JPH0260589A - High level production of foreign protein in e. coli using gene ii promoter of bacteriophage m13 - Google Patents

Abstract

PURPOSE: To efficiently produce a foreign protein by synthesizing an expression vector containing a sequence coding a fragment of gene II of bacteriophage M13 and a foreign protein, introducing the vector into Escherichia coli and then culturing the Escherichia coli.

CONSTITUTION: An expression vector containing a fragment of gene II of bacteriophage M13 and a fragment of sequence coding a foreign protein under control of a gene II promoter of the bacteriophage M13 is prepared. Escherichia coli is transformed by using the expression vector and then, the transformant is cultured in proliferation culture medium kept at 20-40°C to efficiently produce the objective foreign protein. The foreign protein is preferably hepatitis B virus X protein and a fragment containing a sequence coding 29 amino acids at N terminal of bacteriophage M13 gene is preferably used as the fragment of the gene II.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the production of bacteriophage 813 using a fragment of gene L (hereinafter also simply referred to as "gene 1") of bacteriophage 813 and a fragment of a sequence encoding a foreign protein. It relates to an expression vector containing the gene L under the control of the promoter.

More particularly, the present invention provides high levels of production of foreign proteins, especially hepatitis B virus X (III3V The HBVX protein produced by the present invention is easy to purify in large quantities and can be used to induce anti-X antibodies in animals. HBV
Both X protein and anti-XfA are useful as diagnostic reagents for HBV infected patients.

1.1]U[ Hepatitis B virus (IIBV) induces acute and chronic hepatitis and has a deep causal relationship with primary hepatocellular carcinoma in humans (B
easily, R.P., et al., Lancet 2 (198
1) 1129-1132).

The II8V genome contains four open reading frames (ORFs) identified by nucleotide sequence analysis, named regions s, c, p and X (Tioll
ais, P.

et al., Nature 317 (1985) 489-4
95). S and C region gene products and their associated antibodies have been detected in the serum of IIBV-infected patients, and 1I
It has been used as a marker for BVi staining. The X gene is the smallest of the four predicted ORFs, with 1541
can encode polypeptides containing specific amino acids. Recently, human hepatocellular carcinoma in which IIBV DNA has been integrated (Moriar-ty, ^1M0, etc., 5cienc
e 227 (1985) 42! J-433) or human hepatocellular carcinoma transfected with dimeric HBV DNA (Chang, C1 et al., EMflOJ, 6 (
The presence of the X protein in several cell lines (1987) 675-680) was demonstrated. Furthermore,
Anti-X antibodies were detected in the serum of hepatocellular carcinoma patients using an X fusion protein synthesized in Escherichia coli (Kay, 30).
et al., f &out; Elfassi, E., et al., below; Mey
ers, M, L, et al., below: Pfaff, E, et al.
However, it is not yet known whether the presence of II B x Ag or anti-X antibody is associated with the growth of hepatocellular carcinoma.

To study the role of the X protein and its relationship to IIBV infection, sufficient amounts of the X protein are available to screen a large number of patient specimens! g. It is important. To date, the use of E. coli as an expression host for both prokaryotic and eukaryotic genes has been most widespread. For this E. coli use, several high-level expression promoters have been developed, such as the lac, trp, tac and phage λPL promoters (Young, J. F. et al., Pro.
c, Nat l, ^cad, sci, (Is^80
(1983) 6105-6109; Courtne
y, M, etc., Proc, Natl, ^cad, sci
, IJsA81 (1984) 669-673). The monoarmored DN^ bacteriophage M13 of Escherichia coli has been widely used in recombinant DNA technology, primarily for the purpose of nucleotide sequencing (Kieny, M, P, et al.
Gene 26 (1983) 91-99; Yani
scb-Perron, C, et al., Gene 33 (1
985) 103-119). However, no prior art discloses the use of the M13 promoter to express foreign genes.

