JPH0411892A - Protein of streptokinases, corresponding gene, corresponding plasmid recombinant, corresponding character transformant and production thereof - Google Patents

Abstract

NEW MATERIAL:A chemical synthetic gene containing a base sequence capable of coding streptokinase of an amino acid sequence expressed by the formula. USE:Production of streptokinase, etc. PREPARATION:The aforementioned chemical synthetic gene is divided into several oligonucleotide fragments and chemical synthesis thereof is respectively carried out. The resultant fragments are then linked to construct the objective synthetic gene.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel chemically synthesized gene containing a nucleotide sequence encoding the amino acid sequence of streptokinase, its corresponding plasmid recombinants, its corresponding transformants, and its culture. a method for producing streptokinase, a novel streptokinase derivative protein having a modified amino acid sequence, a chemically synthesized gene containing the base sequence that feeds the protein, a recombinant plasmid containing the gene, and a recombinant protein containing the gene. The present invention relates to a transformed transformant and a method for producing a streptokinase derivative protein by culturing the transformant.

BACKGROUND OF THE INVENTION Streptokinase is a protein with a molecular weight of 47,000 that is produced and secreted by hemolytic streptococci and has a thrombolytic effect. The amino acid sequence of the protein is expressed by the following formula (1
), and its producing bacterium, Streptococcus excimilis [5treptococcus equis
imilis H46A (Group C)] DN
From A, the entire base sequence has also been determined.

The above-mentioned streptokinase is also already in clinical use as a thrombolytic agent in Western countries.
It is widely used in the treatment of patients with acute myocardial infarction, etc., and its effectiveness has been sufficiently proven.

However, when streptokinase is produced by culturing the above-mentioned source bacteria, the culture solution contains several extracellular secretions other than streptokinase, and many of these are or have the potential to be toxic to humans. In order to prepare streptokinase for clinical use from the above-mentioned culture solution, it is necessary to perform multi-step purification operations with great care. In addition, streptokinase obtained by culturing the above-mentioned source cells has been reported to cause various shock symptoms due to its antigenicity. Development is required in this industry. Furthermore, it is desired that streptokinase has improved stability in blood and improved ability to specifically dissolve blood clots during its clinical use.

Problems to be Solved by the Invention The object of the present invention is to provide a production method using genetic recombination technology that allows streptokinase to be easily produced in large quantities with high purity, and in particular, to provide a production method using E. coli for this method. A chemically synthesized gene for streptokinase that employs a nucleotide codon that facilitates the production of streptokinase, a corresponding plasmid recombinant into which the gene is inserted (streptokinase expression vector), and a host cell transformed with the fast recombinant (streptokinase expression vector). The purpose of the present invention is to provide a transformant) and its culture production technology.

Furthermore, the purpose of the present invention is to improve the original activity of streptokinase,
Provided is a new streptokinase derivative protein which particularly has thrombolytic activity and has reduced binding to streptokinase-specific antibodies as well as production of specific antibodies (antigenicity) when administered. There is a particular thing.

Another object of the present invention is to provide the above derivative protein which has improved stability in blood and further improved thrombolytic properties.

Furthermore, the present invention provides a method for producing the above-mentioned new streptokinase derivative protein using genetic recombination technology that enables easy production of the protein in high purity and in large quantities, and in particular, a method for producing the new streptokinase derivative protein using Escherichia coli. A chemically synthesized gene that employs a nucleotide codon that is intended to be a chemically synthesized gene, a corresponding plasmid recombinant into which the gene is inserted (expression vector), a host cell (transformant) transformed with several recombinants, and the above protein obtained by culturing the same. The purpose is also to provide manufacturing technology.

Means for Solving the Problems According to the present invention, a chemically synthesized gene encoding a natural streptokinase having the amino acid sequence of the following formula (1), a corresponding plasmid recombinant into which the gene is inserted (a natural streptokinase) Kinase expression vector), a corresponding transformant transformed with several recombinants, and a method for producing native streptokinase by culturing the transformant are provided.

Formula (1): %Formula% Gly-Asp-Thr-11e-Thr-5er-G
ln-Glu-Leu-Leu-Ala-Gln-Al
a-Gln-5er-11e-Leu-Asn-Lys
-Asn-His-Pro-Gly-Tyr-Thr-
11e-Tyr-Glu-Arg-Asp-5er-5
er-11e-Val-Thr-His-Asp-As
n-Asp-11e-Phe-Arg-Thr-I 1
e-Leu-Pro-Met-Asp-Gln-Glu
-Phe-Thr-Tyr-Arg-Val-Lys-
Asn-Arg-Glu-Gln-Ala-Tyr-A
rg-11e-Asn-Lys-Lys-5er-Gl
y-Leu-Asn-Glu-Glu-11e-Asn
-Asn-Thr-Asp-Leu-11e-5er-
Glu-Lys-Tyr-Tyr-Val-Leu-L
ys-Lys-Gly-Glu-Lys-Pro-Ty
r-Asp-Pro-Phe-Asp-Arg-Ser
-His-Leu-Lys-Leu-Phe-Thr-
11e-Lys-Tyr-Val-Asp-Val-A
sp-Thr-Ala-Glu-Leu-Leu-Ly
s-5er-Glu-Gln-Leu-Leu-Thr
-Ala-Ser-Glu-Arg-Asn-Leu-
Asp-Pbe-Arg-Asp-Leu-Tyr-A
sp-Pro-Arg-Asp-Lys-Ala-Ly
s-Leu-Leu-Tyr-Asn-Asn-Leu
-Asp-Ala-Phe-Gly-11e-Met-
Asp-Tyr-Thr-Leu-Thr-Gly-L
ys-Val-Glu-Asp-Asn-His-As
p-Asp-Thr-Asn-Arg-I 1e-I
1e-Thr-Val-Tyr-Met-Gly-Ly
s-Arg-Pro-Glu-Gly-Glu-Asn
-Ala-5er-Tyr-His-Leu-A la
-Tyr-Asp-Lys-Asp-Arg-Tyr-
Thr-Glu-Glu-Glu-Arg-Glu-V
al-Tyr-3er-Tyr-Leu-Arg-Ty
r-Thr-Gly-Thr-Pro-I 1e-Pr
o-Asp-Asn-Pro-Asn-Asp-Lys Also according to the present invention, has streptokinase activity,
C-terminal side 37 of the amino acid sequence shown by the above formula (1) of natural streptokinase (natural streptokinase)
A streptokinase derivative protein is provided that has an altered amino acid sequence in which the third and subsequent positions are deleted, and in which at least one amino acid is deleted, substituted, or inserted.

Amino acid sequences and abbreviations for each amino acid residue, base sequences, nucleobases, and other abbreviations in the above formula (1) and the following specification are IUPAC-
The IUB regulations or symbols commonly used in the field shall be followed, examples of which are listed below.

Ala...Alanine Arg...Arginine A
sn...Asparagine Asp...Aspartic acid Cys...Cystine Gln...Glutamine G
lu...Glutamic acid Gly...Glycine His
... Histidine Ile ... Isoleucine Leu
...Leucine Lys...Lysine Net...
Methionine Phe・・・Phenylalanine Pro・
...Proline Set...Serine Thr...Threonine Trp...Tryptophan Tyr...
Tyrosine Val...Valine A...Adenine T...Thymine G...Guanine C...Cytosine However, the position of each amino acid residue in the amino acid sequence is , deletions and insertions, all shall be displayed according to the amino acid sequence of formula (1) above.

Preferred examples of streptokinase derivative proteins having modified amino acid sequences according to the present invention are as follows.

(1) A polypeptide in which the amino acid sequence of natural streptokinase (shown by the above formula (1)) is deleted from position 373 on the C-terminal side.

(2) In addition to deleting the C-terminal amino acid sequence starting from position 373 of natural streptokinase, at least 4
A polypeptide in which Guy at position 68 is deleted from Arg at position 5.

(3) In addition to deleting the C-terminal position 373 and beyond of the amino acid sequence of natural streptokinase, at least one
A polypeptide in which Phe at position 18 is deleted or substituted with another amino acid residue.

(4) In addition to deleting the C-terminal position 373 and beyond of the amino acid sequence of natural streptokinase, at least 2
A polypeptide in which Lys at position 56 and Lys at position 257 are deleted or substituted with other amino acid residues.

(5) In addition to deleting the C-terminal position 373 and beyond of the amino acid sequence of natural streptokinase, at least 2
A polypeptide in which other amino acid residues are inserted after each of Lys at position 56 and Lys at position 257.

In the above modified amino acid sequence, the other amino acid residues that can be substituted or inserted may be any residues that constitute streptokinase proteins, and are preferably selected from α-amino acids that constitute human protein. Specific examples include Pro, Gin, Thr, Ser
, His etc. More specifically, the amino acid residues for substitution at position 256 are Gin,
Thr Hls etc., and amino acid residues for substitution at position 257 include Pro, Gin, Ser.
, His, and Pro as amino acid residues inserted after positions 256 and 257, respectively.

Further, according to the present invention, a chemically synthesized gene containing a base sequence encoding a streptokinase derivative protein having the above-mentioned modified amino acid sequence, and a corresponding plasmid recombinant into which the gene is inserted (streptokinase derivative protein expression vector) , a transformant transformed by several recombinants, and a method for producing a streptokinase derivative protein by culturing the transformant.

Hereinafter, chemically synthesized genes, expression vectors, and transformants used for the production technology of the natural streptokinase of the present invention, the streptokinase derivative protein of the present invention, and the production technology thereof will be described in detail.

The base sequence of the chemically synthesized gene encoding the amino acid sequence of the natural streptokinase of the present invention is expressed by the formula (1
) can be set in various ways based on the amino acid sequence, in which case it is preferable to adopt the following criteria.

1) Select trinucleotide codons that are frequently used in host cells, such as E. coli.

2) A specific restriction enzyme recognition site is provided within the set base sequence and at both ends thereof, and the site can be arbitrarily manipulated to facilitate linkage with base sequences of other genes, insertion into plasmid vectors, etc. make it a thing.

3) When assembling and linking chemically synthesized genes (DNA fragments), ensure that connections different from the intended connection state do not occur or are kept to a minimum.

4) Prevent any inconvenient sequences, such as transcription termination signals, from occurring in the designed base sequence.

5) Restriction enzyme recognition sites are provided at appropriate positions to facilitate subsequent gene modification.

A preferred example of the chemically synthesized gene (nucleotide sequence) of natural streptokinase designed according to the above is shown in the following formula (2) together with the corresponding amino acid sequence.

