JPH01202287A - Gene for chemical synthesis of cystatin c, corresponding recombinant plasmid, corresponding transformant and production of cystatin c - Google Patents

Abstract

PURPOSE:To produce cystatin C in high efficiency, by introducing a chemical synthesis gene containing a base sequence coding cystatin C having a specific amino acid sequence into a plasmid vector, thereby forming a recombinant. CONSTITUTION:A chemical synthesis gene containing a base sequence coding cystatin C having an amino acid sequence of formula I is inserted into a plasmid vector to obtain a recombinant plasmid. A host cell is transformed with the recombinant plasmid and the obtained transformant is cultured. The objective cystatin C is produced by collecting and purifying the cystatin C manifested by the culture. The purification of the cystatin C is preferably carried out by hydrophobic chromatography, cationic chromatography, etc. The plasmid vector is preferably pBR322 and the host cell is preferably Escherichia coli.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel chemically synthesized gene, more specifically, a chemically synthesized gene containing a base sequence encoding the amino acid sequence of cystatin C, a plasmid recombinant containing the same, The present invention relates to a transformant transformed with the coarsely ground body and a method for producing cystatin C by culturing the transformant.

Prior Art Cystatin C is a type of thiol protease inhibitor, and is a basic protein with a molecular weight of 13,200 and an isoelectric point of 9.5. It was discovered in the urine of patients with renal failure and in the cerebrospinal fluid of the brains of healthy people in 1961, and its amino acid sequence was determined by Grubb et al. in 1982 (proc.

Natl, Acad, Sci, LISA, 79.
3024 (1982)). This amino acid sequence is shown in formula (1) below.

In addition to the above-mentioned cystatin C, various other cystatin groups having similar structures are known to exist in humans and animals, and among the secondary cystatin family, cystatin C is known to be a member of thiol proteases such as papain and cathepsin. It has the strongest inhibitory activity against.

Cystatin C is also localized in human body fluids, particularly semen, brain and cerebrospinal fluid, and abnormal excretion into the urine has been reported in patients with renal failure and systemic lupus erythematosus disease. The significance has not yet been elucidated.

Furthermore, muscular dystrophy, which is an intractable disease, and so-called lysozyme storage disease, in which lipids and mucopolysaccharides accumulate within cells, are both thought to be caused by abnormal hexadylation of intracellular thiol protease activity. In fact, there are reports that the above-mentioned diseases can be cured by inhibiting this enzyme.

In addition, certain thiol proteases play an important role in infection with poliovirus (polio paralysis virus) and influenza virus, and cystatin C has been shown to have an inhibitory effect on infection with second-class viruses in in vitro experiments. It has been reported (J, Cel!, Bioch
em,, 32.91 (198B>).

As described above, cystatin C has great potential for medical use as a therapeutic agent for diseases such as muscular dystrophy, lysosomal storage disease, and various viral diseases, all of which currently have no effective therapeutic agent. However, it is extremely difficult to obtain a sufficient amount of cystatin C by extraction from human biological samples, and therefore, research is currently not being conducted at the biological level to confirm the pharmacological effects of cystatin C on the above-mentioned diseases. In order to carry out such research and to elucidate the physiological functions of cystatin C itself, there is a long-awaited development in the art of a technology for producing cystatin C in large quantities with high purity.

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to easily convert cystatin C into
A production method using genetic recombination technology that can be produced in high purity and in large quantities, in particular a base sequence encoding cystatin C for this method, a chemically synthesized gene containing this, and a product in which the gene is inserted. The object of the present invention is to provide a plasmid recombinant (cystatin C expression vector), a host cell (transformant) transformed with the recombinant, and a culture technique for the same.

Means for Solving the Problems According to the present invention, a chemically synthesized gene containing a base sequence encoding cystatin C represented by the amino acid sequence of formula (1) below, a corresponding plasmid recombinant into which the gene is inserted (cystatin C expression vector), a transformant transformed with the recombinant, and a method for producing cystatin C by culturing the same.

