions (PCR) were from PerkinElmer;
isopropyl-b-D-thiogalactopyranoside (IPTG) was from US
Biochemicals; enzymes for molecular biology were from Promega,
Inc.; all other chemicals were purchased from Sigma (St.
Louis).
[0052] The pSK-mBax plasmid containing the cDNA encoding murine-Bax
was a gift from Dr. John C. Reed from the La Jolla Cancer
Foundation. The plasmid containing murine-Bcl-2 cDNA was provided
by Dr. Timothy J. McDonnell at the University of Texas M. D.
Anderson Cancer Center. Nucleic acid sequences for murine bax and
murine bcl-2 found in GenBank entries L22472 (Oltvai et al., 1993,
Cell, 74:609-619) and M16506 (Negrini et al., 1987, Cell,
49:455-463) were used to design PCR primers that would 1) introduce
an EcoRI site at the 5'-end of the gene to allow cloning into the
vector pASK40 in the correct reading frame, and 2) encode an
additional five histidine residues at the 3'-end of the gene to
facilitate purification of expressed proteins (Hochuli et al.,
1988, Bio/Technology 6:1321-1325). MDH was previously cloned from
genomic E. coli DNA by PCR into pASK40 and pTRC99a (Boernke, et al
(1995) Arch. Biochem. Biophys. 322:43-52).
2TABLE 2 Plasmids used. Plamsid Components Source and/or reference
pASK40 bla, laclq, P/Olac, t lpp, f1-IG, (Skerra et al., supra)
ompA pJF118EH bla, laclq, Ptac (Furste et al., supra) pATP004
GroES-loop encoding leader in this application pJF118EH pATP005
Multicloning site of pUC19 this application substituted for that of
JF118EH in pATP004 pMDH1 E. coli mdh in pASK40 (Boernke et al.,
supra) pMDH13 E. coli mdh in pTRC99a (Boernke et al., supra)
pATP007 E. coli mdh in pATP004 this application pWS213 BCL-2 in
pASK40 this application pATP010 BCL-2 in pATP005 this application
pBAX002 BAX in Xbal/Sal I sites of this application pASK40 (no
leader sequence) pBAX001 BAX in EcoRI/Sal I sites of this
application pASK40 (ompA leader sequence) pATP011 BAX from pBAX001
in pATP005 this application (GroES-loop leader sequence)
[0053] Construction of Vectors for Expressing Chaperonin Binding
Domain Fusion Proteins
[0054] Vectors designed to comprise the chaperonin binding domain
obtainable from GroES were constructed as follows. Oligonucleotides
were synthesized based on the published amino acid sequence of the
E. coli GroES protein. Residues 16 through 33 comprise the
chaperonin binding domain EVETKSAGGIVLTGSAAA. A nucleotide sequence
encoding this sequence and its complement were generated by the
program Lasergene (DNAStar, Inc., Madison, Wis.) using the general
codon preferences for E. coli. A linker was designed to require the
same reading frame as that required in pASK40. Additional
nucleotides encoding the overhang generated by an EcoRI digest of
DNA and an ATG initiation codon were included at the 5'-end of each
to give:
[0055] Oligo ATP6:
5'-MTTATGGMGTTGAMCCAAATGTGCTGGTGGTATCG-TTCTGACCGGTTCTGC-
TGCTGCG-3'
[0056] Oligo ATP7:
5'-MTTCGCAGCAGCAGMCCGGTCAGMCGATACCACCA-GCAGATTTGGTTTCAA-
CTTCCAT-3'
[0057] The design of the linker for attachment of the GroEL-binding
domain of GroES to proteins is shown in FIG. 5.
[0058] The linker was phosphorylated using T4 polynucleotide
kinase, purified with Qiaex resin (Qiagen), and ligated into the
dephosphorylated EcoRI site of the vector pJF118EH. Transductants
of strain JM105 were screened for the presence of an EcoRI site,
which is present in the linker but absent in pJF118EH. Positive
transductants were screened for orientation of the insert. Only one
end regenerates an EcoRI site, and in the correct orientation that
site is adjacent to a multicloning site. EcoRI-EcoRV digests were
analyzed on a 1.5% agarose gel for the presence of the 1,260 base
pair fragment predicted for the correct orientation as opposed to
the 1,200 base pair fragment predicted for the reverse orientation.