Formula (2): % formula % AAA CCG TTCGCT ACT GACTCT
GGCGCT ATGTTT GGCAAG CGA
TGA CTG AGA CCG CGA TACL
ys-Pro-Phe-Ala-Thr-Asp-3e
r-Gly-Ala-Met-TCT CAT AA
A CTCGAG AAG GCA GAT
CTG CTGAGA GTA TTT GA
G CTCTTCCGT CTA GACGAC
5er-His-Lys-Leu-Glu-Lys-A
la-Asp-Leu-Leu-AAA GCA AT
CCAG GAA CAG CTG ATC5CT A
ACTTT CGT TAG GTCCTT GTC
GACTAG CGA TTGLys-Ala-11
e-Gin-Glu-Gin-Leu-11e-Ala
-Asn-GTA CAT TCT AACGACG
ACTACTTT GAG GTACAT GTA A
GA TTG CTG CTG ATG AAA CT
CCATVal-His-Ser-Asn-Asp-A
sp-Tyr-Phe-Glu-Val-ATCGAC
TTCGCT AGCGACGCT ACT ATCA
CCTAG CTG AAG CGA TCG CTG
CGA TGA TAG TGGI 1e-Asp-
Phe-Ala-5er-Asp-Ala-Thr-I
1e-Thr-GACCGT AACGGGCAAA
GTA TACTTCGCT GACCTG GC
A TTG CCG TTT CAT ATG A
AG CGA CTGAsp-Arg-Asn-G
ly-Lys-Val-Tyr-Phe-Ala-As
p-AAA GACGGT TCT GTA A
CT CTT CCG ACT CAATTT
CTG CCA AGA CAT TGA
GAA GGCTGA GTTLys-Asp-
Gly-5er-Val-Thr-Leu-Pro-T
hr-Gln-CCG GTA CAG GAA T
TT CTG CTG TCT GGCCATGG
CCAT GTCCTT AAA GACGACAGA
CCG GTAPro-Val-Gln-Glu-P
he-Leu-Leu-5er-Gly-His-GT
A CGCGTT CGCCCG TACAAA
GAA AAA CCGCAT GCG CAA G
CG GGCATG TTT CTT TTT G
GCVal-Arg-Val-Arg-Pro-Tyr
-Lys-Glu-Lys-Pro-ATCCAG A
ACCAG GCT AAA TCT GTT GAC
GTATAG GTCTTG GTCCGA TTT
AGA CAA CTG CATI 1e-Gln-A
sn-Gln-Ala-Lys-5er-Val-As
p-Val-GAA TACACCGTT CAG T
TCACCCCCG CTG AACCTT ATG T
GG CAA GTCAAG TGG GGCGACT
TGGlu-Tyr-Thr-Val-Gln-Phe
-Thr-Pro-Leu-Asn-CCA GACG
AT GACTTCCGCCCG GGT CTG A
AAGGT CTG CTA CTG AAG GCG
GGCCCA GACTTTPro-Asp-Asp
-Asp-Phe-Arg-Pro-Gly-Leu-
Lys-GACACT AAA CTG CTG AA
A ACCCTG GCT ATCCTG TGA T
TT GACGACTTT TGG GACCGA T
AGAsp-Thr-Lys-Leu-Leu-Lys
-Thr-Leu-Ala-I 1e-GGT GAC
ACCATCACT TCT CAG GAG CTC
CTGCCA CTG TGG TAG TGA AG
A GTCCTCGAG GACGly-Asp-Th
r-11e-Thr-5er-Gln-Glu-Leu
-Leu-GCT CAG GCA CAG TCT
ATCCTG AACAAA AACCGA GTCC
GT GTCAGA TAG GACTTG TTT
TTGAla-Gin-Ala-Gln-5er-I
1e-Leu-Asn-Lys-Asn-CAT CC
G GGCTACACT ATCTACGAA CGC
GACGTA GGCCCG ATG TGA TAG
ATG CTT GCG CTGHis-Pro-G
ly-Tyr-Thr-I 1e-Tyr-Glu-A
rg-Asp-TCT TCCATCGTA ACCC
AT GACAACGACATCAGA AGG TA
G CAT TGG GTA CTG TTG CTG
TAGSer-5er-I 1e-Val-Thr-
His-Asp-Asn-Asp-I 1e-TTCC
GT ACCATT CTG CCG ATG GAC
CAG GAAAAG GCA TGG TAA GA
CGGCTACCTG GTCCTTPhe-Arg-
Thr-I 1e-Leu-Pro-Met-Asp-
Gin-Glu -TTT ACT TACCGT
GTT AAA AACCGCGAA CA
AAAA TGA ATG GCA CAA
TTT TTG GCG CTT GTTP
he-Thr-Tyr-Arg-Val-Lys-As
n-Arg-Glu-Gln-GCT TACCGT
ATCAAT AAA AAA TCCGG
T CTGCGA ATG GCA TAG
TTA TTT TTT AGG CCA
GACAla-Tyr-Arg-11e-Asn-L
ys-Lys-5er-Gly-Leu-AAT G
AA GAG ATT AACAACACT
GACCTG ATCTTA CTT CTCT
AA TTG TTG TGA CTG G
ACTAGAsn-Glu-Glu-11e-Asn-
Asn-Thr-Asp-Leu-11e-TCT
GAA AAG TACTAC GTA CTG
AAA AAA GGTAGA CTT TTC
ATG ATG CAT GACTTT TT
T CCASer-Glu-Lys-Tyr-Tyr
-Val-Leu-Lys-Lys-Gly-GAG
AAG CCG TAT GACCCG TT
CGAT CGT TCTCTCTTTCGGCAT
A CTG GGCAAG CTA GCA
AGAGlu-Lys-Pro-Tyr-Asp-P
ro-Phe-Asp-Arg-5et-CAT C
TG AAA CTG TTCACCATCAA
A TACGTTGTA GACTTT GAC
AAG TGG TAG TTT ATG CA
AHis-Leu-Lys-Leu-Phe-Thr-
I 1e-Lys-Tyr-Val-GACGTCGA
T ACCAACGAA TTA CTG AAG
TCTCTG CAG CTA TGG TTG
CTT AAT GACTTCAGAAsp-
Val-Asp-Thr-Asn-Glu-Leu-L
eu-Lys-5er-GAG CAG CTG
CTG ACCGCT TCCGAA CGT
AATCTCGTCGACGACTGG CGA
AGG CTT GCA TTAGlu-Gln-
Leu-Leu-Thr-Ala-5er-Glu-A
rg-Asn-CTG GACTTCCGCGAT
CTG TACGACCCG CGTGACCTG
AAG GCG CTA GACATG C
TG GGCGCALeu-Asp-Phe-Arg
-Asp-Leu-Tyr-Asp-Pro-Arg-
GACAAA GCT AAA CTG CT
G TACAACAACCTGCTG TTT C
GA TTT GACGACATG TTG
TTG GACAsp-Lys-Ala-Lys-L
eu-Leu-Tyr-Asn-Asn-Leu-GA
T GCT TTCGGT ATCATG G
AG TACACCCTGCTA CGA AA
G CCA TAG TACCTG ATG
TGG GACAsp-Ala-Phe-Gly-
11e-Met-Asp-Tyr-Thr-Leu-A
CT GGT AAA GTA GAA G
ACAACCAT GACGACTGA CCA
TTT CAT CTT CTG TTG
GTA CTG CTGThr-Gly-Lys
-Val-Glu-Asp-Asn-His-Asp-
Asp-ACCAACCGT ATCATCACCG
TA TACATG GGCTGG TTG
GCA TAG TAG TGG CAT
ATG TACCCG Thr-Asn-Arg-Il
e-11e-Thr-Val-Tyr-Met-Gly
-AAA CGT CCG GAA GGT
GAA AAT GCA TCT TACT
TT GCA GGCCTT CCA CTT
TTA CGT AGA ATGLys-A
rg-Pro-Glu-Gly-Glu-Asn-Al
a-5er-Tyr-CAT CTG GCA
TAT GACAAA GACCGT TACA
CCGTA GACCGT ATA CTG
TTT CTG GCA ATG TGGHi
s-Leu-Ala-Tyr-Asp-Lys-Asp
-Arg-Tyr-Thr-GAA GAA GA
A CGT GAA GTT TACTCT
TACCTGCTT CTT CTT GCA
CTT CAA ATG AGA ATG
GACGlu-Glu-Glu-Arg-Glu-
Val-Tyr-Ser-Tyr-Leu-CGCTA
T ACT GGT ACCCCT ATCC
CG GAT AACGCG ATA TGA
CCA TGG GGA TAG GGC
CTA TTGArg-Tyr-Thr-Gly-T
hr-Pro-11e-Pro-Asp-Asn-CC
G AACGAT AAA 3 “GGCTTG
CTA TTT 5'Pro-Asn-Asp-
Lys The base sequence represented by the above formula (2) is shown as a mutually complementary double-stranded DNA sequence encoding the amino acid sequence of the natural streptokinase of the above formula (1). - Full-stranded DNA sequences are also encompassed by the invention.

Furthermore, the base sequence of formula (2) above is designed by preferentially selecting and combining codons that are frequently used by E. coli, taking into account the use of E. coli as a host cell, but the gene of the present invention is limited to this. Not allowed.

Furthermore, the host cell used for flJ in the present invention is not limited to E. coli, as described below, and therefore, depending on the host cell used, it is possible to select and combine codons that are frequently used by the cell. That is, the base sequence in the desired gene is
As long as it has the same genetic information as the nucleotide sequence shown in formula (2) above, that is, it contains the nucleotide sequence encoding the amino acid sequence of streptokinase, and as long as the streptokinase can be expressed and produced by genetic recombination technology, any It may be a chemically synthesized gene (based on the nucleotide sequence of the above formula (2), modification or modification such as alteration, deletion, addition, etc. of the nucleotide sequence is possible. Modification or modification of the secondary nucleotide sequence) For example, the use of a genetic code that encodes the same amino acid sequence encoded by the base sequence of formula (2) above, or actually translating the obtained base sequence into an appropriate vector. This includes the addition of various restriction enzyme recognition sites for linking with various regulatory factors such as promoters necessary for expressing the target protein in microorganisms by inserting it into the microorganism. When constructing the desired expression vector using genetic engineering techniques, the promoter, Shine-Dalgarno sequence (Shine-Dalgarno sequence)
It is necessary to appropriately link various regulatory factors such as liposome binding sites such as o sequence and SD sequence, initiation codon of protein synthesis, termination codon, etc., but operations such as cutting and joining of each base sequence are difficult. Since this is carried out using restriction enzymes, it is necessary to add appropriate restriction enzyme recognition sites before and after the desired gene. An example of such a gene to which a suitable restriction enzyme recognition site is added is shown in the following formula (3).

This has specific restriction enzyme recognition sites added before and after the base sequence of formula (2) above for linking with various regulatory factors such as promoters necessary for the expression of the target protein encoded by the base sequence. It is suitable for subsequent construction of expression vectors. The restriction enzyme recognition site present in the base sequence of formula (3) is shown in the following formula (4). Of course, the restriction enzyme recognition site shown in formula (4) is only an example, and the site to be present in the gene of the present invention can be changed and selected as appropriate depending on the type of expression vector to be constructed.

Formula (3): % formula % GACCGT AACGGGCAAA GTA TACT
TCGCT GACCTG GCA TTG CCG
TTT CAT ATG AAG CGA CTGC
AT CCG GGCTACACT ATCTACGA
A CGCGACGTA GGCCCG ATG TG
A TAG ATG CTT GCG CTGAAA
GACGGT TCT GTA ACT CTT
CCG ACT CAATTT CTG C
CA AGA CAT TGA GAA GGC
TGA GTTTCT TCCATCGTA ACC
CAT GACAACGACATCAGA AGG T
AG CAT TGG GTA CTG TTG CT
G TAGCCG GTA CAG GAA TTT
CTG CTG TCT GGCCATGGCC
AT GTCCTT AAA GACGACAGA C
CG GTATTCCGT ACCATT CTG
CCG ATG GACCAG GAAAAG GCA
TGG TAA GACGGCTACCTG GTC
CTTGTA CGCGTT CGCCCG TAC
AAA GAA AAA CCGCAT GCG
CAA GCG GGCATG TTT CTT
TTT GGCTTT ACT TACCGT
GTT AAA AACCGCGAA CAAAAAA
TGA ATG GCA CAA TTT T
TG GCG CTT GTTATCCAG AA
CCAG GCT AAA TCT GTT G
ACGTATAG GTCTTG GTCCGA T
TT AGA CAA CTG CATGCT
TACCGT ATCAAT AAA AAA TC
CGGT CTGCGA ATG GCA TAG
TTA TTT TTT AGG CCA GAC
GAA TACACCGTT CAG TTCACC
CCG CTG AACCTT ATG TGG C
AA GTCAAG TGG GGCGACTTGA
AT GAA GAG ATT AACAACA
CTTTA CTT CTCTAA TTG TT
G TGAGACCTG ATC CTG GACTAG CCA GACGAT GACTTCCGCCCG
GGT CTG AAAGGT CTG CT
A CTG AAG GCG GGCCCA GACT
TTTCT GAA AAG TACTAC GTA C
TG AAA AAA GGTAGA CTT T
TCATG ATG CAT GACTTT T
TT CCAGACACT AAA CTG
CTG AAA ACCCTG GCT ATCCTG
TGA TTT GACGACTTT TGG GA
CCGA TAGGAG AAG CCG TAT G
ACCCG TTCGAT CGT TCTCTCTT
CGGCATA CTG GGCAAG CTA GC
A AGAGGT GACACCATCACT T
CT CAG GAG CTCCTGCCA
CTG TGG TAG TGA AGA G
TCCTCGAG GACCAT CTG AA
A CTG TTCACCATCAAA TACGT
TGTA GACTTT GACAAG TGG TA
G TTT ATG CAAGCT CAG GC
A CAG TCT ATCCTG AACA
AA AACCGA GTCCGT GTCAGA
TAG GACTTG TTT TTGGACGT
CGAT ACCAACGAA TTA CTG A
AG TCTCTG CAG CTA TGG T
TG CTT AAT GACTTCAGAGAG
CAG CTG CTG ACCCTCGT
CGACGACTGG GCT TCCGAA CGT AATCGA AGG
CTT GCA TTACCG AACGAT A
AA TAA TAG 3'GGCTTG
CTA TTT A'rT ATCAGCT
5'CTG GACTTCCGCGAT CTG
TACGACCCG CGTGACCTG AAG G
CG CTA GACATG CTG GGCCCA Formula (4): %Formula%'MluI The chemically synthesized gene encoding the streptokinase derivative protein having the modified amino acid-following sequence of the present invention is a gene encoding the above-mentioned natural streptokinase. As a basis, according to the modification of its amino acid sequence,
It can be designed by employing a DNA sequence (codon) encoding an amino acid residue corresponding to the modification. Modification of a specific portion of the DNA sequence can be carried out according to various methods known in the art, including cutting and removing a specific region with an appropriate restriction enzyme, or using a chemically synthesized oligo containing a separately synthesized modified portion. Replacement of specific regions with nucleotides (
repair) etc.

The chemically synthesized gene of the present invention is preferably produced by dividing it into several oligonucleotide fragments according to the designed base sequence, chemically synthesizing each, and then ligating the fragments. Each oligonucleotide fragment can be easily synthesized chemically using a commercially available DNA synthesizer or the like according to a conventional method.