Kay, ^3 etc. rThe HBV flBx g
ene expressionn Escherich
ia coli is recognized by
serafrom hepatitis patien
ts”, EMDOJ, 4 (1985) 1287-12
In 92, the HBVX gene was encoded in E. coli using the LacZ promoter with the eight amino acids of β-galactosidase and the HBV X gene encoded by the 145
It is disclosed that the protein was expressed as a fusion protein containing the following amino acids. Meyer, M.L.

etc., rtlepatitis B virus po
lypeptide X:ex-pression i
n Escherichia coli and id
entifica-tion of 5 pecif
ic anLibodies in 5era
from hepaLitis B virus-
infecjed humansJ+J, Virol,
57 (1986) 101-109, La
The HBV X gene is encoded by the IIBV X gene in E. coli using the cZ promoter.
It is disclosed that the protein was expressed as a fusion protein containing the 8 amino acids of β-galactosidase and the 8 amino acids of β-galactosidase. Elfassi, E., et al. 'Detection
of hepatitis B virus
product uSing an open
read:nB frame Esche-ric
hia coli expression vecto
r'', Proc, Natl 8 ca, sci, Us 8 3 (1986) 2219-2222, bl
disclose that the HBVX gene was expressed in E. coli using the a promoter as a fusion protein containing the 133 amino acids encoded by the HBV X gene and portions of the bacterial ompF and β-galactosidase genes. Pfafr, E, et al., rsynthes
: so the X-protein or hep
atitis B virus in vi-tro
and detection of anti-X a
``antibodies inhuman 5era'', V
irology 158 (1987) 456-46
0 discloses that the 11flVX gene was expressed as an MS-X fusion protein in E. coli using the SP6 promoter.

According to the present invention, the X protein is effectively produced at a level of 10% to 20% of the total cellular protein as a fusion protein with the terminal 29 amino acids of a gene controlled by an ongenic promoter of 813. It was discovered that This expression level is slightly higher than that controlled by the SP6 promoter (PfaH, Ekasa, previously cited) (LncZ promoter and bla
Promoters (Kay, ^9, etc., already mentioned, Meyer,
M, L, et al., supra; Elfassi, E., et al., supra).

11 to 11 The object of the present invention is to provide an expression vector containing a fragment of gene II and a fragment j of a sequence encoding a foreign protein under the control of the gene L promoter of bacteriophage M13. It is also an object of the present invention to provide a method for producing high levels of foreign proteins in E. coli using the expression vectors. The method includes the steps of introducing the expression vector into E. coli and culturing it in a growth medium at a temperature of about 20°C to 40°C.

Region X is one of the four open reading frames of hepatitis B virus and encodes a 154 amino acid polypeptide. A 584 bp Bam1ll! of IIBV DNA containing the main part of open reading frame X, encoding 145 amino acids.
The MII fragment is inserted into the internal M2 site on the gene of bacteriophage bar 13. As a result of this insertion,
A 1n-phase gene L-X fusion protein containing 174 amino acids under the control of the gene 1 promoter is obtained. Derived from the above construct (7)
) 7” Lasmid pHLαX, 59 and pHLX.1
Cells incorporating 2d overproduced the 19-kDa fusion protein in E. coli at levels of 10% to 20% of total cellular protein. The fusion protein was recognized by anti-X antibody.

The gene 11-X fusion protein was used to screen for the presence of anti-X antibodies in the serum of HBV-infected patients, and the anti-gene L-x fusion protein antibody was used to detect the presence of X gene products in 1II3V-infected liver biopsies. Identify existence.

It is therefore an object of the present invention to provide an expression vector containing fragment l~ of gene II and a fragment of a sequence encoding a foreign protein under the control of the gene L promoter of bacteriophage M13.

Another object of the present invention is to use an expression vector containing a fragment of gene II and a fragment of a sequence encoding a foreign protein under the control of the gene L promoter of bacteriophage M13, and to introduce the expression vector into Escherichia coli. An object of the present invention is to provide a method for producing high levels of foreign proteins in E. coli, which comprises a step of culturing in a growth medium at a temperature of 20°C to 40°C.