The new vector was designated pATP004.
[0059] Because the multi-cloning site of pATP004 was limited in the
number of sites available for cloning, we exchanged this cluster
for the larger cluster of pUC19 using the enzymes EcoRI and
HindIII. Plasmids obtained from JM105 transductants were screened
for orientation, as above, and for the presence of the KpnI and
XbaI sites present only in the pUC19 multicloning site. The
resulting plasmid was designated pATP005.
[0060] Expression and Analysis of Proteins
[0061] Cultures containing plasmids were grown in LB medium to
A.sub.600 0.2-0.5 then induced with 0.1 mM IPTG. To prepare
cytoplasmic fractions of total proteins, cells were lysed by
treatment with lysozyme. Expression of noncatalytic proteins was
estimated by conventional gel electrophoresis. Gels were scanned
with a UMax PowerlookII scanner and the images were analyzed with
Digital Science 1 D software (Eastman Kodak). For more rigorous
quantitation, 2-dimensional gel electrophoresis was performed.
[0062] Two-Dimensional Gel Electrophoresis
[0063] Cell pellets (7.85 OD.sub.600 units) were lysed in 1 mL of a
solution containing 9 M urea, 2% 2-mercaptoethanol, 4% Nonidet P40,
and 2% ampholytes (BioRad pH 8-10). The resulting homogenates were
centrifuged for 5 min at 435.times.g in a Beckman TL100 tabletop
ultracentrifuge to remove particulates. The supernatants were then
frozen at -70.degree. C. until electrophoresis. Isoelectric
focusing in the first dimension was done essentially as described
by Anderson and Anderson (1978, Anal. Biochem, vol. 85, pages
33-340) using 50% pH 3-10 Biolyte, 25% pH 5-7 Biolyte, and 25% pH
5-7 Servalyte carrier ampholytes. After equilibration in sodium
dodecyl sulphate buffer (O'Farrell, 1975, J. Bio. Chem., vol. 250,
pages 4007-4021) the focused proteins were separated in the second
dimension in slab gels containing a linear gradient of 10-17%
polyacrylamide, essentially as described by O'Farrell, supra, with
modifications described by Anderson and Anderson, 1978, Anal.
Biochem, vol. 85: pages 341-354. After electrophoresis, gels were
fixed and stained in 0.125% (w/v) Coomassie Blue R250 in 2.5%
phosphoric acid and 50% ethanol for approximately 24 h. The gels
were then destained in 20% ethanol. Protein patterns were digitized
using an Eikonix 1412 CCD scanner interfaced with a VAXstation
4000-90. Image processing and generation of parameter lists,
referred to as spot files, were as previously described Anderson,
1982. A master pattern was created using a copy of a 2DE spot file
from the "ethanol and induced" treatment group so that the Bax
protein was represented. Each of the patterns generated for the
experiment, with four replicate patterns within each sample group,
was matched to the master and then examined interactively for the
appearance of new proteins, the loss of normally expressed
proteins, and for statistically significant quantitative
differences between the control patterns and the "induced" or
"ethanol and induced patterns".
Example I
[0064] Construction of Expression Vector
[0065] A synthetic linker designed to encode the chaperonin binding
loop of GroES (EVETKSAGGIVLTGSAAA) was ligated into the EcoRI site
of plasmid pJF118EH. Plasmid DNA was prepared from representative
colonies arising from transformation of E. coli JM105 with this
ligation mixture, and screened for the presence of an Agel site,
which is unique to the introduced linker. Of 10 colonies screened,
all contained the site. The plasmids were then screened for
orientation of the linker, since it could be incorporated in two
directions. The linker was designed so that only one EcoRI site
would be regenerated, which in the desired orientation would be
attached to the multi-cloning site. Plasmids were digested with
EcoRI and EcoRV (present in the tac promoter) and analyzed on a
1.5% gel. The desired orientation, present in 5 of the plasmids,
generated a 1,260 base-pair fragment; those in the wrong
orientation, with the reconstituted EcoRI site at the downstream
side of the inserted linker, generated a fragment of 1,200
basepairs. A representative of the correct orientation was
propagated and designated pATP004.