Ser-3ep-Pro-Gly-Lys-Pro-P
ro-Arg-Leu-Val-Gly-Gly-
Pro-Met-Asp-81a-3er-Val-G
lu-Glu-Glu-Gly-Val-ArO-^r
g-Ala-Leu-Asp-Phe-Ala-'V
al-Gly-Glu-Tyr-Asn-Lys-Al
a-3er-Asn-Asp-)1et-Tyr-11
i 5-3er-^rg-Ala-Leu-Gln-V
al-Val-Ara-Ala-Arg-Lys-Gl
n-11e-Val-Ala-Gly-Val-Asn
-Tyr-Phe-Leu-Asp-Val-Glu-
Leu-Gly-Arg-Dinghr-Thr-Cys-T
hr-Lys-Thr-Gln-Pro-Asn-Le
u-Asp-Asn-Cys-Pro-Phe-old s-
Asp-Gln-Pro-His-Leu-Lys-A
rg-Lys-Ala-Phe-Cys-3er-Ph
e-Gl n-I l e-Tyr-A l a-Va
I-Pro-Trp-Gln-Gly-T
hr-Met-Tyr-Leu-3er-Lys-3e
r-Thr-Cys-Gln-Asp-Ala the above formula (
1) and the amino acid sequences and abbreviations of each amino acid in this specification below, as well as base sequences and nucleobases,
Other abbreviations shall be used in accordance with the IUPAC-IUB regulations or common symbols in the field, examples of which are listed below.

Ala...Alanine Arg...Arginine A
sn...Asparagine ASp...Asparagine destruction CVS...Cystine Gln...Glutamine (
31u...Glutamic acid Gly...Glycine HiS
...Histidine ■le...Isoleucine leu
...Leucine LyS...Lysine Met...
Methionine 1) he...phenylalanine pro
...Proline 3er...Serine Thr...
Threonine Trl)...Tryptophan Tyr/
...Tyrosine Val...Valine A...
Adenine T...Thymine G...Guanine C...Cytosine Below, regarding the cystatin C production technology of the present invention, the amino acid sequence of cystatin C represented by the above formula (1) Design and synthesis of the base sequence encoding the nucleotide sequence and the chemically synthesized gene of the present invention containing the same, construction of a plasmid recombinant vector containing the gene, transformation of host cells by introduction of the vector, culture of the transformed cell, and cystatin The production and purification of C will be sequentially explained.

The nucleotide sequence encoding the amino acid sequence of cystatin C is
Various settings can be made based on the amino acid sequence of formula (1), but in that case, it is preferable to adopt the following criteria.

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

ii) A specific restriction enzyme recognition site is provided within the set base sequence and at both ends thereof, and the site is arbitrarily manipulated to combine or link with the base sequence of other genes, insert a plasmid vector, etc. make it easier.

1ii) 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.

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

A preferred example of the base sequence (gene) encoding the amino acid sequence of cystatin C designed according to the above is shown in the following formula (2).

5° TCT TCCCCG GGT AAA3'
AG8 AGG GGCCCA DingITCCG
CCG CGCCTG GTT GGT G
GCCCG 8TG GACGGCGGCGCG
GACCAA CCA CCG GGCTACCTG
CG Ding CCGTA GAG GAA GAG
GGT GTT CGT CGTCGCAG
G GAT CTCCTT CTCCCA C
AA GCA GCAGCA CTG GAT
TTCGCA GTT GGCGAA TA
CAACCGT'GACCTA AAG CGT
CAA CCG CTT ATG King TGA
AA GCT DING CCAACGACATG TA
CCACTCCCGTTTT CGA AGG
TTG CTG TACATG GTG AG
G GCAGCT CTG CAG GTA GTT
CGCGCT CGT AAA CAGCGA GA
CGTCCAT CAA GCG CGA GCA T
TT GTC＾TCGT＾ GCA GGCGTT
AACTAC-TCCTG GACTAG CAT
CGT CCG CAA TTG ATG AAG
GACCTGGTCGAA CTG GGCCGT A
CT ACT TGCACT AAACAG CTT
GACCCG GCA TGA TGA ACG TG
A TTTACCCAG CCG AACCTG
GACAACTGCCCG TTCTGG GT
CGGCTTG GACCTG TTG ACG
GGCAAGCAT GACCAA CCG
CAT TTA AAA CGT AAA
GCTGTA CTG GTT GGCGTA
AAT TTT GCA TTT CGA
TTCTGCTCCTTCCAG ATCTACGCT
GTT CCGAAG ACG AGG AA
G GTCTAG ATG CGA CAA
GGCTGG CAG GGT ACCATC
ACCCTG TCT AAA TCCACCG
TCCCA TGG TAC GG GACAG
A TTT AGGACT TGCCAG GAC
GCA 3゜TGA ACG GTCCTG CG
T 5° (2) 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 cystatin C of the above formula (1); Each heavy chain DNA sequence also
Also included in the present invention.