Another object of the present invention is that fl in patients with +1 B Vi staining
To screen for the presence of B x A g,
An object of the present invention is to provide anti-X antibodies by immunization with the IIBV X protein produced by the present invention.

The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments of the invention based on the accompanying drawings. It is to be understood that this description is illustrative, not restrictive, and that the scope of the invention is defined by the claims.

According to the present invention, an expression vector containing the gene L promoter of phage 813 is used in Escherichia coli to synthesize a large amount of foreign protein, specifically HBV X protein, which accounts for 10% to 20% of the total cellular protein. It is possible to use.

The present invention provides an expression vector containing a fragment of gene II and a fragment of a sequence encoding a foreign protein under the control of the gene L promoter of bacteriophage 813.

The present invention further provides a fragment of a sequence encoding the N-terminal 29 amino acids of gene II and a sequence encoding a foreign protein, and 31! of bacteriophage M13. (Provides an expression vector under the control of the Tenshi L promoter.

The present invention further provides an expression vector comprising a sequence encoding the N-terminal 29 amino acids of gene II and a [Jamtl I-jizgjl fragment of a sequence encoding IIBV X protein] under the control of the gene II promoter of bacteriophage M13. I will provide a.

The present invention also provides a method for high-level production of a foreign protein in E. coli, using the expression vector, introducing the expression vector into E. coli, and culturing in a growth medium at a temperature of about 20 to 40°C. provide.

According to one embodiment, the present invention provides a method for combining fragments on genes and) the IBV X gene with bacteriophage M1.
(a) tlBV
Isolate a fragment of the XR gene from the clone, (b) insert the fragment obtained in (a) above into the Bam111 site of plasmid pOTSII, and (c) further insert bacteriophage M1 containing the gene L promoter.
3. Insert the Ba+nll Ih or ■DN^ fragment derived from 3 into the BamHI site of (b) above, and (d) delete the HindI [I DNA fragment from (c) above to obtain the DNA fragment obtained in (a) above. Gene L
(e) Introduce the plasmid obtained in (d) above into E. coli; (f) Incubate the E. coli obtained in (e) above at a temperature of approximately 20°C to 40°C.
A method for high level production of ll[lVX protein in E. coli is provided that includes culturing in growth medium at 0°C.

The expression vector construction strategy is shown in Figure 1. In FIG. 18, the white frame part P is PL, nutL, nutR, L
R and c [Phage lambda regulatory region containing R85 (ribonome binding site) is shown. The black part is IIBV x31
! Indicates the region encoding the gene. The spots are lac
The Zα gene is shown. The shaded area is the promoter region and N
The M13 gene region including the sequence encoding the terminal 29 amino acid residues is shown. The primary restriction sites for each construct are abbreviations Ba = Bam1l I, h-muen, E
c=Ec.

Rl, )Id-1!1ndlll. Arrows indicate digestion with the indicated restriction enzymes. Arrows alone indicate connections between related fragments. Figure 1B shows pMLX, 9 and pM
The number below the sequence indicates the number of amino acids of the encoded polypeptide, indicating the LαX, 59-nocloning junction sequence and translation initiation region.

In this implementation RtM, P
Plasmid pOTS1 (De
vare-S, G, et al., Ce1l 36 (1984
) 43-49) were selected for HBV X gene expression (Young, J, E.

et al., previously published; Courtney+M et al., previously published; Fergu
son, B. et al., 5science 224 (1984
) 1343-1346).

The main part of the sequence encoding the X region (codons 10 to 154
) of 584 bp including b! +1 ■−Lee ■DN^ fragment was converted into HBV clone pTWL1 (Lee, Y,
)1. W. et al., Proc., Natl.