[0066] To expand the potential of the vector, the small
multi-cloning site of pAF118EH was excised from pATP004 by
digesting it with EcoRI and HindIII, and replaced with the
EcoRI-HindIII multi-cloning site of pUC19. Of eight colonies
screened, seven contained the inserted multi-cloning site. These
were further shown to have the expected orientation and the
additional restriction sites. A representative colony was
designated pATP005.
Example 11
[0067] Insertion of Genes Into the Expression Vectors.
[0068] The vectors described above initiate protein synthesis at
the ATG codon in the new linker sequence that encodes
EVETKSAGGIVLTGSAAA. The vector's multi-cloning site follows this
linker, and genes are introduced into the cloning sites such that
their reading frame matches that of the linker.
[0069] The gene encoding mouse Bax was cloned from the pSK-mBax
vector and the gene encoding murine Bcl-2 was cloned from cDNA by
PCR as described in Materials and Methods. The bax gene was
subsequently moved into the EcoRI-BamH1 sites of pATP004 to give
the vector pATP001. Genes encoding the BAX homolog Bcl-2 and the E.
coli MDH were also recloned into pATP004 by standard methods.
Example III
[0070] Expression of the Fusion Protein Comprising BAX-Chaperonin
Binding Domain
[0071] Induction of BAX was compared for three classes of genetic
constructs designed to express the BAX gene: 1) without a leader
sequence, 2) with the OmpA leader, and 3) with the chaperonin
binding domain obtainable from GroES.
[0072] The possible effect of the GroES-loop leader sequence on
expression of BAX protein was evaluated in cultures that enhance
the expression of E. coli chaperones in the cell. The rationale was
based on the assumption that folding of expressed fusion proteins
would be mediated through interaction with the chaperonin GroEL.
When grown in the presence of moderate concentrations of ethanol,
E. coli is known to induce higher levels of chaperones and other
stress proteins, Barbarosa, supra. We first evaluated the effect of
various concentrations of ethanol on the growth of the host strain,
JM109. A concentration of 2% ethanol (0.44 M) reduced the growth
rate approximately 2-fold and was chosen for induction studies
(FIG. 2).
[0073] Only those transformants containing BAX as a fusion with the
chaperonin binding domain obtainable from GroES, showed production
of the BAX protein. When those transformants without a leader and
not producing BAX were induced, their growth was unaffected.
Extracts prepared from these cultures showed patterns of proteins
that were identical for the induced and uninduced cultures; there
was no evidence of expression of a heterologous protein, either by
the appearance of a new band of the expected molecular weight or by
alterations in the abundance of E coli host proteins that typically
occur on overexpression.
[0074] Apparently none of the transformants contained a functional
BAX expression system (DNA sequencing analysis of the 5'-end of the
genes indicated that the constructs were as expected in that
region). In striking contrast, induction of transformants
constructed to produce BAX with either of the leader sequences
(OmpA or GroES-loop) caused an immediate cessation of growth.
[0075] Uninduced cells grew normally. In contrast IPTG-induced
cells growth dropped immediately following introduction with IPTG
(FIG. 3). This apparent strong toxicity of BAX was moderated when
cultures were grown in the presence of ethanol, but only in the
case of the BAX chimera containing chaperonin binding domain
obtainable from GroES (solid circles in FIG. 3). Analyses of the
proteins present at the end of this period of induction (FIG. 4)
revealed the presence of a new protein of the anticipated molecular
weight (indicated by the "bar" in the low molecular weight region
(the BAX molecular weight is 21,419 Daltons) of the gel between
lanes 3 and 4 and between lanes 7 and 8). The amount of this new
protein was quite low in each case except that of having the
chaperonin binding domain obtainable from GroES produced in cells
grown in the presence of ethanol (FIG. 4). Densitometry of this
region of the gel indicated that the presence of ethanol resulted
in at least a 15 fold increase in the amount of the BAX produced.
