SEONG BAIK LIN (KR)
CHOI SEONG IL (KR)
SEONG BAIK LIN (KR)
EP0247819A2 | 1987-12-02 |
1. | A expression vector which contains aminoacyltRNA synthetase gene and linker peptide sequence, protease recognition site, tag sequence or restriction enzyme recognition site. |
2. | The expression vector according to Claim 1, wherein aminoacyl tRNA synthetase gene is E. coli aminoacyltRNA synthetase gene. |
3. | The expression vector according to Claim 2, wherein aminoacyl tRNA gene is lys S gene of lysyltRNA synthetase. |
4. | The expression vector pGElysRS according to Claim 3. |
5. | ' The expression vector according to Claim 3, which contains a part of lys S gene encoding the Nterminal domain of lysyltRNA synthetase. |
6. | The expression vector according to Claim 5, which contains the Nterminal domain gene deleted at the amino acid residues 1 to 13. |
7. | The expression vector according to Claim 5, which contains the Nterminal domain gene deleted at the amino acid residues 1 to 29. |
8. | The expression vector according to Claim 5, wherein the N terminal domain gene is OB fold gene. |
9. | The expression vector according to Claim 8, wherein the OB fold gene is the Nterminal domain gene deleted at the amino acids residues 1 to 65. |
10. | The expression vector pGElysN according to Claim 5. |
11. | A is . coli transformant which is prepared by transforming E. coli HMS 174 strain with the expression vector of Claim 10 (accession number : KCTC 0388 BP). |
12. | A expression vector which is prepared by inserting a foreign protein gene into the expression vector of Claim 1. |
13. | The expression vector plysNGMcsf according to Claim 12, wherein the foreign protein is GMcsf protein. |
14. | The expression vector plysNGcsf according to Claim 12, wherein the foreign protein is Gcsf protein. |
15. | The expression vector plysNTIMP2 according to Claim 12, wherein the foreign protein is TIMP2 protein. |
16. | The transformant which is prepared by transforming host cell with the expression vector of Claim 12. |
17. | A process for preparing foreign proteins as soluble forms, wherein the transformant of Claim 16 is cultured and induced for the expression of protein. |
FOREIGN PROTEINS AS SOLUBLE FORMS
Field of the Invention
The present invention relates to novel expression vectors which can
produce foreign proteins as soluble forms by using lysyl tRNA synthetase
and a process for preparing foreign proteins by using the expression vectors.
More particularly, the present invention relates to the expression
vectors which can provide foreign proteins as fused and soluble forms by
exploiting the structure and expression pattern of lysyl-tRNA synthetase
and the process for preparing foreign proteins in E. coli effectively, which
can be utilized industrially to produce active proteins in mass.
Background of Invention
With the advance of genetic engineering, heterologous proteins
which are used industrially as medicine and the like, have been produced by
utilizing animal cells, yeasts and prokaryotes such as E. coli. Especially
E. coli has been exploited as a popular host cell to produce foreign proteins
since it grows fast and has been studied more thoroughly than any other
organisms.
Unfortunately, E. coli lacks cellular components necessary for
posttranslational modification processes like glycosylation, disulfide-
crosslinking or the like. And foreign proteins produced massively and
excessively in E. coli are sequestered into inclusion bodies, which can be
easily separated. But in order to obtain active proteins, these inclusion
bodies should be solubilized to form primary structure by using high
concentration of urea, guanidium HCI or the like and then refolded
removing the above reagents.
Generally, the refolding process for preparing a active protein can
not be always performed successfully since its result varies according to the
cases. For example, proteins having high molecular weight, such as
antibodies, tissue plasmingen activator, factor VIII and so on, are not
refolded easily to become active proteins. And, it is difficult to produce a
recombinant protein on a large scale.
Therefore, it is very important to express foreign proteins as soluble
forms in E. coli for improving the problems caused in above cases.
Presently, following methods have been exploited to express
foreign proteins as soluble forms effectively.
First, there is a method in which N-terminus of foreign protein is
linked to signal peptide so as to secrete foreign protein into periplasm of E.
coli as a soluble form (Stader, J. A. and Silhavy, T. J., 1970, Methods in
Enzymol, 165 : 166-187). Since the foreign proteins are not expressed
effectively by the process, this method is not useful industrially.
Second, there is a method in which foreign proteins are expressed
with chaperone genes such as groES, groEL, dnaK and the like to obtain
soluble proteins (Goloubinoff, P., Gatenby, A. A. and Lorimer, G. H.,
1989, Nature, 337 : 44-47). But this method is not general to prevent the
formation of inclusion body since it is available on only specific proteins.
Third, there is a method in which target proteins are fused at the C-
terminus with fusion partner proteins which can be expressed highly in E.
coli. Since the target proteins are linked at the C-terminus of fusion
partners, translation initiation signal of the fusion partner protein can be
exploited usefully. And the solubility of the fused foreign protein
increases so that large amount of foreign proteins can be obtained as soluble
forms in E. coli.