Sci, Counc, B. ROC9 (1985) 11
0-118; Lo, S, J, Kasa, Biochem, B
iophys, Res, Commun, 129 (1
985) 797-803) and inserted into the unique Bam1 (I site) of pOTSl. A plasmid containing the sequence encoding the X protein under the control of the λPL promoter was isolated and named pHLX.9. However, In pHLX.9 cells, one of the X gene products
No significant band measured at 16 kDa representing 45 amino acids was detected (Figure 2, lanes 5 and 6).
.

1. A 683-bp Ba derived from M13mp18 that sequentially encodes the α-peptide sequence of acZ, a transcriptional regulatory element, and a 29-amino acid sequence encoding a gene.
mtlIjllll DNA fragment, pMLX
, 9(') was inserted into the Bam1l 1 site. The resulting plasmid pHLαX, 59, contains two new ORFs.
, namely, a LacZα-peptide sequence (157 amino acid residues) controlled by the λPL promoter and a gene-x fusion sequence (174 amino acid residues) controlled by the gene L promoter.
and was played.

Next, the 1.9 kb tlindlD containing the complete λPL promoter and the sequence encoding the N-terminus of the α-peptide
The N^ fragment was deleted from pMLαx,59 and the plasmid pHLX. I got 12d.

As shown in lanes 3.4 and 7 of Figure 2, pMLαX
, 59 and pHLX. 12d both synthesized a 19 kDa protein. Judging from the relative staining density of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this protein accounts for 1% of the total cellular protein.
This corresponds to 0% to 20%. The presence of X protein in this 19-kDa polypeptide was demonstrated by immunoblot I analysis using antibodies against synthetic X peptide (Mori et al.
This was confirmed by the MS2/X fusion protein (Pfaff, E., et al., previously cited) or the MS2/X fusion protein (Pfaff, E., et al., previously cited).

Plasmid pOTS1, pMLαX, 59* TahapH
LX. N5151 (λc I Ta20
5) Cells are grown at 28°C (lanes 1.3 and 5).
) or grown overnight at 28°C and induced for 1 hour at 42°C (lanes 2.4 and 6 > incorporated pHLX.12d) [l3101i cells were grown without induction at 42°C.
Grown at 7°C. Total cellular proteins of cells that had integrated various plasmids were analyzed by 0.1% 5DS-15% PAGE.
and stained with Coomassie brilliant blue.

The results are shown in Figure 2, lanes 1 and 2 indicate plasmid p
Analysis of total cellular protein in cells that have integrated OTS1, lanes 3 and 4 show total cellular protein in cells that have integrated plasmid pMLαx,59, lanes 5 and 6.
indicates total cellular protein of cells that have integrated plasmid pMLLX.9, lane 7 shows plasmid pHLX.9. 12
Total cellular protein of cells HB 10II incorporating d is shown. The numbers at both ends are protein standard (x 10-'),
Phosphorylase 94000, bovine serum albumin 670
00, ovalbumin 43000, carbonic anhydrase 30000, soybean trypsin inhibitor 200
00, α-lactalbumin 14400.

pHLX. II 8101 cells containing 12d were first treated with Triton X-1
00, and rsurfaceBoteins o
f M co lasma h one
umoniae identi-fied from
an Escbericl+ia coli-exp
reaction plasmid 1ibrarH1Pr
oc, Natl, Sci, Counc, B, ROC9 (
1985) K11nke disclosed in 110-118
Extracted using urea methods such as rL, M, and Q. Proteins in the final 7M urea solution were analyzed by SDS-PAGE (Laemmli,
U. et al., Nature 227 (1970) 6
80-685). 19-kDa gene-■
Gel sections containing the -X fusion protein were homogenized and used as antigens to immunize rabbits.

To test the specificity of the immunized rabbit serum, synthetic
Western blot tests using antibodies against the peptides were performed simultaneously. According to these results, as shown in Figure 3, the induced antibody of the present invention recognized the 19-k Da 3fi gene LX fusion protein similarly to the anti-synthetic X-peptide antibody, but it recognized the bacterial protein in the pond. It turns out that it doesn't recognize it.