Under these conditions inclusion of ethanol induced GroEL
production approximately 1.5-1.8 fold.
[0076] Due to the presence of other proteins of similar molecular
weight, the estimates of relative production of the BAX and GroEL
proteins from 1-D gels are inaccurate. Therefore the extracts were
separated by 2-dimensional gel electrophoresis (DE). Samples were
separated by 2-DE and the resulting protein patterns were analyzed
for qualitative and quantitative differences as described in
Materials and Methods. The extent of the overexpression of BAX and
its enhancement caused by inclusion of ethanol in the culture was
found to be greater than that suggested by the 1-dimensional gel
analysis. In the absence of IPTG inducer, no protein attributable
to BAX was detectable.
[0077] The protein pattern analysis indicated that of all the
proteins separated on 2DE, only four were overexpressed: GroEL,
Hsp70, the fusion of chaperonin binding domain and BAX, and serine
hydroxymethyl transferase (Table 3).
3TABLE 3 Qualitative analysis of protein expression. condition avg.
density relative amt DnaK control nos. 1 +IPTG (A) nos. 0.7 +IPTG,
+EtOH (B) nos. 2.1 GroEL control nos. 1 +IPTG (A) nos. 0.85 +IPTG,
+EtOH (B) nos. 2.8 SHMT control nos. 1 +IPTG (A) nos. 0.6 +IPTG,
+EtOH (B) nos. 1.6 fusion with BAX control 0 0 +IPTG (A) 3089 --
+IPTG, +EtOH (B) 45494 14.7 rel to -EtOH
[0078] No density above background was detected for the fusion with
BAX in the absence of IPTG inducer. The small amount formed when
IPTG was present was increased 14.7 fold when cultures were grown
with 2% ethanol. Under these conditions, GroEL and Hsp70 were
enhanced 3 fold above the amount observed when only IPTG was
present.
[0079] Various other examples and modifications of the foregoing
description and examples will be apparent to a person skilled in
the art after reading the disclosure without departing form the
spirit and scope of the invention, and it is intended that all such
examples or modifications be included within the scope of the
appended claims. All publications and patents referenced herein are
hereby incorporated in their entirety.
Sequence CWU 1
1
44 1 54 DNA Escherichia coli 1 gaagttgaaa ccaaatctgc tggtggtatc
gttctgaccg gttctgctgc tgcg 54 2 18 PRT Escherichia coli 2 Glu Val
Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala 1 5 10 15
Ala Ala 3 19 PRT Escherichia coli 3 Glu Val Glu Thr Lys Ser Ala Gly
Gly Ile Val Leu Thr Gly Ser Ala 1 5 10 15 Ala Ala Lys 4 19 PRT