The method includes, for example, the solid phase phosphotriester method [N
ature, 310.105 (1984)),
Solid-phase Phosph T7 Midide method [S, L, Beauc
age and M, H.

Caruthey, Tetrahedron Le
tters, 22.1859 (1981)]. Isolation and purification of the synthesized oligonucleotide fragments can be carried out by conventional methods such as high-performance liquid chromatography, and confirmation of each purified base sequence can be carried out by
For example, the Maxam-Gilbert method [A, M, Maxa
m and W, G11bert.

Proc, Natl Acad, Sci, US
A, 74.560 (1977) -A, M, Ma
xam and W, G11bert, Methods
inEnzymol,, 65.499 Aca
d, Press (1980)).

The hydroxyl group at the 5' end of the chemically synthesized oligonucleotide thus obtained was phosphorylated using T4 polynucleotide kinase, annealed, and ligated using T4 DNA ligase to create a plurality of blocks. The target gene of the present invention can be constructed by linking the subunits and linking the subunits.

The above-mentioned subunits and desired genes can be stored, amplified, etc. by once being incorporated into an appropriate vector, such as pBR322, and are preferably excised from the vector before subsequent manipulations. Specific examples of each of the above operations are as shown in Examples below.

In constructing the above-mentioned plasmid vector, usual operations, methods, etc. in gene recombination technology can be employed. This includes cutting of DNA using various restriction enzymes, S1 nuclease, etc., ligation of DNA using T4 DNA ligase, isolation of DNA by agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc., purification, and phenol extraction. This includes recovery and purification of DNA by methods. The presence of the plasmid vector in the host cell can also be confirmed using the alkaline-8DS extraction method (H, C).

Birnboim and J., Doly, Nuc.
leic Ac1ds Res,, 7゜1513 (
1979), etc., by separating plasmid DNA, treating it with various restriction enzymes, and examining the presence or absence of secondary restriction enzyme recognition sites and the length of the generated NA fragments. Furthermore, for example, gene base sequences can be directly analyzed by the dideoxy method (F, Sanger et al., Proc.
, Natl, Acad, Sc1. , USA,
The above-mentioned confirmation can also be made by an analysis method such as 74°5463 (1977).

The origin vector used for constructing a plasmid recombinant incorporating the gene of the present invention is pBR32.
2 and various plasmid vectors derived therefrom are suitable, but the present invention is not limited to these, and various conventionally known vectors such as bacteriophage and various virus vectors including animal and plant viruses, various plasmids, cosmids, etc. can also be used.

In order for the transformant obtained by introducing the vector carrying the gene of the present invention to express the target streptokinase or its derivative protein, the vector carrying the gene of the present invention may contain, in addition to the gene of the present invention,
Various regulatory factors necessary for its expression, such as promoters, transcription termination signals, poly A chain addition signals (when eukaryotic cells are used as host cells), transcription factors, and translation factors such as bosome binding sites. It is necessary to have factors such as Such regulatory factors are well known depending on the host cell. For example, promoters include Hasase L promoter, Ha (promoter tac promoter, UL promoter, Wholesale L promoter, etc.) in E. coli, and 5POI promoter, 5PO2 promoter, etc. in Bacillus subtilis. ,!
In yeast and other eukaryotic cells, PH
Examples include the 05 promoter, the PGK promoter, and the promoter derived from 竫赳. These regulatory factors can be obtained by selecting a plasmid containing them as a source vector when constructing the vector of the present invention, or by isolating or chemically synthesizing them from a plasmid containing them according to a conventional method. By integrating them into an appropriate vector, each of them can be present in the vector of the present invention, and thus a desired expression vector can be obtained.

The resulting expression vector (recombinant main body) for streptokinase or its derivative protein is a streptokinase (natural type or derivative protein) in which a promoter and a ribosome binding site are linked upstream of the gene of the present invention, and a transcription termination signal is linked downstream. ) It possesses protein expression information, and by introducing it into a suitable host cell and transforming the cell, the target streptokinase or its derivative protein can be expressed (produced and accumulated) in the cell. It can be done.

In particular, when Escherichia coli is used as a host cell, the expression system vectors include both a system in which the target protein is directly expressed within the bacterial body and a system in which the target protein is secreted into the periplasmic layer. In the secretory expression system described above, it is necessary to construct a vector in which a gene encoding a signal peptide is linked upstream of the chemically synthesized gene of the present invention. This secretory expression system will be described in detail below.

Here, the signal peptide is a highly hydrophobic amino acid sequence of several to several tens of amino acids that exists at the amino terminus of various secretory proteins, and it guides the above protein from the cytoplasm of the cell to the outside of the membrane. In addition, it is itself separated from the target secretory protein by a signal peptidase.

The use of such signal peptides in genetic recombination methods and the advantages thereof are known, for example, in JP-A-61
It is detailed in the publication No.-149089.

The signal peptide used in the present invention may be the same as those described in the above-mentioned publications, or may be different from those described therein. Specific examples include Escherichia coli β-lactamase (blayu), alkaline phosphatase (Kokutan''-), and Escherichia coli outer membrane protein (Kano to Snore, Satoshi P''-1).
Examples include signal peptides such as (m-, etc.).

The base sequence encoding the secondary signal peptide can be chemically synthesized or naturally occurring.

Specific examples of vectors containing the base sequence encoding the signal peptide include, for example, pKT shown in Examples below.
I can give an example of N.

pKTN is a vector containing a base sequence encoding the E. coli anal signal peptide, and more specifically, has genetic information in which a tac-promoter, an SD sequence, and a base sequence encoding the blal mini-signal peptide are arranged in the same direction. The E. coli JM103 strain harboring the pKTN is “Esch
erichia coli, JM-103゜pKTN
-2-2J is indicated, and the number 1398 (FE
RM BP-1398).

A suitable example of the secretion expression system is the plasmid psKXT constructed using the pKTN described above, and the details of its construction are as shown in Examples below. The secretory expression vector pSKXT is a base sequence in which the first codon encoding the first amino acid of the streptokinase gene is directly linked after the base sequence encoding the L signal peptide, that is, the combination of the signal peptide and streptokinase. It has a nucleotide sequence that encodes a fusion protein, and there is no risk of the reading frame being shifted (.) A fusion protein of a signal peptide and streptokinase is expressed in E. coli cells by the action of the tac promoter, and it passes through the inner membrane. This allows only the target protein to be secreted and accumulated in the periplasmic layer.

E. coli JM109 harboring this plasmid psKXT
The strain is “Escherichia coli, JM-
109゜pSKXT J, and its deposit number is FER-KEN Article No. 2464 (FER).
M BP-2464).

The desired expression vector obtained as described above is introduced (transformed) into an appropriate host cell.
The ability to produce the streptokinase or its derivative protein of the present invention can be imparted to the host cell. The host cells used here are not particularly limited, and may be any of a variety of known cells, such as Durham-negative bacteria such as Escherichia coli, Gram-positive bacteria such as Bacillus subtilis, actinomycetes, yeast, animal and plant cells, etc. Escherichia coli is particularly preferred, and among them, 12 strains (7
) H8101 strain (H, W, Boyer and D,
Rowland-Dussoix, J., Mo.
1. Biol, 41.459 (1969))
and strain JM109 (J, Messing et al.
,, Nucleic Ac1ds Res,, 9.3
09 (1981)) is preferred.

Methods for introducing the vector of the present invention into the above-mentioned host cells and for transformation thereof include commonly used methods;
For example, a method in which host cells are treated at low temperature in an aqueous solution containing galcium chloride and a vector is added to the solution [E,
Lederberg and S.

Cohen, J. Bacteriol, 119
．． 1072 (1974)).

The present invention also provides a host cell transformed according to the above-mentioned method (a transformant into which an expression vector for the native streptokinase of the present invention or its derivative protein has been introduced).

The transformant of the present invention can be cultured using a normal cell culture medium. Media that can be used for the above culture include:
For example, various types of media such as L''broth''broth medium 9 can be mentioned.In addition, these media can also contain various commonly known carbon sources, nitrogen sources, inorganic salts, vitamins, and natural products. Extracts, physiologically active substances, etc. can also be added.

Cultivation can be carried out by various methods employing conditions such as pH, temperature, aeration, and stirring that are suitable for host cell growth. For example, in the case of E. coli, the pH range is about 5 to 8, especially pH
7 is appropriate, and it is desirable to culture at a temperature of about 20 to 43° C. under aeration and stirring conditions, and there is no particular limitation on the scale of the culture. Furthermore, in order to increase the expression level of the target protein, or to promote or suppress the secretion of the target protein, the above-mentioned medium composition, culture conditions, etc. can be changed as appropriate.

Through the above culture, E. coli transformed with, for example, the streptokinase or its derivative protein secretion expression vector of the present invention secretes and accumulates the protein of interest in the periplasm layer, which can be isolated, recovered, and purified by conventional methods. These operations can be carried out using gel filtration, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography, etc. alone or in appropriate combinations, for example, on the periplasmic layer prepared by the osmotic shock method.

Particularly, those secreted into the periplasmic layer or the culture supernatant have the advantage of being relatively easy to separate and purify.

Thus, according to the present invention, streptokinase and its derivative proteins can be produced by genetic recombination technology.

The obtained protein can be easily confirmed using, for example, the presence of a single peak in high-performance liquid chromatography or the presence of a single band in polyacrylamide gel electrophoresis as indicators. Furthermore, the target highly purified streptokinase or its derivative protein can be identified using methods similar to those used for conventional structural analysis of polypeptides or proteins, such as 5
This can be carried out by molecular weight analysis by DS-PAGE, isoelectric point measurement by isoelectric focusing, measurement of amino acid composition by an amino acid analyzer, amino acid sequence analysis by a protein sequencer, etc.

In addition, the streptokinase derivative protein having a modified amino acid deletion sequence of the present invention can be produced more advantageously by first cleaving the native streptokinase expression vector obtained according to the above-described method with an appropriate restriction enzyme to obtain a specific region. After removing this, the gene is repaired using a chemically synthesized oligonucleotide fragment having the desired base sequence, and this is again inserted into an appropriate vector in the same manner to construct a modified desired derivative protein expression vector. , can be produced by introducing this into host cells and culturing the cells. According to this method, amino acid residues can be deleted or substituted at any position in the natural streptokinase of formula (1), and amino acid residues can be inserted at any position. These operations are as described above, and can be carried out in accordance with the methods detailed in the Examples below.

Thus, by genetic recombination technology, natural streptokinase and its derivative proteins can be easily produced with high purity and in large quantities. In particular, the streptokinase derivative protein obtained by the present invention has lower antigenicity and improved stability in blood, as compared to natural streptokinase, and has a higher selectivity of thrombolytic activity. Specificity (thrombus selectivity) is also excellent. Therefore, it can be used more effectively in various pharmaceutical applications where natural streptokinase is used.

EXAMPLES Hereinafter, in order to explain the present invention in more detail, examples of expression of natural streptokinase will be given as Examples 1 to 4, and then examples of production of the streptokinase derivative protein of the present invention will be given as Examples 5 to 12.

It should be noted that each method and operation used in each example was performed as follows unless otherwise specified.

1. DNA cutting operation using restriction enzymes The restriction enzymes used were those manufactured by Takara Shuzo Co., Ltd., Toyobo Co., Ltd., and Nippon Gene Co., Ltd., and the reaction solution used was one with a composition specified by each company. The reaction temperature was 37°C, and the mixture was allowed to stand in a hot bath for 3 hours to react.

The standard amount of restriction enzyme used was 1 unit per μg of DNAI, and the final reaction volume was 100 μl. A sterilized 1.5xl Eppendorf tube was used as the reaction vessel.

2. Phenol extraction method After the enzymatic reaction was completed, this extraction method was used to deactivate the enzyme and stop the reaction. That is, half of the volume of TE buffer saturated phenol [1mM ED] was added to the reaction solution.
A 10mM) lithium-hydrochloric acid (pH 8,0) buffer containing TA saturated with phenol was added, thoroughly shaken, and then centrifuged (1200 rpm, 5 molecules sj
) to collect the aqueous layer containing DNA (7), further added the same amount of ether, and after shaking and stirring in the same manner, centrifugation was performed to collect the aqueous layer. This operation was repeated 2 to 3 times. The collected aqueous layer was added with 0.1 volume of 3M sodium acetate buffer (pH
4,8) and 2.5 times the volume of cold ethanol, shake and stir, leave at -80℃ for 30 minutes or more, and centrifuge (120℃).
00 revolutions/min for 5 minutes) to precipitate and collect DNA.

3. Linking of DNA fragments using T4 DNA ligase 67mM Tris-HCl (pH 7.6), 6.7mM magnesium chloride, 10mM dithiothreitol and 1mM
Dissolve the DNA in an aqueous solution containing ATP, add T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) in an amount of 1 unit per 1 μg of DNA, and react at 12°C for 5 hours or more or at 4°C overnight to bind the DNA. . The final reaction volume was 100 μl. After the reaction was completed, DNA was collected using the phenol extraction method described above.