Immunoplot of plasmids pUc19, pOTSl, pHLX.X. 9MariLtpMLαX.

Total cellular protein of N5151 (λc Its857) cells containing 59 was separated in 0.1% 5DS-15% P^O as described above. Dissolved protein in Towbin
, tl, etc. (Proc.

Natl, ^cad, sc i, USA76 (19
79) 4350-4354) and Gershoni
, J.M., et al. (8 na1.Biochem, 124
(1982) 396-405) and electrophoretically transferred to a nitrocellulose sheet at a ratio of 1:1.
000 dilution of (a>rabbit control serum, (b) M
Rabbit antiserum against S2/X fusion protein, and (c
) Hybridized with rabbit antiserum against synthetic X peptide. Antigen-bound antibodies were detected with a peroxidase-conjugated goat antibody to rabbit immunoglobulin, and the complex was
．． Color was developed with 3°-diaminobenzidine. 3rd result
As shown in the figure.

Lane 1 does not show analysis of pUc19 X gene expression. Lanes 2 and 4 are pHLX. 9 is shown. Lane 3 shows analysis of pMLαX,59. Lane 5 is pOTSl
shows the analysis of The protein Mr standard identified in FIG. 2 is shown on the far right (K=kDa).

pHLX. Cellular proteins of HDIOI cells containing 12d were separated by 0.1% 5DS-15% PAGE. 1.5
(a) rabbit control serum, (b) synthetic X at 0 dilution
Rabbit antiserum against peptide and (c) gene 11-
Western plots similar to those described above were performed using rabbit antiserum against the X fusion protein. The results are shown in Figure 4.

Lane 1 shows total protein reactivity. Lane 2 is pH
LX. Figure 2 shows the reactivity of proteins extracted with 7M urea from II B 101 cells containing 12cl. Lane 3 is the negative control, N51 containing pOTSl.
51 (λc I Ta205) shows the total protein reactivity of cells.

The same protein Mr standard as in Figure 2 is shown on the far right (
K; kDa).

These results indicate that the gene L promoter of M13 can act as a strong promoter for producing large amounts of gene L-x fusion protein. As far as we know, this is the first step in M1 for expressing foreign genes in E. coli.
3 genes [This is the first successful example using a promoter.

The U-X fusion protein can be used to screen for the presence of anti-X antibodies in the serum of IIBV infected patients. Anti-gene 11-X fusion protein antibodies are also useful for detecting and identifying the presence of the X gene product in 1Ir3V infected liver biopsies.

The invention has been described above in terms of specific embodiments. However, it will be apparent to those skilled in the art that many variations, modifications and variations of these descriptions are possible.

Explanatory diagram of the construction strategy and structure of pM[,X,12d, 2nd
The figure is a photograph of the particle structure obtained as a result of analyzing the total cellular protein of cells that have incorporated various plasmids by 0.1% 5OS-15% PAGE and staining with Coomassie brilliant blue. A photograph of the particle structure obtained as a result of immunoplot analysis of expression, FIG.
HLX. 12d is an explanatory diagram (photograph) showing the reactivity of the X gene product with antiserum against synthetic X bebutide and gene 11-X fusion protein.

Claims (26)