Acyrthosiphon pisum 4 Glu Val Glu Ser Lys Ser Ala Gly Gly Ile Val
Leu Thr Gly Ser Ala 1 5 10 15 Ala Gly Lys 5 19 PRT Haemophilus
ducreyi 5 Glu Val Glu Thr Cys Ser Ala Gly Gly Ile Val Leu Thr Gly
Ser Ala 1 5 10 15 Thr Val Lys 6 19 PRT Pseudomonas aeruginosa 6 Glu
Glu Glu Thr Lys Thr Ala Gly Gly Ile Val Leu Pro Gly Ser Ala 1 5 10
15 Ala Glu Lys 7 19 PRT Allochromatium vinosum 7 Glu Glu Glu Arg
Leu Ser Ala Gly Gly Ile Val Ile Pro Asp Ser Ala 1 5 10 15 Thr Glu
Lys 8 19 PRT Coxiella burnetii 8 Glu Glu Glu Arg Thr Ser Ala Gly
Gly Ile Val Ile Pro Asp Ser Ala 1 5 10 15 Ala Glu Lys 9 19 PRT
Legionella micdadei 9 Glu Glu Glu Arg Thr Thr Ala Gly Gly Ile Val
Ile Pro Asp Ser Ala 1 5 10 15 Thr Glu Lys 10 19 PRT Sinorhizobium
meliloti 10 Glu Ser Glu Glu Lys Thr Lys Gly Gly Ile Ile Ile Pro Asp
Thr Ala 1 5 10 15 Lys Glu Lys 11 19 PRT Legionella pneumophila 11
Glu Glu Glu Arg Thr Thr Ala Gly Gly Ile Val Ile Pro Asp Ser Ala 1 5
10 15 Thr Glu Lys 12 19 PRT Brucella abortus 12 Glu Ser Glu Ala Lys
Thr Ala Gly Gly Ile Ile Ile Pro Asp Thr Ala 1 5 10 15 Lys Glu Lys
13 19 PRT Bradyrhizobium japonicum 13 Asp Ala Glu Glu Lys Thr Ala
Gly Gly Ile Ile Ile Pro Asp Thr Val 1 5 10 15 Lys Glu Lys 14 19 PRT
Agrobacterium tumefaciens 14 Glu Ser Glu Ala Lys Thr Lys Gly Gly
Ile Ile Ile Pro Asp Thr Ala 1 5 10 15 Lys Glu Lys 15 19 PRT
Clostridium acetobutylieum 15 Glu Ala Glu Glu Thr Thr Lys Ser Gly
Ile Val Leu Pro Ser Ser Ala 1 5 10 15 Lys Glu Lys 16 19 PRT Amoeba
proteus 16 Glu Glu Glu Arg Thr Thr Ala Gly Trp Ile Val Ile Pro Asp
Ser Ala 1 5 10 15 Thr Glu Lys 17 19 PRT Sinorhizobium meliloti 17
Glu Ser Glu Glu Lys Thr Lys Gly Gly Ile Ile Ile Pro Asp Thr Ala 1 5
10 15 Lys Glu Lys 18 19 PRT Lactococcus lactic 18 Glu Glu Glu Glu
Lys Ser Met Gly Gly Ile Val Leu Thr Ser Ala Ser 1 5 10 15 Gln Glu
Lys 19 19 PRT Streptomyces albus 19 Asp Ala Glu Gln Thr Thr Ala Ser
Gly Leu Val Ile Pro Asp Thr Ala 1 5 10 15 Lys Glu Lys 20 19 PRT
Thermoactinomyces sp. 20 Glu Thr Glu Glu Lys Thr Ala Ser Gly Ile
Val Leu Pro Asp Thr Ala 1 5 10 15 Lys Glu Lys 21 19 PRT Bacillus
subtilis 21 Glu Ser Glu Glu Lys Thr Ala Ser Gly Ile Val Leu Pro Asp
Ser Ala 1 5 10 15 Lys Glu Lys 22 19 PRT Bacillus stearothermophilus
22 Glu Thr Glu Glu Lys Thr Ala Ser Gly Ile Val Leu Pro Asp Thr Ala
1 5 10 15 Lys Glu Lys 23 19 PRT Mycobacterium tuberculosis 23 Glu
Ala Glu Thr Thr Thr Ala Ser Gly Leu Val Ile Pro Asp Thr Ala 1 5 10
15 Lys Glu Lys 24 19 PRT Bradyrhizobium japonicum 24 Asp Ala Glu