Lac Z or Trp E protein have been utilized as a fusion partner
protein in order to produce fusion proteins in E. coli. But active-form
proteins can not be obtained easily since most fusion proteins were
expressed in the forms of inclusion body. Therefore, many researches
have been accomplished to obtain novel fusion partner proteins which
facilitates the production of active-form proteins. Practically, some fusion
partner proteins have been developed, such as glutathione-S-transferase
(Smith, D. B. and Johnson, K. S., 1988, Gene, 67 : 31-40), maltose-binding
protein (Bedouelle, H. and Duplay, P., 1988, Euro. J. Biochem., 171 : 541-
549), protein A (Nilsson, B. et al., 1985, Nucleic Acid Res., 13 : 1151-
1162), Z domain of protein A (Nilsson, B. et al., 1987, Prot. Eng., 1 :
107-113), protein Z (Nygren, P. A. et al., 1988, J. Mol. Recog., 1 : 69-
74) and thioredoxin (Lavallie, E. R. et al., 1993, Bio/Technology, 11 :
187-193) .
Although foreign proteins have been expressed by linking the
fusion partner described above and prepared as soluble forms, some were
expressed as inclusion body or partly as soluble proteins according to the
fusion partner protein.
Particularly, thioredoxin has been known to be the most successful
protein as a fusion partner. However, in the case of thioredoxin E. coli
transformant should be cultured at low temperature such as 15°C in order to
express most fusion proteins as soluble forms. Since E. coli grows very
slowly at that temperature, the process using the thioredoxin may be
inefficient.
Lysyl-tRNA synthetase (hereinafter it refers to "Lys RS") and its
gene have been investigated as described below, which is preferable for the
fusion partner protein and expressed highly in E. coli.
Although in E. coli aminoacylation is performed by using a specific
aminoacyl-tRNA synthetase, two lysyl-tRNA synthetases which are
encoded from lys S gene and lys U gene are involved in the aminoacylation
independently, lys S gene is expressed constitutively in normal condition
and lys U gene is induced by heat shock, low pH, anaerobiosis, L-alanine,
L-leucine, L-leucyldipeptide. And amino acid sequences derived from the
two genes show 88% of homology.
In addition, the X-ray crystallographical structure of lysyl-tRNA
synthetase which is expressed from lys U gene (hereinafter it refers to
"Lys U") was illucidated at the 2.8 A° resolution level (Onesti, S., Miller.
A. D. and Brick, P., 1995, Structure, 3 : 163-176). Lys U protein is
composed of homodimer which has N-terminal domain contacting with
tRNA and C-terminal domain of dimer interface showing the enzyme
activity (see Fig. 1).
In addition, nuclear magnetic resonance (NMR) structure of N-
terminal domain (31-149 amino acid residues) of lysyl-tRNA synthetase
which is expressed from lys S gene (hereinafter it refers to "Lys S") was
revealed by Frederic Dardel group (Stephane, C. et al., J. Mol. Biol., 253 :
100-113). As Lys U protein and Lys S protein share a high degree of
identity in the amino acid sequences, the N-terminal structures of the two
enzymes are identified to be very similar.
In detail, the N-terminal domain of lysyl-tRNA synthetase has
secondary structure of five stranded antiparallel β barrel which is composed
of α-helix (H4) located between 3rd and 4th β-sheet and contiguous 3 α-
helices. The post-part of N-terminal domain corresponds to OB fold
(A1A2A3H4A4A5) which is found in proteins binding with
oligosaccharides or oligonucleotides commonly. It has been reported that
OB fold was discovered in aspartyl-tRNA synthetase of yeast, β-subunit of
heat labile enterotoxin, berotoxin and staphylococcal nuclease (Murzin, A.
G., 1993, EMBO J., 12 : 861-867).
The N-terminal domain of Lys RS protein of which the structure is
described above shows the following characteristics as a fusion partner
protein.
When lys S gene was expressed in E. coli, Lys S protein has
accumulated to 80% of total soluble proteins. Since Lys S protein is
composed of homodimer of which the contact region is located at the C-
terminus of monomer, the fusion protein using intact Lys S protein or C-
terminal domain of Lys S protein as a fusion partner makes heterodimer
with Lys S protein of E. coli.
But such a heterodimer is fatal to E. coli . Thus the C-terminal
domain of Lys S protein is not appropriate as a fusion partner protein and
only the N-terminal domain can be exploited as a fusion partner protein.
Practically, only N-terminal domain of Lys S protein (hereinafter it refers to
"Lys N") can be used to express foreign proteins well, to approximately
40% of the total proteins and produced mostly as a soluble form.
As mentioned above, OB fold located in the N-terminal domain of
Lys RS protein has a secondary structure which facilitates protein folding
and increases the solubility of fusion proteins expressed.