4. Transformation method E. coli strain HB-101 or JM-109 as host cell
A stock was used.

The host cell line was grown in L-broth medium (1% Bactodryptone, 0.5% Bactoyst extract, 0.5%
The cells were grown by shaking culture at 37° C. in sodium chloride until the absorbance of 600 nanoliters reached 0.25. Centrifuge 10xl of this culture solution (7000 rpm, 5 minutes)
The bacterial cells were collected and cooled with water.

Add 5xl of 0.1M magnesium chloride to this, suspend and wash, followed by centrifugation (7000 rpm, 1
5 minutes) to collect the bacterial cells, and further cooled with water in a mixed solution of 0.1M calcium chloride and 0.05M magnesium chloride.
xi was added and suspended. This was left in water for 30 minutes or more, and then centrifuged (7000 rpm, 1 minute) to collect the bacterial cells, which were again suspended in 0.5xl of the same solution. 20 μl of a reaction composition of DNA bound using T4 DNA ligase was added to 0.2 z/ml of this suspension, and the mixture was cooled on ice for 30 minutes.

Next, heat the L-br in a hot bath at 42.5°C for 30 seconds.
oth medium 1. OzA' was added, and the mixture was left standing in a 37°C hot bath for 1 hour.

The thus obtained transformed strain was selected using the following antibiotic resistance as an indicator. That is, L-br containing 1.5% agar
Add 0.2 of each of the reaction composition solutions obtained above to a plate culture medium prepared by adding 50 μg/xi of ampicillin to oth medium.
Spread each xll and culture this overnight at 37°C.
Growing colonies were isolated.

5. Isolation and purification of plasmid The strain carrying the plasmid was treated with ampicillin 50ttg/
37 with 400xl of L-broth medium supplemented with xi
Shaking culture was carried out at ℃ for 12 to 16 hours. This is centrifuged (6
000 revolutions/min for 10 minutes) to collect bacterial cells, and add solution I [50mM glucose, 10mM EDTA
, 25mM Tris-HCl (pH 8,0) and 2/x
1.4xl of lysozyme, for sterilized stool] was added and suspended, and the mixture was left in water for 30 minutes. Furthermore, solution II [0,
2N sodium hydroxide and 1% sodium dodecyl sulfate]
28xl! After stirring, add solution M [3M sodium acetate (pH 4, 8), for stool after sterilization] 2111 and leave it in water for more than 60 minutes, then centrifuge (8000 rpm). , 10 minutes) to obtain a precipitate.

To this solution IV [0.1 M sodium acetate and 0.05
Add 1111 of M Tris-HCl (pH 8,0) to dissolve, and then add 2.5 times the volume of cold ethanol to -80
It was left at ℃ for 30 minutes. Centrifugation (12,000 degrees)
rotation/min for 15 minutes) and collect the precipitate.

The resulting precipitate was then added with 4xl of a solution of TE buffer [10mM] Lis-HCl (pH 7,5) and 1mM EDTA.
Add and dissolve 4.62 g of cesium chloride, stir and dissolve, and then add 5 x/x ethidium bromide.
i-liquid 0.42xl! added. The resulting solution was centrifuged (3,000 rpm, 10 minutes) to remove suspended matter, and the solution was ultracentrifuged (50,000 rpm, 15 hours). After completion of this ultracentrifugation, the plasmid DNA portion that emitted fluorescence upon irradiation with ultraviolet rays was collected. This was extracted 5-6 times with isopropanol saturated with 5M sodium chloride solution to remove ethidium bromide. Finally, Biogel A5 Q (BiOgel A-50, manufactured by Bio-Rad) column chromatography (2,5an
X15~2Oan column size, elution solvent = TE buffer + 0.5M sodium chloride solution, detection by UV 254nH) to remove cesium chloride and contaminating RNA, etc., and remove plasmid DNA by the phenol extraction method described above.
was recovered.

The amount of purified plasmid DNA obtained was determined at OD 260nm
0D26o=0,022 by measurement 1μglxiDN
Calculated in terms of A amount.

6. Synthesis of oligonucleotides Chemical synthesis of NA was performed using Applied Biochemicals model 381A DNA synthesizer (AppliedBio Chemicals).
chemicals, Model 381A type
[solid-phase β-cyanoethylphosphoramidide method, 0.2
μM column used]. Its operation followed the company's manual. The oligonucleotide obtained by the synthesizer has a 3'
Column carrier resin on the terminal side, protecting group on each active group, 5
Since the dimethoxyltrityl group remained attached to the 'terminal side, it was necessary to remove these groups, and this operation was also performed according to the same manual.

Next, in order to efficiently remove side reactants, free protecting groups, deprotecting bases, etc. from the target oligonucleotide, high performance liquid chromatography was used to fractionate and purify the oligonucleotide until a single peak was obtained. The column used here was YMC
A-PACK AM-303005 (manufactured by Yamamura Chemical Research Institute, 4, 6 x 250 mm, Waterst
ype), and the elution solvent was 5% → 40% acetonitrile 10. Gradient elution was performed using LM triethylammonium acetate aqueous solution (pH 7,2). The system used was a Pon 1002M1 UV-visible detector UV-8000 and a controller CCP manufactured by Tosoh Corporation.

7. Agarose gel electrophoresis The agarose gel concentration was 0.9 or 1.6%. Agarose I (manufactured by Doguchi Kagaku Kenkyusho) was weighed to the above concentration, and THE buffer (0,089M Tris-boric acid, 0.0%
．． 02M EDTA) was added and dissolved by heating to form a gel. The electrophoresis is performed using a mini gel electrophoresis system.
Using Mupid 2 (manufactured by Cosmo Bio Co., Ltd.),
Further, TBE buffer was used as the migration buffer. After electrophoresis, 0.5μg/xi! DN that emits fluorescence by applying a gel to ethidium bromide solution and irradiating it with ultraviolet light
Fragment A was confirmed. To elute DNA from the gel, cut out the desired band with a knife, etc., and use a dialysis tube (3500
MW cut), filled with TE buffer, and electrophoresed for 30 minutes in the same mini gel electrophoresis device. The DNA eluted above was concentrated to dryness, a small amount of distilled water was added thereto, and the DNA was recovered according to the phenol extraction method described above.

8. Base sequence analysis The base sequences of the constructed streptokinase and its derivative proteins were determined using the M-13 dideoxy method [J. Messing
,, Methods in Enzymology,
101.20 (1983)). The operation is performed using Toyobo Co., Ltd.'s M-13 sequencing kit [[a
-32P using dCTP, manufactured by Amajam Co., Ltd.],
I followed the kit's manual. As the electrophoresis gel, Fuji Photo Film Co., Ltd. (%) was used.

Example 1 Streptokinase expression vector psKX
Preparation of T (1) Construction of psKX The construction of plasmid pSKX, in which the chemically synthesized gene for natural streptokinase was cloned, will be described in detail below.

■ Synthesis of oligonucleotides In constructing the entire base sequence shown in formula (3) above, including the streptokinase structural gene, first convert the same base sequence into the following
52 oligonucleotide fragments of 43 to 56 chain length shown in Tables to Table 4 (A-1 to A-14B-1, , B-12,
C-1 to C-12 and D-1, D-14), and each fragment was chemically synthesized by solid phase β-cyanoethylphosphoramidide method.

Table of Contents ■ Linkage of synthetic oligonucleotides Construction of the entire base sequence shown in formula (3) above was carried out separately for subunits SKA, 5KBSSKC, and SKD.

Subunit SKA has NakaLI from the EcoRI restriction enzyme recognition site of the above entire base sequence as shown in the following formula (5).
, , . . . . . . . . . . . . . . . . . μie I restriction enzyme recognition site included. Subunit SKB is composed of an EcoRI restriction enzyme recognition site and a SalE restriction enzyme recognition site including Nhe I and Sac I restriction enzyme recognition sites, as shown in the following formula (6). The subunit SKC is Eco as shown in the following equation (7).
It consists of a RI restriction enzyme recognition site to a Gilier I restriction enzyme recognition site including Sac I to it II restriction enzyme recognition sites. In addition, subunit SKD has 4at from the EcoRI restriction enzyme recognition site as shown in the following formula (8).
Subunit SKA (EcoRI) A-1 5' AATTCGGATCCATGATCGCGG
GCCCGGAATGGCTGCTGGA3'
GCCTAGGTACTAGCGCCCGGGCCTT
ACCGACGACCTCCGTCCGTCTGTTA
ACAACTCCCCAGCTGGTTGTTTCCGT
AGGCAGGCAGACAATTGTTGAGGGT
CGACCAACAAAGGCAT-→
A-7TAACGTACA
TTCTAACGACGACTACTTTGAGGTA
ATCGACATTGCATGTAAGATTGCTG
CTGATGAAAACTCCATTAGCTGA-''
8---H---1-11-1-TTCGCTAGC
G 3'AAGCGATCGCAGCT
5 GCTGGCACTGTTGAAGGTACTAACC
AGGACATCTCTCTGACGACCGTGAC
AACTTCCATGATTGGTCCTGTAGAG
AGACTAATTTTTCG'AAATCGACCT
GACCTCTCGTCCGGCCCATGGTTAA
AAAGCTTTAGCTGGACTGGAGAGCA
GGCCGGGTACC-----=--------
A-12TGGTAAAACCGAACAGG
GCCTGTCCCCCGAAATCTAAACCGAC
CATTTTGGCTTGTCCCGGACAGGGG
CTTTAGATTTGGCTT CG CTA C
T G A C T CTGGC G CTATGT C
T CATAAA C TCGAGAAAGCGAT
GACTGAGACCGCGATACAGAGTATT
TGAGCTCTAGGCAGATCTGCTGAAA
GCAATCCAGGAACAGCTGATCGCTC
CGTCTAGACGACTTTCGTTAGGTCC
TTGTCGACTAGCG1--1-→
A-9〈Subunit SKB) (EcoRI) B-1 AATTCGCTAGCGACGCTATCAC
CGACCGTAACGGCAAGCGATCGCTG
CGATGATAGTGGCTGGCATTGCCGT
TAGTATACTTCGCTGACAAAGACGG
TTCTGTAACTCTTCCGTCATATGAA
GCGACTGTTTCTGCCAAGACATTGA
GAAGGCACTCAACCGGTACAGGAAT
TTCTGCTGTCTGGCCATGTACTGAG
TTGGCCATGTCCTTAAAGACGACAG
ACCGGTACATGGCGTTCGCCCGTAC
AAAGAAAAACCGATCCAGAACCAGG
CCGCAAGCGGGCATGTTTcTTTTTG
GcTAGGTcTTGGTccGB-10
81111111. B-4 TAAATCTGTTGACGTAGAATACACC
GTTCAGTTCACCCCGATTTAGACAA
CTGCATCTTATGTGGCAAGTCAAGT
GGGGCATCGTAACCCATGACAACGA
CATCTTCCGTACCATTCTGCTAGCA
TTGGGTACTGTTGCTGTAGAAGGCA
TGGTAAGACGCTGAACCCAGACGAT
GACTTCCGCCCGGGTCTGAAAGACA
GACTTGGGTCTGCTACTGAAGGCGG
GCCCAGACTTTCTGTCGATGGACCA
GGAATTTACTTACCGTGTTAAAAAC
CGCGAGCTACCTGGTCCTTAAATGA
ATGGCACAATTTTTTGGCGCTCTAAA
CTGCTGAAAACCCTGGCTATCGGTG
ACACCATCACGATTTGACGACTTTTT
GGGACCGATAGCCACTGTGGTAGTG
B-7 TTCTCAGGAGCTCG AAGAGTCCTCGAGCAGCT〈Subunit SKC〉 (EcoRI) C-1 AATTCGAGCTCCTGGCTCAGGCACA
GTCTATCCTGAACAAGCTCGAGGAC
CGAGTCCGTGTCAGATAGGACTTGT
TAAACCATCCGGGCTACACTATCTA
CGAACGCGACTCTTCCTTTGGTAGG
CCCGATGTGATAGATGCTTGCGCTG
AGAAGGC - 11 ---- ACAAGCTTACCGTATCAATAAAAAA
TCCGGTCTGAATGAATGTTCGAATG
GCATAGTTATTTTTTTAGGCCAGACT
TACTTGAGATTAAACAACACTGACCT
GATCTCTGAAAAGTACTACGCTCTA
ATTGTTGTGACTGGACTAGAGACTT
TTCATGATGC←----11111-・C-
8 TACTGAAAAAAAGGTGAGAAGCCGTA
TGACCCGTTTCGATCGATGACTTTTT
TCCACTCTTCGGCATACTGGGCAAG
CTAGCTTCTCCATCTGAAAACTGTTCA
CCATCAAATACGTTGACGTCAAAGAG
TAGACTTTGACAAGTGGTAGTTTAT
GCAACTGCAGC-7 G CAGCT (SalI) subunit 5KD) (EcoRI) AATTCGACGTCGATACCAACGAATT
ACTGAAGTCTGAGCAGCTGCAGCTA
TGGTTGCTTAATGACTTCAGACTCG
TGCTGCTGACCGCTTCCGAACGTAA
TCTGGACTTCCGCGATCGACGACTG
GCGAAGGCTTGCATTAGACCTGAAG
GCGCTACTGTACGACCCGCGTGACA
AAGCTAAAACTGCTGTACAACAGACA
TGCTGGGCGCACTGTTTCGATTTGA
CGACATGTTGTACCTGGATGCTTTTC
GGTATCATGGACTACACCCTGACTG
GTGGACCTACGAAAGCCATAGTACC
TGATGTGGGACTGACCTAAAGTAGA
AGACAACCATGACGACACCAACCGT
ATCATCATTTCATCTTCTGTTGGTA
CTGCTGTGGTTGGCATAGTAGACCG
TATACATGGGCAAACGTCCGGAAGG
TGAAAATGCATTGGCATATGTACCC
GTTTGCAGGCCTTCCACTTTTACGT
ACTTACCATCTGGCATATGACAAAG
ACCGTTACACCGAAGAGAATGGTAG
ACCGTATACTGTTTCTGGCAATGTG
GCTTCTAGAACGTGAAGTTTACTCT
TACCTGCGCTATACTGGTACCTCTT
GCACTTCAAATGAGAATGGACGCGA
TATGACCATGGCCTATCCCGGATAA
CCCGAACGATAAAATAATAGGGATAG
GGCCTATTGGGCTTGCTATTTATTA
TCAGCT (SalI) Each subunit was divided into blocks 1 to 8 and constructed separately. Construction of each block is carried out as follows,
Its outline is shown in Figure 1. In FIG. 1, the black dots at the ends of the solid lines representing chemically synthesized oligonucleotides indicate the introduction of a 5'-terminal phosphate group.