[Claims] (1) An expression vector comprising a fragment of the gene II of bacteriophage M13 and a fragment of a sequence encoding a foreign protein under the control of the gene II promoter of bacteriophage M13. (2) Claim 1, wherein the fragment of the gene \II\ contains a sequence encoding the N-terminal 29 amino acids of the gene \II\ of bacteriophage M13.
The expression vector described in. (3) The foreign protein is hepatitis B virus (HBV).
) The expression vector according to claim 1, which is an X protein. (4) The sequence encoding the foreign protein is HBVX
\Bam\H I -\Bg of the protein-coding sequence
The expression vector according to claim 1, which is a l\II fragment. (5) The sequence encoding the N-terminal 29 amino acids of the bacteriophage M13 gene ¥II and the sequence encoding the HBVX protein ¥Bam¥H I -¥Bgl¥
2 fragment under the control of the bacteriophage M13 gene\II\ promoter. (6) Plasmid pMLX. The expression vector according to claim 5, characterized in that it is 12d. (7) Using the expression vector according to any one of claims 1, 2, 3, 4, 5, or 6, introducing the expression vector into E. coli and growing it in a growth medium at a temperature of about 20°C to 40°C. A method for producing a high level of foreign protein in Escherichia coli, the method comprising culturing. (8) The method according to claim 7, wherein the foreign protein is an HBVX protein. (9) The method according to claim 7, wherein the E. coli into which the expression vector has been integrated is cultured in Luria broth at 37°C supplemented with ampicillin. 10. The method of claim 7, wherein the foreign protein produced is at a level of 10% to 20% of total cellular protein. (11) The method according to claim 10, wherein the foreign protein is a gene\II\fusion protein. (12) The gene\II\fusion protein is gene\II\
-X fusion protein, the method according to claim 11. (13) The method according to claim 11, wherein the gene\II\ fusion protein contains the N-terminal 29 amino acids of gene\II\ of bacteriophage M13. (14) The method according to claim 12, wherein the gene\II\ fusion protein contains the N-terminal 29 amino acids of gene\II\ of bacteriophage M13. (15) The method according to claim 12, wherein the gene\II\-X fusion protein contains the N-terminal 29 amino acids of gene\II\ and the 145 amino acids of HBVX protein. (16) Use of the HBVX protein produced by the method according to claim 7 for inducing anti-X antibodies in animals. (17) Use of the HBVX protein and the anti-X antibody according to claim 16 as a diagnostic reagent for HBV-infected patients. (18) A fragment of gene II of bacteriophage M13 and the HBVX gene were added to bacteriophage M
Using a plasmid containing 13 genes under the control of the II promoter as an expression vector, (a) isolate a fragment of the HBVX gene from the clone, (b) insert the fragment obtained in (a) above into the plasmid pOTS1. (c) \Bam\H I -\Bgl derived from bacteriophage M13, which further contains the gene \II\ promoter;
Insert the ¥II DNA fragment into the ¥Bam¥HI site of (b), and (d) insert the ¥Hind¥IIID fragment from (c) above.
Create a plasmid by deleting the NA fragment and containing the fragment obtained in (a) above under the control of the gene\II\ promoter, and (e) introduce the plasmid obtained in (d) above into E. coli. (f) The E. coli obtained in (e) above is heated to a temperature of approximately 20°C to 4°C.
A method for producing high levels of HBVX protein in E. coli, the method comprising the step of culturing in a growth medium at 0°C. (19) The expression vector is bacteriophage M1
The sequence encoding the N-terminal 29 amino acids of gene 3 II and the HBVX gene BamH I -Bg
19. The method according to claim 18, characterized in that the method contains the l\II fragment under the control of the gene\II\ promoter of bacteriophage M13. (20) The expression vector is plasmid pHLX. 12
20. The method according to claim 19, characterized in that: d. (21) The E. coli into which the expression vector has been incorporated,
The method according to claim 18, characterized in that the culturing is carried out in Luria broth at 37°C supplemented with ampicillin. (22) The method of claim 18, wherein the HBVX protein produced is at a level of 10% to 20% of total cellular protein. (23) The method according to claim 22, wherein the HBVX protein is a gene\II\-X fusion protein. (24) The method according to claim 23, wherein the gene\II\-X fusion protein contains the N-terminal 29 amino acids of the gene, \II\, and the 145 amino acids of the HBVX protein. . (25) Use of the HBVX protein produced by the method according to claim 18 for inducing anti-X antibodies in animals. (26) Use of the HBVX protein and the anti-X antibody according to claim 25 as a diagnostic reagent for HBV-infected patients.