The present inventors have researched to develop a fusion partner
protein which is useful to produce heterologous proteins by recombinant
DNA technology. Thus we have demonstrated that the N-terminal domain
of lysyl-tRNA synthetase can be utilized as a fusion partner protein to
produce foreign proteins massively in a soluble form. And by using the
lysyl-tRNA synthetase, we have developed novel E. coli expression vectors
and a process for preparing active foreign proteins effectively.
Summary of the Invention
The object of the present invention is to provide expression vectors
containing total or part of aminoacyl-tRNA synthetase gene. The
aminoacyl-tRNA synthetase gene can be obtained from all kinds of cells.
The expression vectors of the present invention are composed of
linker peptide sequence, tag sequence, protease recognition site, restriction
enzyme recognition site for inserting foreign gene or the like, in addition to
the aminoacyl-tRNA synthetase gene.
In addition, the object of the present invention is to provide the E.
coli expression vectors containing total or part of lysyl-tRNA synthetase
gene. The lysyl-tRNA synthetase gene can be selected among lys S gene
or lys U gene.
Particularly, the present invention provides the expression vector
pGE-lysRS containing intact lys S gene.
In addition, the object of the present invention is to provide the
expression vectors containing the N-terminal domain gene of lysyl-tRNA
synthetase.
The present invention provides the expression vectors containing
the N-terminal domain of lysyl-tRNA synthetase which is deleted at the
amino acid residues 1 to 13. And the present invention also provides the
expression vectors containing the N-terminal domain gene of lysyl-tRNA
synthetase which is deleted at the amino acid residues 1 to 29.
In addition, the present invention provides the expression vector
containing only OB fold gene of lysyl-tRNA synthetase. For the purpose,
the expression vectors contain the N-terminal domain gene of lysyl-tRNA
synthetase which is deleted at the amino acid residues 1 to 65.
Particularly, the present invention provides the E. coli expression
vector pGE-lysN. E. coli HMS 174 strain was transformed by the
expression vector pGE-lysN and the transformant has been deposited with
Korea Research Institute of Bioscience and Biotechnology, Korea, on
September 26, 1997 (accession number : KCTC 0388 BP).
The object of the present invention is to provide a process for
preparing useful foreign proteins as soluble forms of fusion protein by
inserting the foreign genes into the above expression vectors.
Particularly, the present invention provides the expression vector
plysN-GMcsf by inserting GMcsf (human granulocyte and macrophage
colony stimulating factor) gene into the expression vector pGE-lysN. Host
cell was transformed with the expression vector and induced to express
GMcsf protein as a fusion protein.
At that time, all kinds of E. coli strain can be used, which is
appropriate for the expression of the fusion protein. Preferably, E. coli
HMS 174 strain can be used as a host cell.
Particularly, the present invention provides the expression vector
plysN-Gcsf by inserting Gcsf (human granulocyte colony stimulating
factor) gene into the expression vector. By using the above process, Gcsf
protein is prepared.
Particularly, the present invention provides the expression vector
plysN-TIMP2 by inserting TIMP2 (human tissue inhibitor of
metalloprotease 2) gene into the expression vector. By using the above
process, TIMP2 protein is prepared.
Brief Description of the Drawings
Fig. 1 depicts the secondary structure of lysyl-tRNA synthetase
(Lys U).
Stick is helix structure and arrow is β-sheet structure.
Fig. 2 depicts a strategy for constructing the expression vector
pGE-lysRS into which lys S gene is inserted.
Fig. 3 depicts the expression of Lys S protein by performing SDS-
polyacrylamide gel electrophoresis, which used E. coli HMS 174 strain
transformed with the expression vector pGE-lysRS of the present invention,
lane 1 : standard protein marker;
lane 2: total proteins of E. coli induced for the protein expression ;
lane 3: total proteins of E. coli transformant;
lane 4: total proteins of E. coli transformant induced for the protein
expression ;
lane 5: supernatant of disrupted E. coli induced ;
lane 6: supernatant of disrupted E. coli transformant;
lane 7: supernatant of disrupted E. coli transformant induced;
lane 8: precipitate of disrupted E. coli induced;
lane 9: precipitate of disrupted E. coli transformant;
lane 10: precipitate of disrupted E. coli transformant induced
Fig. 4 depicts a strategy for constructing the expression vector
pGE-lysN which uses the N-terminal domain of Lys S protein as a fusion
partner protein.
Fig. 5 depicts a strategy for constructing the E. coli expression
vector pLysN-GMcsf which expresses GMcsf protein by using the
expression vecotr pGE-lysN.