Among the 52 chemically synthesized oligonucleotides A-1 to D-14, A-I A-8B-I B-7C-I
For 44 bottles excluding C-7, D-1 and D-8, 1 to 3 μg/xi warm solution of 20 μg each! In addition to this, 5 units of T4 DNA polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) and 5μ of the reaction buffer specified by the company!
and 1μA' of 100mM ATP, and further water to bring the final reaction volume to 50μ! The mixture was reacted at 37°C for 1 hour to introduce a phosphate group into the 5' end of each oligonucleotide.

Next, for each block to be constructed, take 2 μl of the DNA solution of each oligonucleotide as a raw material (for example, in the case of block 1, add oligonucleotides A-1, A-2, A-
3, A-12, A-13 and A-19, and 100m
M ATP 1 μA' and ligase reaction buffer [660
10 μl of lithium hydrochloric acid (pH 7,6), 66 mM magnesium chloride, 100 mM dithiothreitol solution]
was added, the final reaction volume was adjusted to 100 μl, and the mixture was heated in a 100° C. bath for 2 minutes and then naturally cooled. Next, T4
2.5 units of DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was allowed to react overnight at 4°C to ligate.

After each ligation reaction product was extracted with phenol, 10% polyacrylamide gel electrophoresis was performed, and the double-stranded DNA portion, which was a block of the desired size, was scraped off and eluted.

Through the above operations, each of blocks 1 to 8 was constructed.

(2) Preparation of pSKA, psKBSpsKc and psKD A vector psKA was prepared by incorporating the subunit SKA consisting of block 1 and block 2 constructed in (1) above into plasmid pBR322 by the following procedure. Similarly, pSKB.

pSKC and psKD were respectively produced.

The outline is shown in Fig. 2. In the figure, Ap represents ampicillin resistance, Tc represents atracycline resistance, or
i indicates the replication start point. In addition, the restriction enzyme recognition sites in each base sequence are indicated by the following abbreviations, and the same applies to each subsequent figure.

A, -Aat II A p, -1 Wholesale I
13,,,Ba! aHIE・・・Elbow ORI
E V...elbow deaf V' N...1 bu? INa,-
N&e I p , -1PBt I 3,
-5al l5c11.5acI (2) Preparation of -1psKA First, pBR322 was treated with restriction enzymes EcoRI and Sal, and then subjected to 0.9% agarose gel electrophoresis to obtain a DNA fragment of about 3.7 kb. This DNA fragment and the previously obtained blocks 1 and 5. Tsuku 2, T4D
NA ligase, ATP, and a ligase reaction buffer were mixed and reacted at 4° C. overnight to ligate the DNA. Using this reaction solution, Escherichia coli HB-101 was isolated by the calcium method.
Strain transformation was performed. DNA was collected from the resulting colonies by a simple boiling method and screened.
Colonies containing the desired pSKA were selected.

Further, purified DNA was collected by alkaline-8DS extraction method, CsC7 equilibrium density ultracentrifugation method, and Biogel A-50 column chromatography method, and restriction enzymes (Hiasa 11 Salmon II, NakaaI, μKo-■, Jilu I, Xho II
, Hinf I, etc.) to create a restriction enzyme cleavage map.

As a result, it was confirmed that the selected colony contained the target psKA.

Furthermore, the base sequence of the SKA introduction part of pSKA was confirmed using M-
This was carried out by the 13 dideoxy method. That is, EcoRI-5
t1. The I fragment was subcloned into M-13"p18 or "I)194: and the base sequence was analyzed.

As a result, it was confirmed that it had the desired sequence.

■-Creation of 2psKB, pSKC and pSKD Above ■-
In the same manner as in 1, subunit SKB consisting of block 3 and block 4 was transformed into Escherichia coli HB-101 strain, colony screening and plasmid DNA collection and purification were performed to construct and confirm pSKB.

Similarly, the subunit SKC consisting of block 5 and block 6 was transformed into Escherichia coli HB-101 strain, colony screening and plasmid DNA collection.
Purification was performed to construct and confirm pSKC, and subunit SKD consisting of block 7 and block 8 was transformed into Escherichia coli HB-101 strain, and colony screening and plasmid DNA collection and purification were performed.

(2) Preparation of pSKAB This plasmid is an intermediate vector for constructing the desired pSKX, and the outline of its preparation is shown in FIG. 2, and was carried out as follows.

The psKA obtained in step ① above was treated with restriction enzymes Pst I and Nhe.
107 by agarose gel electrophoresis.
A 2 bp DNA fragment was isolated and purified.

Similarly, pSKB obtained in step ① above was treated with the same restriction enzyme to isolate and purify a 3250 bp DNA fragment.

Both of the above DNA fragments were ligated using T4 DNA ligase, and the reaction product was used to transform Escherichia coli HB-101 strain to obtain a colony containing the desired psKAB. Plasmid DNA was collected from the obtained colony, purified, a restriction enzyme cleavage map was created, and psKAB of 4323bl) was confirmed.

■ Construction of pSKABX To construct the streptokinase secretion expression system vector, use the Net
A plasmid vector with a base sequence that does not have a codon is required. Therefore, p
sKABX was produced as follows.

Its outline is shown in FIG.

That is, pSKAB was treated with restriction enzymes EC0RI and Wholesale LI, and newly chemically synthesized oligonucleotides E-1 and E-2 were added to perform a ligation reaction in the presence of T4 DNA ligase, and E. coli was transformed with this reaction solution. Then, vector DNA was collected from the resulting colony, purified, and a restriction enzyme cleavage map was created to obtain the target psKABX.

In addition, the linker for secretion expression system vector construction obtained using the above oligonucleotides E-1 and E-2 and the like.
The base sequence of is as follows.

Linker number of bases Base sequence E 1 1 g AATTCGATATCGC
GGGCCE -210CGCGATATCG <Linker for secretion expression system vector construction> AATTCGA
TATCGCGGGCCGCTATAGCGC (EcoRI) (ApaI) The thus obtained plasmid vector can be used to construct a streptokinase secretion expression system.

The base sequences of the introduced portions of E-1 and E-2 of pSKABX were obtained using the M-13 dideoxy method [J, Messing.

Methods in Enzymology, 10
1.20 (1983)) It was confirmed that the above was true.

■ Preparation of pSKX The outline of the preparation of this pSKX is shown in FIG.

Treat psKABX with restriction enzymes Sac I and Pst Engineering I to generate a DNA fragment of 1352bI) and pSK
A 309 bp DNA fragment is generated by treating C with restriction enzymes Sac I and Aat II, and 3305 is generated by treating psKD with restriction enzymes Aat II and Aat II.
A ligation reaction was performed with a bp DNA fragment, and the reaction product was used to transform Escherichia coli. Vector DNA was collected and purified in the same manner as described above, and a restriction enzyme map was created and analyzed.

As a result, it was confirmed that the target pSKX (4966 bp) was obtained.

Example 2 Construction of a Streptokinase Secretory Expression Vector This example is an example of the construction of a vector that secretes and expresses streptokinase into the periplasmic layer of E. coli. The streptokinase gene is inserted, and the streptokinase structural gene is linked so that the codon frame of the streptokinase structural gene is not shifted along the codon flow with respect to the amino acid sequence of the bla signal peptide, and the desired streptokinase secretion expression is achieved. The vector was constructed.

The outline of its construction is as shown in FIG.

In the figure, the abbreviations at the restriction enzyme recognition sites are the same as above, and Sp bla represents the bla signal peptide, and S
D is the liposome binding site, PtaC is! The promoters are shown, the white part shows the υ-promoter, the hatched part shows the bla signal peptide, and the black part shows the base sequence part encoding the streptokinase chemical synthesis gene.

■ Preparation of pSKXT A DNA fragment of 1251bl) generated by treating pSKX with restriction enzymes EcoRI and Giri I and 37 generated by treating the same pSKX with restriction enzymes EcoRI and Giri I.
An 11bp DNA fragment! pKTN2-2, which has a promoter and a concave signal peptide portion (E. coli JM-103 strain carrying this has been deposited as FAIKEN Deposit No. 9146), was digested with restriction enzymes EcoRI and μhe.
A 460 bp DNA fragment generated by treatment with I,
Each of the DNA fragments was collected by agarose gel or polyacrylamide gel electrophoresis, ligated with 2 or 3 types of DNA fragments, and then transformed into E. coli strain JM-109.

Vector DNA was collected from the resulting colony, purified,
Create a restriction enzyme cleavage map and select the desired vector pS.
I got KXT.

According to the E. coli JM-109 strain carrying psKXT, a protein in which the blal signal peptide and streptokinase are fused is produced within the cells by the action of the ゛υ煕-promoter, and then the protein is produced by the action of the anal signal peptide. The signal peptide is transferred to the intracellular membrane, where the signal peptide is cleaved by the action of proteases present in the inner membrane.
Only streptokinase can be secreted and produced between the outer and inner membranes, ie, in the periplasmic layer.

Example 3 Expression and confirmation of streptokinase ■ Cultivation of Escherichia coli JM-109 strain carrying the streptokinase secretion expression vector pSKXT E. coli J carrying the vector pSKXT obtained in Example 2
M-109 strain was cultured by shaking culture as follows using M-9 zamino acid liquid medium having the composition shown in Table 5 below.

Table 5 However, "" in the table indicates that the product has been separately sterilized using an autoclave (121
℃, 15 minutes).

Furthermore, if necessary, filter-sterilized ampicillin was added at a final concentration of 50 μg/zl.

Preculture solution 111 was added to a Sakaro flask containing 100zA' of the same medium, and cultured with reciprocal shaking at 37°C.

About 3 hours after the start of culture (OD600 = about 0.3), I
PTG (isopropyl-β-D-thiogalactoside, manufactured by Sigma) was added to a final concentration of 0.25 μ/xi, and the culture was continued.

(2) Extraction of target product from bacterial cells The cells were cultured under the conditions (2) above, and the culture was stopped approximately 4 hours after the addition of IPTG, and each fraction was obtained by the following operations.

That is, first, the culture solution was centrifuged (5000 rpm, 10
1 minute) to separate the bacterial cells and the culture supernatant. The culture supernatant thus obtained is used as a medium fraction.

In addition, 30mM Tris-HCl (pH 8.0) was added to the obtained bacterial cells.
- Add 20% sucrose buffer to the same amount as the culture solution and suspend.
Furthermore, the final concentration is 0. Add an amount of EDTA aqueous solution to become OIM, and shake at 24°C at 180 rpm on a rotary shaker.
The mixture was shaken and stirred for 10 minutes, and then centrifuged (6000 rpm, 10 minutes). The supernatant thus obtained is used as a sucrose buffer fraction.

Furthermore, the same amount of ice-cold water as the culture solution was added to the sediment after the above centrifugation to resuspend it, and the mixture was allowed to stand in water for 15 minutes, stirred occasionally, and then centrifuged (10,000 rpm, 5 minutes). ) to separate the supernatant and sediment. The supernatant thus obtained is designated as the periplasm fraction.

■ Streptokinase activity measurement The plasminogen activator activity of streptokinase was measured using the method of Jackson et al.
n, et al, Methods in Enz
The assay was carried out by measuring the activity of plasmin generated by plasminogen activation of streptokinase, according to Cymology, 80°387 (1981)).