Glu Lys Thr Ala Gly Gly Ile Ile Ile Pro Asp Thr Ala 1 5 10 15 Lys
Glu Lys 25 19 PRT Staphylococcus aureus 25 Glu Gln Glu Gln Thr Thr
Lys Ser Gly Ile Val Leu Thr Asp Ser Ala 1 5 10 15 Lys Glu Lys 26 19
PRT Mycobacterium bovis 26 Glu Ala Glu Thr Thr Thr Ala Ser Gly Leu
Val Ile Pro Asp Thr Ala 1 5 10 15 Lys Glu Lys 27 19 PRT
Mycobacterium lepvae 27 Glu Ala Glu Thr Met Thr Pro Ser Gly Leu Val
Ile Pro Glu Asn Ala 1 5 10 15 Lys Glu Lys 28 19 PRT Clostridium
perfringens 28 Glu Ala Glu Glu Thr Thr Lys Ser Gly Ile Ile Val Thr
Gly Thr Ala 1 5 10 15 Lys Glu Arg 29 19 PRT Synechococcus PCC7942
29 Glu Ala Glu Glu Lys Thr Ala Gly Gly Ile Ile Leu Pro Asp Asn Ala
1 5 10 15 Lys Glu Lys 30 19 PRT Synechococcus PCC6301 30 Glu Ala
Glu Glu Lys Thr Ala Gly Gly Ile Ile Leu Pro Asp Asn Ala 1 5 10 15
Lys Glu Lys 31 19 PRT Synechocystis PCC6803 31 Pro Ala Glu Glu Lys
Thr Ala Gly Gly Ile Leu Leu Pro Asp Asn Ala 1 5 10 15 Lys Glu Lys
32 19 PRT Chlamydophila pheumoniae 32 Glu Glu Glu Ala Thr Ala Arg
Gly Gly Ile Ile Leu Pro Asp Thr Ala 1 5 10 15 Lys Lys Lys 33 19 PRT
Leptospiya interrogans 33 Gln Glu Ala Glu Glu Lys Ile Gly Ser Ile
Phe Val Pro Asp Thr Ala 1 5 10 15 Lys Glu Lys 34 19 PRT
Chlamydophila psittaci 34 Glu Glu Asp Ser Thr Ala Arg Gly Gly Ile
Ile Leu Pro Asp Thr Ala 1 5 10 15 Lys Lys Lys 35 19 PRT Chlamydia
trachomatis 35 Glu Glu Ala Ser Thr Ala Arg Gly Gly Ile Ile Leu Pro
Asp Thr Ala 1 5 10 15 Lys Lys Lys 36 19 PRT Rattus norregiens 36
Ala Ala Glu Thr Val Thr Lys Gly Gly Ile Met Leu Pro Glu Lys Ser 1 5
10 15 Gln Gly Lys 37 19 PRT Bos taurus 37 Ala Ala Glu Thr Val Thr
Lys Gly Gly Ile Met Leu Pro Glu Lys Ser 1 5 10 15 Gln Gly Lys 38 18
PRT Orienta tsutsugamushi 38 Gln Asn Asp Glu Ala His Gly Lys Ile
Leu Ile Pro Asp Thr Ala Lys 1 5 10 15 Glu Lys 39 19 PRT
Spirillospora sp. 39 Glu Val Glu Asn Lys Thr Ser Gly Gly Leu Leu
Leu Ala Glu Ser Ser 1 5 10 15 Lys Glu Lys 40 18 PRT Arabidopsis
thaliana 40 Ile Gln Pro Ala Lys Thr Glu Ser Gly Ile Leu Leu Pro Glu
Lys Ser 1 5 10 15 Ser Lys 41 54 DNA Artificial Sequence
oligonucleotide 41 gaagttgaaa ccaaatctgc tggtggtatc gttctgaccg
gttctgctgc tgcg 54 42 61 DNA Artificial Sequence oligonucleotide 42
aattcgcagc agcagaaccg gtcagaacga taccaccagc agatttggtt tcaacttcca
60 t 61 43 20 PRT Artificial Sequence linker 43 Met Glu Val Glu Thr
Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser 1 5 10 15 Ala Ala Ala
Asn 20 44 61 DNA Artificial Sequence linker 44 aattatggaa
gttgaaacca aatctgctgg tggtatcgtt ctgaccggtt ctgctgctgc 60 g 61
* * * * *