Fig. 6 depicts the expression of GMcsf protein by performing
SDS-polyacrylamide gel electrophoresis, which used E. coli HMS 174
strain transformed with the expression vector pLysN-GMcsf of the present
invention.
lane 1 : standard protein marker;
lane 2: total proteins of E. coli transformant;
lane 3: total proteins of E. coli transformant induced for the
expression;
lane 4: precipitate of disrupted E. coli transformant;
lane 5: precipitate of disrupted E. coli transformant induced;
lane 6: supernatant of disrupted E. coli transformant;
lane 7: supernatant of disrupted E. coli transformant induced;
Fig. 7 depicts the expression of GMcsf protein for comparison by
performing SDS-polyacrylamide gel electrophoresis, which used
thioredoxin as a fusion partner protein and E. coli GI724 strains
transformed with the expression vector pTRXFUS-GMcsf and pTRXFUS
respectively.
lane 1 : standard protein marker;
lane 2: supernatant of disrupted E. co/z/pTRXF S-GMcsf
transformant induced for the protein expression;
lane 3: precipitate of disrupted E. < >///pTRXFUS-GMcsf
transformant induced;
lane 4: supernatant of disrupted E. co///pTRXFUS transformant
induced;
lane 5: precipitate of disrupted E. co///pTRXFUS transformant
induced;
Fig. 8 depicts a strategy for constructing the E. coli expression
vector pLysN-Gcsf which expresses Gcsf protein by using the expression
vector pGE-lysN.
Fig. 9 depicts the expression of Gcsf protein by performing SDS-
polyacrylamide gel electrophoresis, which used E. coli HMS 174 strain
transformed with the expression vector pLysN-Gcsf
lane 1 : standard protein marker;
lane 2: total proteins oϊE. coli transformant;
lane 3: precipitate of E. coli transformant;
lane 4: supernatant of E. coli transformant;
lane 5: total proteins of is. coli transformant induced for the protein
expression;
lane 6: precipitate of disrupted E. coli transformant induced;
lane 7: supernatant of disrupted E. coli transformant induced
Fig. 10 depicts a strategy for constructing the E. coli expression
vector pLysN-TIMP2 which expresses TIMP2 protein by using the
expression vector pGE-lysN.
Fig. 11 depicts the expression of TIMP2 protein by performing SDS-
polyacrylamide gel electrophoresis, which used E. coli HMS 174 strain
transformed with the expression vector pLysN-TIMP2.
lane 1 : standard protein marker;
lane 2: total proteins ofE. cø///pGE-lysN transformant;
lane 3: total proteins of is. co///pGE-lysN transformant induced for
the protein expression;
lane 4: precipitate of disrupted E. co///pGE-lysN transformant
induced;
lane 5: supernatant of disrupted E. co// ' /pGE-lysN transformant
induced;
lane 6: total proteins of E. coli transformant
lane 7 : total proteins of E. coli transformant induced;
lane 8 : precipitate of disrupted E. coli transformant induced;
lane 9 : supernatant of disrupted E. coli transformant induced
Description of The Preferred Embodiments
The present invention provides expression vectors which produce
useful foreign proteins as soluble forms by exploiting the structural
characteristics of aminoacyl-tRNA synthetase. All kinds of aminoacyl-
tRNA synthetase genes can be used to prepare expression vectors of the
present invention as fusion partner proteins.
The present invention provides expression vectors which use lysyl-
tRNA synthetase (Lys RS) which has been studied well as a fusion partner.
At that time, Lys RS protein gene can be selected among lys S gene and lys
U gene.
Lys RS protein gene can be obtained by performing polymerase
chain reaction (PCR) which utilized E. coli chromosomal DNA as a
template.
Particularly, lys S gene obtained by the above process has been
inserted into the plasmid vector such as pGEMEX M -l (Promega) so as to
construct the expression vector pGE-lysRS of the present invention (see Fig.
2). E. coli strains proper for the expression have been transformed with
the expression vector pGE-lys RS and induced to express Lys RS protein.
As a result, Lys RS protein was expressed well, to 80% of total soluble
proteins of the host cell. Generally E. coli transformants are cultured at
37°C in order to express Lys RS protein of the present invention. But
soluble proteins are expressed efficiently at low temperature such as 15°C -
30°C which facilitates the increase of the soluble protein ratio.
The present invention provides expression vectors which uses the
N- terminal domain of Lys RS protein as a fusion partner protein.
In order to produce useful foreign proteins effectively, the
expression vector of the present invention contains linker peptide sequence,
tag sequence, protease recognition site, restriction enzyme recognition site
and so forth selectively, in addition to the N-terminal domain of Lys RS
protein. Therefore, fusion proteins expressed by using the expression
vectors can be produced as forms of soluble proteins in the host cells and
separated easily and only the foreign proteins can be purified by digesting
the fusion proteins with specific protease.
Particularly, the N-terminal domain gene of Lys RS protein can be
obtained by performing polymerase chain reaction which utilizes the
expression vector pGE-lysRS as a template. And the N-terminal domain
gene obtained by the above process has been inserted into the plasmid
vector pGEMEX™-ΔNdeI to construct the expression vector pGE-lys N of
the present invention (see Fig. 4).
The E. coli HMS 174 strain was transformed by the expresseion
vector pGE-lysN of the present invention and the transformant has been
deposited with Korea Research Institute of Bioscience and Biotechnology,
Korea, on September 26, 1997 (accession number : KCTC 0388 BP).