The activity of plasmin is dependent on the synthetic substrate S-2251[D
-Val-Leu-Lys-p-nitroaniline, manufactured by Kabi Vitram Co.], and measured the increase in absorption of p-nitroaniline liberated by plasmin at 405 nll'. Details of this measurement method are shown below.

The measurement sample was diluted with 0.025% Triton
Place 25 μl of pH 7.5) in a test tube and incubate for 5 minutes at 37°C, then add 50 mM containing bovine serum albumin (hereinafter abbreviated as BSA) at a concentration of 1/17+.
) 80Mg/xi in Lis-HCl buffer (pH 7,5)
25 μl of human plasminogen (manufactured by Sigma) diluted to the desired concentration was added and incubated at 37°C.

After 15 minutes, 50mM containing 1.6M sodium chloride
2.5■/xl in Tris-HCl buffer (pH 7,5)
S-2251 diluted to 25μ! 10 at 37℃
After incubating for minutes, 1.5xl of 0.2M acetic acid aqueous solution was added to stop the reaction, and the absorbance at 405n'' was measured.

The activity unit of the measurement sample is 100 international units/μ in the standard substance.
After measuring according to the above method using streptokinase derived from Streptococcus equisimilis H46A (Group C) having a specific activity of 1.5 g, it was determined from the standard curve.

■ Western blotting Streptokinase in the bacterial cells obtained in the above (■) was determined by Western blotting using a streptokinase-specific antibody [H, Towbin, T, 5taehelina
nd J., Gordon, Proc., Natl., A.
cad, sci,, U, S, A,.

76, 4350 (1979)). The details are shown below.

A specific antiserum for streptokinase was prepared by immunizing a rabbit with a purified product of streptokinase derived from Streptococcus equisimilis H46A (group C) as an antigen. This is lyophilized streptokinase 1■ dissolved in 0.5xl of physiological saline, followed by 0.5xl of Freund's complete adjuvant! The same amount of emulsified streptokinase was injected intradermally into the backs of the rabbits every two weeks for a total of four immunizations. After 10 days, whole blood was collected and serum was separated. The antiserum thus obtained was used for detection of streptokinase.

A sample for polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (hereinafter referred to as rSDS-PAGEJ) is, for example, bacterial cells obtained from a 1 xi culture solution in a 10 mM lithium-hydrochloric acid buffer containing 2% SDS and 2% dithiothreitol. Suspend in 1 zl of solution (pH 8,0) and incubate at 100°C for 50 minutes.
After heating for a minute, the mixture was centrifuged (12,000 rpm, 10 minutes) and the resulting supernatant was prepared.

5DS-PAGE was performed using Laemmli's method (U, K.

Laemmli, Nature, 227.680
(1970)) containing 0.1% SDS.
Using a 5% polyacrylamide gel, the sample prepared by the above method was run on 2111A/an for about 3 hours. To detect streptokinase, proteins in the gel after electrophoresis are electrophoretically transferred to a nitrocellulose membrane (manufactured by Bio-Rad), and streptokinase is specifically stained from within the protein using a specific antibody. carried out.

For the above staining, first, the nitrocellulose membrane after transfer was dyed with 0.1
Bovine serum albumin (hereinafter referred to as rBsA) was dissolved to a concentration of 10% in Lis-HCl buffer (pH 7.5, hereinafter referred to as rTBSJ) containing 5M sodium chloride.
After shaking for 1 hour at room temperature, 0.1% B
The above streptokinase antiserum diluted 1000 times with SA-TBS was reacted at room temperature for 3 hours, and after the reaction, T
Washed 4 times with BS and further washed with 0.1% BSA-TBS for 200 min.
After treatment with 0x diluted peroxidase-conjugated anti-rabbit IgG (manufactured by Kappel) for 60 minutes and washing again, 0.03
0.02M citrate buffer (pH 6.5) containing % hydrogen peroxide and 0.5/11 4-chloro-1-naphthol
It was carried out by developing color.

■ Measurement of streptokinase expression level Escherichia coli JM-1 harboring the secretory expression vector pSKXT
The 09 strain was cultured and fractionated according to the above (1) and (2), and then measured according to the above (2) to determine the streptokinase expression level.

As a result, it was not detected in the medium supernatant, and 200% was detected in the bacterial cell fraction.
An expression level of 11 international units was observed. Most of it was present in the veriplasm fraction. Furthermore, when analyzed according to the above ①, its immunoactivity was found to be 47,000 yen, which has the same molecular weight as the natural type.
appeared in the position of

From the above results, the streptokinase detected by the present invention is translated within the bacterial body, passes through the intracellular membrane due to the action of the signal peptide, undergoes processing, and the signal peptide is separated, resulting in the same streptokinase as the natural type. was determined to be secreted.

Example 4 Production and identification of recombinant streptokinase ■ Extraction from bacterial cells 2.44' of culture solution obtained by the culture method shown in ■ in Example 3
The cells were centrifuged (8,000 rpm, 10 minutes) to collect bacterial cells, which were then treated according to the osmotic shock method described in Example 3 (2) to extract the periplasmic fraction.

■ Purification of streptokinase Streptokinase is recovered from the above fraction extract by a combination of separation methods such as ammonium sulfate salting out, isoelectric focusing, gel filtration, ion exchange chromatography, and hydrophobic chromatography. This allows a single purified product to be obtained. As an example, methods using hydrophobic chromatography and anion exchange chromatography will be described in detail below.

First, ammonium sulfate was added to the periplasmic fraction 600z/ to a concentration of 0.6M;
It was passed through a butyl Toyopearl column (manufactured by Tosoh Corporation, 3×20an) equilibrated with ammonium sulfate. Column 0.
After washing with 3M ammonium sulfate 500yA', 50mM
) The fraction containing streptokinase activity was eluted by flowing Lis-HCl buffer (pH 8.5). Next, the obtained elution fraction was dialyzed against 50 mM Tris-HCl buffer (pH 8.5), and then DEAE-5PW (manufactured by Tosoh Corporation, 2.
15x15an) using a preparative HPLC system (manufactured by Tosoh Corporation, model HLC-837). The separation conditions were a flow rate of 411/min, and a linear concentration gradient from 0.1M to 0.3M sodium chloride using 50mM) Lis-HCl buffer (pH 8.5) as the eluent. The butyl toyopearl elution fraction after the above dialysis was subjected to indie-excidin using this HPLC system, and the main protein peak portions were pooled.

Streptokinase activity was eluted in a peak that coincided with the protein peak. Furthermore, the pool of eluted active fractions was transferred to a Sephadex G-25 column (manufactured by Pharmacia, 2.5X
After desalting with 25an), DEAE was added again under the same conditions as above.
-5PW column.

Finally, the streptokinase active fraction was desalted using the same Sephadex G-25 column and then lyophilized to obtain a final purified product.

■ Identification of the final purified product ■-I Molecular weight measurement by 5DS-PAGE The purified sample obtained in the above (■) was subjected to 5DS-PAGE in the presence and absence of a reducing agent.
Analyzed by PAGE.

As a result, Streptococcus equisimilis H46
A single band was shown at the same molecular weight position of 47,300 as streptokinase derived from A (group C).

(2)-2 Amino Acid Analysis The purified sample was hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours, and amino acid analysis was performed by the ninhydrin method using a Hitachi Model 835 amino acid automatic analyzer.

The results are shown in Table 6 below.

For comparison, Table 6 also shows the same results for streptokinase derived from Streptococcus equisimilis H46A (group C) (indicated as "natural form" in Table 6).

Table 6 -3 Amino acid sequence analysis The sequence analysis of the amino terminal 20 amino acid residues of the same purified sample was performed using the method of Hewick et al. [: R, M, He1w1ck.

et al., J. Biol. Chem., 2
56.7990 (1981)].

As a result, the sequence of 20 amino acid residues from the amino terminus in the purified sample was identified to be identical to that of streptokinase (natural product) derived from Streptococcus equisimilis H46A (group C).

The analysis of the same amino acid sequence made it possible to identify Trp, which is the 6th amino acid from the N-terminus, which could not be detected in the amino acid analysis of ①-2 above.

The amount of recombinant streptokinase obtained according to the above method was determined to be 2.5 μ by amino acid analysis of the final purified sample.

In addition, the specific activity of plasminogen activator activity is 1
01.5 international units/μg, and that of the above natural product (1
00 international units/μg).

From the above results, the streptokinase obtained above is Streptococcus equisimilis H46A (group C
)-derived streptokinase (natural substance) was confirmed to be the same substance.

Example 5 Preparation and expression of streptokinase derivative protein expression vector pSKXT (1-372) Streptokinase expression vector pSK obtained in Example 2
Using XT, streptokinase derivative protein (1-
372) was constructed as follows. The outline is as shown in FIG.

■ Chemical synthesis of DNA fragments In order to produce a derivative protein (1-372) lacking the C-terminal side of streptokinase, the following newly chemically synthesized oligonucleotide (DNA) fragments (F-1 and F
-2) was used.

ATACATGGGCAAACGTTAATAGTGT
ACCCGTTTGCAATTATCAGCT(Acc
I) F-2 (Sal I) 2nd class DNA
The method for synthesizing the fragment was the same as described above.

The above derivative protein expression vector was first constructed using psKXT
The restriction enzyme recognition site Acc I (present at position 367 of streptokinase; there are three other locations) and the same Sal
Yu I (present after the same codon.

This was done by extracting the fragment (existing at one other location) and inserting the above fragments F-1 and F-2.

■ Construction of p S K X T (1-372) psK
XT with the restriction enzymes pst I and Mlu! A 1635 bp DNA fragment generated by treatment with pst and the restriction enzyme pst
D of 2962 bp generated by processing with I and Sal I
The NA fragment and a 677 bp DNA fragment generated by further treatment with restriction enzymes -I and MluI were collected by agarose gel electrophoresis, and the chemically synthesized NA fragment obtained in ① above and After ligation reaction, Escherichia coli JM109 strain was transformed with the reaction product, vector DNA was collected from the resulting colonies, purified, a restriction enzyme map was created, and the desired vector pS K
372) I got it.

■ Expression and confirmation of derivative protein (1-372) Above ■
The E. coli JM-109 strain carrying the vector pSK
Shaking culture was carried out in the same manner as described above.

About 4 hours after the addition of I PTG, the culture was stopped, the bacteria were collected, and the bacterial cells and the culture supernatant were separated in the same manner as in Example 3 (■).
A sucrose buffer fraction and a periplasmic fraction were obtained.

For each fraction, streptokinase activity measurement and Western blotting were performed in the same manner as in Example 3 (1) and (2) to determine the expression level.

As a result, it was not detected in the medium supernatant, and approximately 10
The expression level of 00 countries/ill was observed.

Most of it was present in the veriplasm fraction. Furthermore, as a result of Western blotting analysis, the immunoactivity appeared at a position with a calculated molecular weight of about 42,300.

From the above results, streptokinase derivative protein (1
-372) was translated within the bacterial body, passed through the intracellular membrane under the action of the signal peptide, was processed, and the signal peptide was cleaved and secreted.

■ Recombinant streptokinase derivative protein (1-3
72) Production and identification Culture solution 2.41 was centrifuged (800
0 revolutions/min for 10 minutes) to collect the bacterial cells, extract the periplasmic fraction by the osmotic shock method, and extract the streptokinase derivative protein (1-372) from the extract in the same manner as in Example 4. The product was collected and purified, and finally freeze-dried to obtain a purified product.

The purified product was analyzed by 5DS-PAGE in the presence of a reducing agent, and a single band was found at the desired molecular weight of about 42,300.

Furthermore, 6N hydrochloric acid was added to the same purified product to hydrolyze it at 110°C for 24 hours, and amino acid analysis was performed by the ninhydrin method using a Hitachi Model 835 amino acid automatic analyzer. The results are shown in Table 7 below.

Table 7 Furthermore, sequence analysis of 20 amino acid residues from the amino terminus of the purified product was carried out according to the method of Haywick et al., and sequence analysis of 5 residues from the carboxyl terminus was carried out using carboxypeptidases A and B. From these results, we were able to identify the desired streptococcal derivative protein (1-372).

The amount recovered of the recombinant streptokinase derivative protein (1-372) obtained according to the above method was 9 μm based on amino acid analysis of the final purified sample.

Furthermore, the specific activity of plasminogen activator activity was 107 in terms of molar ratio, taking that of the natural form as 100, and was equivalent to that of natural streptokinase.

Example 6 Construction and expression of streptokinase derivative protein expression vector p3KXT (1-372,118 missing) Using streptokinase derivative expression vector pSKXT (1-372) obtained in Example 5 , this streptokinase derivative protein (1-372/118 missing)
An expression vector was constructed as follows. The outline is as shown in FIG.

■ The derivative protein expression vector for chemical synthesis of DNA fragments is psKXT (1-3
72) by replacing the restriction enzyme recognition site between Nhe I and Hinf I with the following newly chemically synthesized oligonucleotide (DNA) fragments (E1 to E4).