The expression vector constructed by the above process has the
following characteristics. The expression vector of the present invention
contains T7 promoter which regulates transcription of the fusion protein.
In addition to T7 promoter, all kinds of promoters which can be used in E.
coli strans, such as tac promoter, λ pL promoter and the like, is available for
the expression vector of the present invention.
The expression vectors of the present invention have been
constructed in order to exploit the N-terminal domain of Lys RS protein as a
fusion partner protein effectively.
In the N-terminal domain of Lys RS protein, helix 1 structure exists.
Since the helix 1 structure is very close to linker peptide, it may prevent
enteropeptidase from digesting fusion protein and affect protein folding.
Ln order to provide the suitable expression vector for the production of
foreign proteins, helix 1 structure can be removed from the expression
vector.
The present invention provides the expression vector removed at the
helix 1 structure to prepare foreign proteins more efficiently.
Preferably, the expression vector of the present invention contains
the N-terminal domain of Lys RS protein which is deleted at the amino acid
residues 1 to 13. Preferably the expression vector also contains the N-
terminal domain of LysRS protein which is deleted at the amino acid
residues 1 to 29.
In addition, preferably the expression vector of the present
invention contains OB fold gene which is involved in folding process of Lys
RS protein. Particularly, the expression vector contains the N-terminal
domain of Lys RS protein which is deleted at the amino acid residues 1 to
65 corresponding to helix structure 1, 2 and 3. The expression vectors
above are suitable for the production of fusion proteins as soluble forms.
The expression vector of the present invention can also contain OB
fold domain gene of other proteins in addition to the N-teπninal domain
gene of Lys RS protein. In detail, OB fold genes found in aspartyl-tRNA
synthetase of yeast, B subunit of thermolabile enterotoxin, berotoxin and
Staphylococcal nuclease can be utilized for the construction of the
expression vector.
The expression vector of the present invention contains linker
peptide connecting fusion partner protein and foreign protein. Particularly,
the amino acid residues 147 to 154 of Lys RS protein is used as a linker
peptide. This linker peptide is very useful since it is protruded on the
protein surface and the length of linker peptide can be controlled according
to the foreign proteins expressed. The expression vector can also contain
useful linker peptides of other proteins in addition to Lys S protein
described above.
The expression vector of the present invention also contains
histidine tag of 6 histidine residues after the above linker peptide. This
histidine tag enables the fusion proteins expressed with the expression
vector to be purified easily. Practically, histidine tagged fusion protein can
be separated and purified easily by using nickel chelating column
chromatography and the like.
In addition to hisitidine tag, polyarginine or consensus biotinylation
sequence can be inserted into the expression vector. Fusion proteins
produced by using the above expression vector can be separated and
purified from various affinity column chromatographies. The tag
sequences described above can be located in any available region of C-
terminus or N-teπninus of the fusion protein.
The expression vector of the present invention contains protease
recognition site in order to separate only foreign protein from fusion protein
expressed and purified. In detail, the expression vector of the present
invention contains enteropeptidase recognition site (DDDDK sequence)
after 6 histidine residues, which enables fusion protein to be separated into
fusion partner protein and foreign protein easily. At that time,
enteropeptidase digests the C-terminus of the above enteropeptidase
recognition site.
In addition, the above protease recognition site can be substituted
with thrombin recognition site (LVPRGS sequence) or Xa factor
recognition site (IEGR sequence) in order to produce foreign proteins
efficiently.
The expression vector of the present invention contains restriction
enzyme sites after the above protease recognition site in order to insert
foreign protein genes conveniently. In detail, the expression vector pGE-
lysN of the present invention contains restriction enzyme recognition sites
Kpnl - BamHI - EcoRI - Sail - Hindlll. All kinds of restriction recognition
sites which is used conveniently in cloning foreign genes can be inserted in
addition to the above reconition sites.
Various foreign proteins which are expressed as inclusion bodies in
E. coli can be prepared as soluble forms efficiently by using the expression
vectors of the present invention.
Particularly, the present invention provides the expression vectors
which uses the N-terminal domain of lysyl-tRNA synthetase (Lys N) in
order to produce human granulocyte and macrophage colony stimulating
factor (GMcsf), human granulocyte colony stimulating factor (Gcsf) and
human tissue inhibitor of metalloprotease (TIMP 2) and the like massively.
The present invention constructs the expression vector which
produces GMcsf protein as a soluble form by using Lys N protein. In
detail, GMcsf gene was obtained by performing polymerase chain reaction
which utilized the expression vector pTRXFUS-GMcsf as a template. And
the GMcsf gene obtained above has been inserted into the expression vector
pGE-lysN to construct the expression vector plysN-GMcsf of the present
invention (see Fig. 5).
In order to examine the availability of Lys N as a fusion partner
protein, GMcsf protein fused with Lys N protein has been compared with
GMcsf protein fused with thioredoxin according to their expression. For
the previous comparison, the expression vector pTRXFUS-GMcsf which
contains GMcsf gene and thioredoxin gene and produces their fusion
protein has been constructed (see Fig. 7).
In addition, the present invention constructs the expression vector
which produces Gcsf protein as a soluble form. In detail, Gcsf gene was
obtained by performing polymerase chain reaction which utilized the
expression vector pTRXFUS-Gcsf as a template. And the Gcsf gene has
been inserted into the expression vector pGE-lysN to construct the
expression vector plysN-Gcsf of the present invention (see Fig. 8).
In addition, the present invention constructs the expression vector
which produces TIMP 2 protein as a soluble protein. In detail, TIMP 2
gene was obtained by performing polymerase chain reaction which utilized
the vecor pGETIMP 2 as a template. And the TIMP 2 gene has been
inserted into the expression vector pGE-lys N to construct the expression
vector pGElysN-TIMP 2 of the present invention (see Fig. 10).
The E. coli strains proper for the expression have been transformed
with the above expression vectors. Transformants have been cultured at
37°C and as results foreign proteins fused with Lys N protien, namely, Lys
N-GMcsf protein, Lys N-Gcsf protein and Lys N-TIMP 2 protein as soluble
forms were expressed at the ratio of 5 - 30% of total soluble proteins (see
Fig. 6, Fig. 9 and Fig. 11). On the other hand, when thioredoxin was used
as a fusion partner protein, fusion protein was expressed as an inclusion
body (see Fig. 7). Therefore, Lys N protein of the present invention is
identified to be a more outstanding fusion partner protein than thioredoxin.
Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on
consideration of this disclosure, may make modification and improvements
within the spirit and scope of the present invention.
Examples
<Example 1> Cloning of lys S gene and construction of the expression
vector pGE-lysRS
In order to clone lys S gene which is necessary to construct the
expression vector of the present invention, polymerase chain reaction (PCR)
was performed, which utilized primer 1 of SEQ ID. NO: 1, primer 2 of SEQ
ID. NO: 2 and E. coli chromosomal DNA as a template (see Sequence
Listing). Amplified lys S gene was digested with resctriction enzyme Ndel
and Hindlll .
For the convenience of cloning process, among two Ndel sites of
plasmid pGEMEX™-l (Promega) Ndel site located on DNA sequence 3251
was removed from the plasmid as shown in Fig. 2 to construct the plasmid
P GEMEX™-ΔNdeI. The plasmid pGEMEX™-ΔNdeI was digested with
restriction enzyme Ndel and Hindlll. The plasmid and PCR product
digested above were electrophoresed on 1% agarose gel and the gel which
contained the DNA fractions appearing at long wavelenth of UV was cut.
Each DNA fraction was eluted from the gel by Jetsorb Kit (GENOMED)
and was ligated.
As a result, the expression vector pGE-lysRS containing lys S gene
was constructed.
<Example 2> Expression of Lys S protein
is. coli HMS 174 strain was transformed with the expression vector
pGE-lysRS constructed in Example 1. The E. coli transformant selected
was inoculated into 1.5 ml of LB medium containing ampicillin 100 μg/ml,
chloramphenicol 30 μg/ml. The transformant was cultured overnight at
37°C, and the growing culture was again inoculated into 50 ml of LB media.
When the concentration of is. coli was 0.5 at OD 60 o, IPTG was added into
the E. coli culture in order to induce the expression of protein and again the
E. coli culture was incubated for more 5 hours. The above culture broth
was centrifuged for 10 minutes at 5,000g, and cell pellet was suspended in
10 ml of phosphate buffered saline (PBS) buffer. The cells were disrupted
and the crude exrtact prepared in the above process was centrifuged for 15
minutes at 15,000g in order to separate supernatant from precipitate. This
precipitate was again suspended in 10 ml of PBS buffer. 28μl of above
each sample was mixed with 7μl of 5 X SDS loading buffer, and boiled for
5 minutes. 10 μl of the above mixture was loaded onto 12% SDS-
polyacrylamide gel, electrophoresed at 120 V and identified with the
protein band by using Coomasie blue dye.
As a result, as is shown in lane 7 of Fig. 3, the expression vector of
the present invention expresses Lys S protein highly at the ratio of 80% of
total soluble proteins (see Fig. 3).
<Example 3> Construction of the expression vector pGE-lysN
In order to construct the expression vector using the N-terminal
domain of Lys S protein as a fusion partner protein, polymerase chain
reaction was performed, which utilized primer 1 of SEQ ID. NO: 1, primer
3 of SEQ ID. NO: 3 and the expression vector pGE-lysRS constructed in
Example 1 as a template (see Sequence Listing).
Amplified gene in the above reaction was digested with restriction
enzyme Ndel and Hindlll and the plasmid vector pGEMEX™-ΔNdeI was
also digested with Ndel and Hindlll. And above products were ligated
after elution (see Fig. 4).