E2 TACGCTAAAGACGGATCCGTAACTC
TTCCGATGCGATTTCTGCCTAGGCA
TTGAGAAGGCTGA (BamHI) E3
Each DNA fragment such as (Hinf I) was separately chemically synthesized in the same manner as above. In addition, - for later analysis, pSKXT
Restriction enzyme recognition site Bam that does not exist in (1-372)
HI was set.

■ p S K X T (1-372, 118 missing)
Construction of 1522b generated by treating pSK
p DNA fragment and restriction enzymes Pst I and Sac I
A DNA fragment of 3488 bl) produced by treatment with restriction enzymes di-2 and Hinf I and a 212 bp DNA fragment produced by further treatment with restriction enzymes Ditri-■ and Hinf I were collected by agarose gel electrophoresis, respectively. A ligation reaction was carried out with the four chemically synthesized NA fragments obtained in (However, oligomers E2 and E (4 were phosphorylated at their 5 ends)
After that, E. coli strain JM109 was transformed with the reaction product, vector DNA was collected from the resulting colonies, purified, a restriction enzyme map was created, and the target vector pSKX
T (1-372,118 missing) Got it.

■ Expression and confirmation of derivative protein (1-372/118 missing) The vector p S K X 'l' (1-
Escherichia coli JM-109 strain harboring 372,118 deletion) was cultured with shaking in the same manner using the above-mentioned M-9 zamino acid liquid medium.

After adding PTG, bacteria were collected in the same manner and streptokinase activity (plasminogen activator activity) was measured. As a result, it was not detected in the medium supernatant, and approximately 250
The expression level of Kokuyō Seiichi/zl was observed. Most of it was present in the veriplasm fraction. Furthermore, as a result of Western blotting analysis, the immunoactivity appeared at a calculated molecular weight of 42,200.

■ Recombinant streptokinase derivative protein (1-3
Production and identification of 72,118 (missing) In the same manner as in Example 5 (■), the culture solution 1.61 was centrifuged to collect the bacterial cells, the periplasmic fraction was extracted by the osmotic shock method, and the desired product was extracted from the extract. The derivative protein (1-372/118 missing) was recovered and purified.

5DS-PAG in the presence of a reducing agent for the purified product obtained.
As a result of analysis using E, a single band was observed at the desired molecular weight position of 42,200.

In addition, 6N hydrochloric acid was added to the same purified product for hydrolysis at 110°C for 24 hours, and amino acid analysis was performed using a Hitachi 835 automatic amino acid analyzer. The results are shown in Table 8 below.

Table 8 The amount recovered of the recombinant streptokinase derivative protein (1-372, missing 118) obtained according to the above procedure was 2.4 μm based on amino acid analysis of the final purified product.

Furthermore, the specific activity of plasminogen activator activity was 100 in molar ratio, taking that of the natural form as 100, and was equivalent to that of the natural streptokinase.

Example 7 Streptokinase derivative protein expression Hector p S K X T (1-372, 45-68
The streptokinase derivative protein (1''''372, 45-68
An expression vector for (deletion) was created as follows.

Its outline is as shown in FIG.

■ Chemical synthesis of DNA fragment The derivative protein expression vector used is pSKXT (1-37
Restriction enzyme recognition sites (7524) in 2) were created by replacing the region between V and I with the following new chemically synthesized oligonucleotide (DNA) fragments (row 1 and row 2).

(Nsp(7524) V) o -1CGAAA
TCGACCTGACCTCTGCTATGTCTCA
TAAACTTTAGCTGGACTGGAGACGA
TACAGAGTATTTGAGCT Low 2
(Xho I) This DNA fragment contains a portion where positions 45 to 68 of streptokinase are deleted, and secondary DNA fragments were separately chemically synthesized in the same manner as above.

■ Construction of p SK X T (1-372, 45-68 missing) 1) 1325 bp DNA generated by treating SK Fragment and restriction enzyme Pst
3864 bp D generated by processing with I and Xho I
NA fragments were collected by agarose gel electrophoresis,
After ligating these and the two chemically synthesized NA fragments obtained in ① above, E. coli strain JM109 was transformed with the reaction product, vector DNA was collected from the obtained colony, purified, and restriction enzymes were added. Create a map and select the desired vector pS
KXT (1-372, 45-68 missing) was obtained.

■ Expression and confirmation of derivative protein (1-372/45-68 missing)
Escherichia coli strain 7M-109, which harbors 2,45-68 deletion), was similarly cultured with shaking using the above-mentioned M-9 forcezamino acid liquid medium, and after harvesting, streptokinase activity (plasminogen activator As a result of measuring the
Approximately 1,200 countries are removed from the bacterial fraction/1! The expression level was observed, and most of it was present in the veriplasm fraction. Furthermore, as a result of Western blotting analysis,
The immunoreactivity appeared at a calculated molecular weight of 39,600.

■ Recombinant streptokinase derivative protein (1-3
Production and identification of 72,45 = 68 missing) In the same manner as in Example 5 (■), bacteria were collected from the culture solution 800zA', the periplasmic fraction was extracted by the osmotic shock method, and the target derivative protein (1 -372,45''''68 missing) was collected and purified to obtain a lyophilized purified product.

The amino acid analysis results of the purified product obtained are shown in Table 9 below.

The amount recovered of the recombinant streptokinase derivative protein (1-372, 45-68 missing) obtained according to the above table was 2.5 μm based on amino acid analysis of the final purified product.

Furthermore, the specific activity of plasminogen activator activity was 112 in molar ratio, taking that of the natural form as 100, and was equivalent to that of natural streptokinase.

Example 8 Streptokinase derivative protein expression vector pSK
57GIn substitution) Tosono expression Streptokinase derivative expression vector obtained in Example 5 1) S K
T (1-372) for use, this streptokinase derivative protein (Gin at positions 256 and 257, respectively)
An expression vector was prepared as follows.

Its outline is as shown in FIG.

■ Chemical synthesis of DNA fragment The derivative protein expression vector used is psKXT (1-37
The following new chemically synthesized NA) fragment (bars 1 to 4) is inserted between the restriction enzyme recognition site Hind III and the restriction enzyme recognition site Hind III of 2).
It was created by replacing it with .

()find III) bar IAGCTTA
CCGTATCAACCAGCAGTCTGGTCTG
AATGAAGATGGCATAGTTGGTCGTC
AGACCAGACTTACTTC Bar 4 This DNA fragment is located at position 256 and 2 of streptokinase.
Each contains a portion in which position 57 is substituted with Gin, and is divided into four parts, bar 1 to bar 4, and chemically synthesized in the same manner as above. Note that a new restriction enzyme recognition site EcoRV was set for later analysis.

■ p S K X T (1-372,256GIn
, 257GIn substitution) Construction of p 3 K X 7 (1-372) with the restriction enzyme Hin
608b produced by processing with d III and Nco I
p DNA fragment and restriction enzymes Nco I and 5naB
The DNA fragments of 4612bl) produced by treatment with I were collected by agarose gel electrophoresis, and these were subjected to a ligation reaction with the four chemically synthesized NA fragments obtained in step ① above. After phosphorylating the 5' ends of oligomer bars 2 and 4), E. coli strain JM109 was transformed with the reaction mixture, vector DNA was collected from the obtained colonies, purified, a restriction enzyme map was created, and the target vector was obtained.

■ Derivative protein (1-372, 256GIn, 25
7GIn substitution) Expression and confirmation E. coli JM-109 strain harboring the vector obtained in ① above was similarly cultured with shaking using the M-9 zamino acid liquid medium described above, and after addition of IPTG, the bacteria were collected. , streptokinase activity (plasminogen activator activity)
As a result of the measurement, it was found that the expression level was about 70 cells/xll in the bacterial cell fraction without being detected in the medium supernatant, and most of it was present in the cell plasm fraction. Further analysis by Western blotting revealed that the immunoactivity appeared at a position with a calculated molecular weight of about 42,300.

■ Recombinant streptokinase derivative protein (1-3
Production and identification of 72,256GIn, 257GIn substitution) In the same manner as in Example 5 (■), culture solution 2.41 was centrifuged to collect bacteria, the periplasmic fraction was extracted by the osmotic shock method, and the desired product was extracted from the extract. Derivative protein (1-372,2
56GIn, 257GIn substitution) was recovered and purified to obtain a freeze-dried purified product.

5DS-PAG in the presence of a reducing agent for the purified product obtained.
Analysis using E4 showed a single band at the desired molecular weight of about 42,300.

In addition, the results of amino acid analysis of the same purified product are shown in Section 1 below.
Shown in Table 0.

Table 10 The recovery amount of the recombinant streptokinase derivative protein (1-372, 256GIn, 257GIn substitutions) obtained according to the above procedure was 4.5 μm based on amino acid analysis of the final purified product.

Further, the specific activity of plasminogen activator activity was 7 in molar ratio, taking that of the natural product as 100.

Example 9 Streptokinase derivative protein expression vector pSKXT (1-372,118 missing,
256GIn, 257GIn substitution) and its expression Streptokinase derivative expression vector p3 K XT (1-372,118 missing) obtained in Example 6 and streptokinase derivative expression vector p obtained in Example 8
S K X T (1-372,256GIn, 257
GIn substitution) This streptokinase derivative protein (1-372゜118 deletion, 256GIn,
257GIn substitution) was prepared as follows.

The outline is as shown in FIG.

■ psK xT, (1-372, 118 missing, 25
Construction of p3 K X 7 (1-372,118 missing) with restriction enzyme! ! A 515 bp DNA fragment generated by treatment with ILI I and Hlnd III and p S K X T
14 produced by treating (1-372,256GIn, 257GIn substitution) with restriction enzyme Pst I and phase LII
A 43 bp DNA fragment and a 3335 bp DNA generated by further treatment with restriction enzymes pst engineering I and Hlnd III
A fragment was collected by agarose gel electrophoresis, and these DNA fragments were subjected to a ligation reaction. E. coli JM109 strain was transformed with the reaction product, vector DNA was collected from the resulting colonies, purified, a restriction enzyme map was created, and the target vector p3KXT (1-3
72,118 missing, 256 GIn.

257G1n substitution) obtained.

■ Derivative protein (1-372, 118 missing, 256
Expression and confirmation of GIn, 257GIn substitution) Escherichia coli JM-109 strain harboring the vector obtained in ① above was cultured with shaking in the above-described M-9 zamino acid liquid medium in the same manner.

After adding I PTG, bacteria were collected in the same way and streptokinase activity (plasminogen activator activity) was measured. As a result, it was not detected in the medium supernatant, and approximately 160
The expression level of Kokuyō Seiichi/zl was observed. Most of it was present in the veriplasm fraction. Further analysis by Western blotting revealed that the immunoreactivity appeared at a position with a calculated molecular weight of approximately 42,200.

■ Recombinant streptokinase derivative protein (1-3
72,118 missing, 256GIn, 257GIn substitution) Production and identification In the same manner as in Example 5 (■), culture solution 1.21 was centrifuged to collect bacterial cells, and the periplasmic fraction was extracted by the osmotic shock method. , target derivative protein (1-37
2, 118 deletion, 256GIn, 257GIn substitution) were recovered and purified.

5DS-PAG in the presence of a reducing agent for the purified product obtained.
As a result of analysis using E, a single band was observed at the desired molecular weight of approximately 42,200.

Further, 6N hydrochloric acid was added to the same purified product to hydrolyze it at 110°C for 24 hours, and amino acid analysis was performed using a Hitachi 835' automatic amino acid analyzer. The results are shown in Table 11 below.

Table 1: Recombinant streptokinase derivative protein obtained as described above (1-372,118 missing, 256GIn,
The recovered amount of 257GIn substitution) was 5.4 ■ from amino acid analysis of the final purified product.

Further, the specific activity of plasminogen activator activity was 9.98 in terms of molar ratio, taking that of the natural product as 100.

Example 1o Preparation of streptokinase derivative protein expression vector pS K S K X T (1-372,4
5-68 missing) and p S K X T (1-372
, 256GIn, 257GIn substitution), the present streptokinase derivative protein (1-372, 45-68
Expression vectors for (deletion, 256GIn, 257GIn substitution) were prepared as follows.

The outline is as shown in FIG.

■ Construction of p SK A 1563 bp DNA fragment generated by processing and p SK
(1-372,256GIn, 257GIn substitution) is treated with restriction enzymes Pst I and Mlu I to produce 3
A 661 bp DNA fragment was collected by agarose gel electrophoresis, the DNA fragments were ligated, the E. coli strain JM109 was transformed with the reaction product, and vector DNA was collected from the resulting colony and purified. Then, create a restriction enzyme map and aim (7) vector p SKX.
T (1-372, 45-68 missing.

256GIn, 257GIn substitution) were obtained.

■ Derivative protein (1-372, 45-68 missing, 25
6GIn, 257Gin substitution) Expression and confirmation E. coli strain JM-109 carrying the vector obtained in ① above was similarly cultured with shaking using the M-9 zamino acid liquid medium described above.

After addition of PTG, bacteria were collected in the same manner and streptokinase activity (plasminogen activator activity) was measured. As a result, it was not detected in the medium supernatant, and approximately 40 plasminogens/ill were detected in the bacterial cell fraction. The expression level was observed. Most of it was present in the veriplasm fraction. Furthermore, as a result of Western blotting analysis, the immunoactivity appeared at a position with a calculated molecular weight of about 39,800.