As a result, the expression vector containing the N-terminal domain
gene of Lys S protein was constructed and named as the expression vector
pGE-lysN (accession number : KCTC 0388 BP).
<Example 4> Construction of the expression vector plysN-GMcsf and
expression of fusion protein LysN-GMcsf
In order to express human GMcsf protein as a soluble protein in E.
coli, which has been expressed independently as an inclusion body in E.
coli, GMcsf gene was cloned into the expression vector pGE-lysN of the
present invention (see Fig. 5).
In order to obtain GMcsf gene, PCR was performed by utilizing
primer 4 of SEQ ID. NO: 4, primer 5 of SEQ ID. NO: 5 and the expression
vector pTRXFUS-GMcsf as a template (see Sequence Listing).
Amplified gene by the above reaction was digested with restriction
enzyme Kpnl and Hindlll and the expression vector pGE-lysN of the
present invention was also digested with Kpnl and Hindlll. And the above
products were ligated after elution. As a result, the expression vector
which produces GMcsf protein fused with LysN protein was constructed
and named as the expression vector plysN-GMcsf.
In addition, E. coli was transformed with the expression vector
plysN-GMcsf. As a result, fusion protein was expressed as is shown in
Fig. 6 and the size is 33kDa as is predicted. In addition, most LysN-
GMcsf fusion protein was expressed highly at the ratio of 10% of total
soluble proteins (see Fig. 6).
<Example 5> Construction of the expression vector pTRXFUS-GMcsf
and expression of thioredoxin-GMcsf
In order to examine the availability of Lys N of the present
invention as a fusion partner protein, as a control experiment the effect of
fusion partner protein, thioredoxin on the expression of GMcsf fusion
protein was examined,
The expression vector pTRXFUS-GMcsf which expresses GMcsf
fusion protein was constructed by subcloning GMcsf gene into Kpnl and
BamHI site of the expression vector pTRXFUS using thioredoxin as a
fusion partner protein (see Fig. 7).
When the E. coli transformed with the expression vector of the
present invention was cultured at 37°C, fusion proteins were expressed as
inclusion bodies (see Fig. 7, lane 3).
As a result, thioredoxin was less effective than Lys N protein as a
fusion partner protein.
<Example 6> Construction of the expression vector plysN-Gcsf and
expression of LysN-Gcsf fusion protein
In order to express human Gcsf (granulocyte colony stimulating
factor) as a soluble protein, which has been expressed as an inclusion body
independently in E. coli, Gcsf gene was cloned by performing the same
method as Example 4.
Polymerase chain reaction was performed by utilizing primer 6 of
SEQ ID. NO: 6, primer 7 of SEQ ID. NO : 7 and the plasmid vector
pTRXFUS-Gcsf as a template (see Sequence Listing). Gcsf gene
amplified by the above reaction was phosphorylated by T4 polynucleotide
kinase, and the expression vector pGE-lysN was also digested with EcoRV,
and then treated by CIP (calf intestine phosphatase). The two resultants
were ligated after elution by performing the same method of Example 1.
As a result, the expression vector plysN-Gcsf was constructed which
expresses Gcsf-LysN fusion protein (see Fig. 8).
In addition, fusion protein was expressed by fransfoiming E. coli
with the expression vector plysN-Gcsf. As a result, fusion protein was
expressed as is shown in Fig. 9, and the size of protein is 36kDa as is
predicted. Particularly, Lys-Gcsf fusion protein was expressed as a
soluble protein, and occupied 30% of total soluble proteins.
<Example 7> Construction of the expression vector plysN-TIMP2 and
expression of fusion protein LysN-TIMP2
In order to express human TIMP2 (tissue inhibitor of
metalloprotease 2) as a soluble protein, which has been expressed as an
inclusion body in E.coli, TIMP2 gene was inserted into the expression
vector pGE-lysN of the present invention.
In order to clone TIMP2 gene, polymerase chain reaction was
performed by utilizing primer 8 of SEQ ID. NO: 8, primer 9 of SEQ ID.
NO: 9 and the plasmid vector pGE-TIMP2 (see Sequence Listing).
Amplified TIMP2 gene by the above reaction was digested with restriction
enzyme EcoRV and Hindlll, and the expression vector pGE-lysN of the
present invention was also digested by EcoRV and Hindlll. Above two
resultants were ligated after elution by performing the same method as
Example 1. As a result, the expresion vector plysN-TIMP2 which
expresses fusion protein LysN-TIMP2 was constructed (see Fig. 10).
In addition, fusion protein was expressed by transforming E. coli
with the expression vector plysN-TIMP2. As a result, fusion protein was
expressed as is shown in Fig. 1 1, and the size of protein is 41kDa as is
predicted. Particularly, Lys N-TIMP2 fusion protein was expressed as a
soluble protein, to 5% of total soluble proteins (see Fig. 11, lane 9).