■ Recombinant streptokinase derivative protein (1-3
72, 45-68 missing, 256GIn, 257GI
Production and identification of n-substitution) In the same manner as in Example 5, 0.81 of the culture solution was centrifuged to collect the bacterial cells, the periplasmic fraction was extracted by the osmotic shock method, and the target derivative protein was extracted from the extract. (1-37
2,45-68 missing, 256GIn, 257Gl
n substitution) was recovered and purified.

5DS-PAG in the presence of a reducing agent for the purified product obtained.
As a result of analysis using E, a single band was observed at the desired molecular weight of about 39,800.

In addition, 6N hydrochloric acid was added to the same purified product for hydrolysis at 110°C for 24 hours, and amino acid analysis was performed using a Hitachi 835 automatic amino acid analyzer. The results are shown in Table 12 below.

Table 12 Recombinant streptokinase derivative protein obtained as above (1-372, 45-68 missing, 256GIn
, 257GIn substitution) was found to be 0.56 ■ by amino acid analysis of the final purified product.

Further, the specific activity of blasminogen activator activity was 6.30 in terms of molar ratio, taking that of the natural product as 100.

Example 11 Streptokinase derivative protein expression vector pSKXT (1-372°45-68
Missing, 118 missing, 256 Gln, 257 Gin
Construction of the streptokinase derivative expression vector p3KXT (1-372,1
18 missing) and psKX '1' (1-372,45
-68 deletion, 256GIn, 257GIn substitution)
This streptokinase derivative protein (1-3
72, 45-68 missing, 118 missing, 256 GIn, 2
57G1n substitution) was prepared as follows.

Its outline is as shown in FIG.

■ Construct p sK A 515 bp DNA fragment generated by treatment with p SK
(1-372, 45-68 missing, 256Gln, 2
57GIn substitution) with the restriction enzyme Pst I and! ! IL
A 1371 bp DNA fragment generated by treatment with II and a 3335 bp DNA fragment generated by further treatment with restriction enzymes Pst I and HindIII were collected by agarose gel electrophoresis, and
After ligation reaction of A fragment, E. coli JM109 with the reaction product
The vector DNA was extracted from the obtained colonies by transforming the strain.
A was collected and purified, a restriction enzyme map was created, and the target vector pSKXT (1-372, 45-68
missing, 118 missing, 256GIn, 257GIn replacement)
I got it.

Escherichia coli JM-109 strain carrying the above vector is “E
scherichia coli, JM-109, p
sKXT(1-372゜Δ45-68.Δ118.QQ
) It has been deposited with FERM BP-28 under the designation J and its deposit number is FERM BP-28.
28).

■ Derivative protein (1-372, 45-68 missing, 11
Expression and confirmation of B deletion, 256Gin, 257GIn substitution) Escherichia coli JM-10 carrying the vector obtained in ① above
Nine strains (FERM BP-2828) were cultured with shaking in the same manner using the M-9 zamino acid liquid medium described above.

After adding PTG, bacteria were collected in the same manner and streptokinase activity (plasminogen activator activity) was measured. As a result, it was not detected in the medium supernatant, and approximately 170
The expression level of Kokuyo Seiyu/xl was observed. Most of it was present in the veriplasm fraction. Furthermore, as a result of Western blotting analysis, the immunoactivity appeared at a calculated molecular weight of 39,700.

■ Recombinant streptokinase derivative protein (1-3
72, 45-68 missing, 118 missing, 256 GIn
, 257GIn substitution) Production and Identification In the same manner as in Example 5 (2), culture solution 1.21 was centrifuged to collect bacterial cells, the periplasmic fraction was extracted by the osmotic shock method, and the desired derivative was extracted from the extract. Protein (1-37
2, 45-68 missing, 118 missing, 256 GIn.

257GIn substitution) was recovered and purified.

5DS-PAG in the presence of a reducing agent for the purified product obtained.
As a result of analysis using E, a single band was observed at the desired molecular weight position of 39,700.

In addition, 6N hydrochloric acid was added to the same purified product for hydrolysis at 110°C for 24 hours, and amino acid analysis was performed using a Hitachi 835 automatic amino acid analyzer. The results are shown in Table 13 below.

Table 13 Recombinant streptokinase derivative proteins obtained as above (1-372, 45-68 missing, 118 missing, 2
The recovered amount of 56Gin/257GIn substitution) was 2.4■ from amino acid analysis of the final purified product.

Further, the specific activity of plasminogen activator activity was 7.74 in terms of molar ratio, taking that of the natural product as 100.

Example 12 Streptokinase derivative protein expression vector pSK XT (1-372°256, 25
7Lys-Pro-Lys-Pro modification) and its expression Using the streptokinase derivative expression vector psKXT (1''''372) obtained in Example 5, the present streptokinase derivative protein (-Lys-Pro at positions 256 and 257) was prepared.
-Pro-Lys-Pro-2 modified expression vector was prepared as follows.

Its outline is as shown in FIG.

■ Chemical synthesis of DNA fragment The derivative protein expression vector used is pSKXT (1-37
It was produced by replacing the region between restriction enzyme recognition site Hind III and I in 2) with the following new chemically synthesized NA fragments (nee 1 to knee 4).

(Hind III) Knee-1AGCTT
ACCGTATCAACAAAACCGAAACCGTC
TGGTCTGAATGAAATGGCATAGTTG
TTTGGCTTTGGCAGACCAGACTTAC
TT knee 4 This DNA fragment is located at positions 256 and 2 of streptokinase.
57th place (y) Lys-Lys to Lys-Pro-Lys
-Pro includes the part replaced with -Pro, and carries out the changes of the present invention that involve amino acid substitution and insertion. The oligonucleotides were divided into four oligonucleotides, Knee 1 to Knee 4, and each was chemically synthesized in the same manner as above.

For later analysis, a new restriction enzyme recognition site EcoR was added.
V was set.

■ p S K X T (1-372,256,25
7Lys-Pro-Lys-Pr.

Construction of 1) S K X T (1-372) with restriction enzyme H1
eos generated by processing with nd III and Nco I
A 4612 bp DNA fragment and a 4612 bp DNA fragment generated by treatment with the restriction enzymes μCOI and Parallel I were collected by agarose gel electrophoresis.
After ligation reaction with this chemically synthesized NA fragment (however, the 5' ends of oligomers 2 and 4 were phosphorylated), E. coli strain JM109 was transformed with the reaction product, and the vector DNA was extracted from the resulting colonies. was collected, purified, and a restriction enzyme map was created to obtain the desired vector.

■ Derivative protein (1-3'r2, 256, 25'
7Lys-Pro-Lys-Pro modification) Expression and confirmation E. coli JM-109 strain harboring the vector obtained in ① above was similarly cultured with shaking using the M-9 lysamino acid liquid medium. After adding PTG, collect bacteria and detect streptokinase activity (plasminogen activator activity)
As a result of the measurement, it was not detected in the medium supernatant, but an expression level of about 7 x 11 was observed in the bacterial cell fraction, and most of it was present in the cell plasm fraction. Further analysis by Western blotting revealed that the immunoreactivity appeared at a position with a calculated molecular weight of about 42,500.

■ Recombinant streptokinase derivative protein (1-3
72, 256, 257 Lys-Pro-Lys-Pro
Production and Identification of (Modified) In the same manner as in Example 5 (2), culture solution 2.41 was centrifuged to collect the bacterial cells, the periplasmic fraction was extracted by the osmotic shock method, and the target derivative protein ( 1-37
2, 256, 257Lys-Pro-Lys-Pro modification) were recovered and purified to obtain a freeze-dried purified product.

5DS-PAG in the presence of a reducing agent for the purified product obtained.
As a result of analysis using E, a single band was observed at the desired molecular weight of approximately 42,500.

In addition, the results of amino acid analysis of the same purified product are shown in Section 1 below.
It is shown in Table 4.

Table 1: Recombinant streptokinase derivative proteins (1-372, 256, 257Lys-Pro-
Lys-Pr.

The recovered amount of (change) was determined to be 0.00% by amino acid analysis of the final purified product.
It was 46■.

Further, the specific activity of plasminogen activator activity was 17.7 in terms of molar ratio, taking that of the natural product as 100.

[Brief explanation of the drawing]

FIG. 1 shows a schematic diagram of constructing blocks 1 to 8 from synthetic oligonucleotides and constructing subunits SKA, SKB, SKC, and SKD of the streptokinase gene. Figure 2 shows the plasmid ps carrying each of the above subunits.
KA, psKB, psKc and pSKD were constructed, and streptokinase cloning vector psKX was created from them via plasmid vector psKAB and pSKABX.
A schematic diagram of constructing the is shown. Figure 3 shows the above vector pSKX and plasmid pKTN2.
-2 and streptokinase secretion expression vector psK
A schematic diagram of constructing XT is shown. FIG. 4 is a schematic diagram of the construction of a streptokinase derivative protein (1-372) expression vector according to Example 1. FIG. 5 is a schematic diagram of the construction of a streptokinase derivative protein (1-372/118 deletion) expression vector according to Example 2. FIG. 6 is a schematic diagram of the construction of a streptokinase derivative protein (1-372/45-68 missing) expression vector according to Example 3. FIG. 7 is a schematic diagram of the construction of a streptokinase derivative protein (1-372,256GIn, 257GIn substitution) expression vector according to Example 4. FIG. 8 shows the streptokinase derivative protein according to Example 5 (1-372,118 missing, 256Gln, 257G
FIG. 2 is a schematic diagram of the construction of an expression vector (ln substitution). FIG. 9 shows the streptokinase derivative protein according to Example 6 (1-372, 45-68 missing, 256GIn,
257GIn substitution) Schematic diagram of construction of expression vector. FIG. 10 shows the streptokinase derivative protein according to Example 7 (1-372, 45-68 missing, 118 missing, 25
6GIn. 257GIn substitution) Schematic diagram of construction of expression vector. FIG. 11 shows the streptokinase derivative protein (1-372, 256, 257Lys-Pro-L) according to Example 8.
ys-Pro modification) is a schematic diagram of the construction of the expression vector. (Above) Figure ii Essence I Figure Dan grain ε Figure connection Figure connection 10 Figure far special connection Figure connection diagram procedure amendment (voluntary) Name of the invention Streptokinase-type protein, corresponding gene, correspondence Relation to the case concerning plasmid recombinants, corresponding transformants, and those who amend the manufacturing method Patent applicant Otsuka Pharmaceutical Factory Co., Ltd.

Claims (17)

[Claims] (1) A chemically synthesized gene containing a base sequence encoding streptokinase having the following amino acid sequence. [There is a gene sequence. ] [There is a gene sequence. ] (2) The chemically synthesized gene according to claim (1), wherein the nucleotide sequence encoding streptokinase is as follows. [There is a gene sequence. ] [There is a gene sequence. ] (3) The chemically synthesized gene according to claim (1), which has the following base sequence. [There is a gene sequence. ] [There is a gene sequence. ] (4) A recombinant plasmid obtained by inserting the chemically synthesized gene according to claim (1) into a plasmid vector. (5) A transformant transformed with the plasmid recombinant according to claim (4). (6) C-terminal side 3 of the amino acid sequence of streptokinase that has streptokinase activity and is represented by the following formula (1)
A streptokinase derivative protein having a modified amino acid primary sequence that is deleted from position 73 onward and in which at least one amino acid is deleted, substituted, or inserted. Formula (1): [There is a gene sequence. ] [There is a gene sequence. ] (7) The streptokinase derivative protein according to claim (6), which further has a modified amino acid primary sequence in which Arg at position 45 to Gly at position 68 are deleted. (8) The streptokinase derivative protein according to claim (6), further having a modified amino acid primary sequence in which Phe at position 118 is deleted or substituted with another amino acid residue. (9) The streptokinase derivative protein according to claim (6), further having a modified amino acid primary sequence in which Lys at position 256 and Lys at position 257 are deleted or substituted with other amino acid residues. (10) The streptokinase derivative protein according to claim (6), which further has a modified amino acid primary sequence in which other amino acid residues are inserted after each of Lys at position 256 and Lys at position 257. (11) The streptokinase derivative protein according to claim (6), which has a modified primary amino acid sequence that is a combination of at least two of the modifications described in claims (7) to (10). (12) A chemically synthesized gene encoding a streptokinase derivative protein having the modified amino acid primary sequence according to claim (6). (13) A recombinant plasmid obtained by inserting the chemically synthesized gene according to claim (12) into a plasmid vector. (14) A transformant transformed with the recombinant according to claim (13). (15) The transformant according to claim (14), wherein the host cell is E. coli. (16) A method for producing streptokinase, which comprises collecting and purifying the streptokinase expressed by culturing the transformant according to claim (5). (17) A method for producing a streptokinase derivative protein, which comprises collecting and purifying the streptokinase derivative protein expressed by culturing the transformant according to claim (14).