The expression vectors of the present invention expresses lysyl-
tRNA synthetase and foreign proteins fused with the N-terminal domain of
lysyl-tRNA synthetase as soluble forms, which makes their protein
activities maintained. Thus the present invention is outstanding in view of
recombinant DNA technology.
Practically, the expression vector of the present invention expresses
Lys RS protein highly at the ratio of 80% of total soluble proteins, and also
expresses foreign proteins fused with Lys N protein highly at the ratio of 5-
30%. In addition, Lys N protein is more effective than thioredoxin
developed already.
Particularly, the expression vectors of the present invention can
produce foreign protein efficiently, for example GMcsf, Gcsf and TIMP2
proteins. In addition to the previous proteins, the expression vector of the
present invention is useful to produce foreign proteins which are difficult or
impossible to be obtained as active forms and have high molecular weights,
such as antibodies, tissue plasminogen activator and factor VIII.
In addition, the expression vector of the present invention is
constructed to make foreign proteins genes inserted, fusion proteins purified
easily and protease recognition site digested specifically, which facilitates
the production of intact target proteins. Thus the expression vector is very
useful to produce various foreign proteins.
Sequence Listing
(1) General Information
(iii) Number of sequences : 9
(2) Information for SEQ ID NO: 1 :
(i) Sequence Characteristics :
(A) Length : 36 nucleic acids
(B) Type : nucleic acid
(C) Srrandedness : single
(D) Topology : linear
(ii) Molecular type : oligonucleotide
(xi) Sequence Description : SEQ ID NO: 1
GACTACCATA TGTCTGAACA ACACGCACAG GGCGCT 36
(2) Information for SEQ ID NO: 2:
(i) Sequence Characteristics :
(A) Length : 42 nucleic acids
(B) Type : nucleic acid
(C) Strandedness : single
(D) Topology : linear
(ii) Molecular type : oligonucleotide
(xi) Sequence Description : SEQ ID NO: 2
GACTACAAGC TTCTATTATT TTACCGGACG CATCGCCGGG AA 42
(2) Information for SEQ ID NO: 3:
(i) Sequence Characteristics :
(A) Length : 96 nucleic acids
(B) Type : nucleic acid
(C) Strandedness : single
(D) Topology : linear
(ii) Molecular type : oligonucleotide
(xi) Sequence Description : SEQ ID NO: 3
GACTACAAGCTTGTCGACGATATCGGATCC GGTACCCTTGTCATCGTCATCGTGGTGGTG
60 GTGGTGGTGCGGCAGCGGAC GCAGTGCTTTGGTCAG 96
(2) Information for SEQ ID NO: 4:
(i) Sequence Characteristics :
(A) Length : 33 nucleic acids
(B) Type : nucleic acid
(C) Strandedness : single
(D) Topology : linear
(ii) Molecular type : oligonucleotide
(xi) Sequence Description : SEQ ID NO: 4
GACAAGGGTACCGCACCCCGCTCGCCCAGCCCC 33
(2) Information for SEQ ID NO: 5:
(i) Sequence Characteristics :
(E) Length : 33 nucleic acids
(F) Type : nucleic acid
(G) Strandedness : single
(H) Topology : linear
(ii) Molecular type : oligonucleotide
(xi) Sequence Description : SEQ ID NO: 5
GAGCGCAAGC TTTCACTCCT GGACTGGCTC CCAGCA 33
(2) Information for SEQ ID NO: 6:
(i) Sequence Characteristics :
(A) Length : 33 nucleic acids
(B) Type : nucleic acid
(C) Strandedness : single
(D) Topology : linear
(ii) Molecular type : oligonucleotide
(xi) Sequence Description : SEQ ID NO: 6
GACAAGGGTACCAACCCCCCTGGGCCCTGCCAGC 33
(2) Information for SEQ ID NO: 7:
(i) Sequence Characteristics :
(E) Length : 36 nucleic acids
(F) Type : nucleic acid
(G) Strandedness : single
(H) Topology : linear
(ii) Molecular type : oligonucleotide
(xi) Sequence Description : SEQ ID NO: 7
GACAAGAAGC TTTCATCAGG GCTGGGCAAG GTGGCG 36
(2) Information for SEQ ID NO: 8:
(i) Sequence Characteristics :
(I) Length : 33 nucleic acids
(J) Type : nucleic acid
(K) Strandedness : single
(L) Topology : linear
(ii) Molecular type : oligonucleotide
(xi) Sequence Description : SEQ ID NO: 8
GTCATCGATATCTGCAGCTGCTCCCCGGTGCAC 3 3
(2) Information for SEQ ID NO: 9:
(i) Sequence Characteristics :
(A) Length : 36 nucleic acids
(B) Type : nucleic acid
(C) Strandedness : single
(D) Topology : linear
(ii) Molecular type : oligonucleotide
(xi) Sequence Description : SEQ ID NO: 9
GTCATCAAGC TTTCATTATG GGTCCTCGAT GTCGAG 36
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