Furthermore, the base sequence of formula (2) above was designed by preferentially selecting and combining codons that are frequently used by E. coli, taking into consideration the use of E. coli as a host cell. Genes are not limited to this. Furthermore, the host cells used in the present invention are not limited to E. coli, as described below, and therefore, depending on the host cell used, the genes of the present invention can be expressed by selecting and combining codons that are frequently used by the cells. Can be built.

That is, the nucleotide sequence of the gene of the present invention may be any sequence as long as it contains the nucleotide sequence encoding the amino acid sequence of cystatin C and can express and produce cystatin C by genetic recombination technology. (1
), it is possible to modify or modify the base sequence by slight changes, deletions, additions, etc. Base sequences that have undergone such alterations or modifications also have the formula (
As long as it has the same genetic information as the base sequence shown in 1), it is included in the present invention. Modification or modification of a secondary base sequence is known in the art, and specifically, modification or modification of a secondary base sequence is known in the art. It is to adopt.

In addition, the modification (addition) of the base sequence described above involves actually inserting the resulting base sequence into an appropriate vector and linking it with various regulatory factors such as promoters necessary for expression in microorganisms. , addition of various restriction enzyme recognition sites, etc.

That is, the gene of the present invention can be used to construct a desired cystatin C expression vector by genetic engineering techniques, using the promoter, Shine-Dalgarno sequence (s
It is necessary to appropriately link various regulatory factors such as liposome binding sites such as h1ne-oa + garno sequence, SD sequence, protein synthesis initiation codon, termination codon, etc.
Operations such as cutting and joining each base sequence are performed using restriction enzymes, so the desired gene can be
Appropriate restriction enzyme recognition sites must be added before and after the gene, and genes to which appropriate restriction enzyme recognition sites are added are also included in the present invention.

An example of this is shown in the following equation (3). This is obtained by adding specific restriction enzyme recognition sites before and after the base sequence of formula (2) for linking with various regulatory factors such as promoters necessary for the expression of the base sequence. It is particularly suitable for use in subsequent construction of expression vectors. The restriction enzyme recognition site present in the base sequence represented by formula (3) is shown in formula (4). Of course, the restriction enzyme recognition site shown in formula (4) is just an example, and the restriction enzyme recognition site that should be present in the gene of the present invention may be appropriately changed or selected from various known sites depending on the type of cystatin C expression vector to be constructed. can.

5' AA TTCGGA TCCATG TCT
TCCCCG GGT AAA3° G CCT
AGG TACAGA AGG GGCCCA TT
TCCG CCG CGCCTG GTT GGT G
GCCCG ATG GACGGCGGCGCG G
ACCAA CCA CCG GGCTACC1
GGCG Ding CCGTA GAG GAA G
AG GGT GTT CGT CGTCGC
AGG CAT CTCCTT CTCCCA CAA
GCA GCAGCA CTG GAT TTCGC
A GTT GGCGAA TACAACCGT G
ACCTA AAG CGT CAA CCG
CTT 8TG TTGAAA GCT
TCCAACGACATCDINGACCAC,TCCCGT
TTT CGA AGG TTG CTG TACAT
G GTG AGG GCAGCT CTG CAG
GTA GTT CGCGCT CGT AAA CA
GCGA GACGTCCAT CAA GCG
CGA GCA TTT GTCATCG18 GCA GGCGTT 8ACTACTTCCT
G GACTAG CAT CGT CCG
CAA TTCATG AAG GACCTG
GTCGAA CTG GGCCGT ACT ACT
TGCACT AAACAG CTT GACC
CG GCA TGA TGA ACG T
GA TTTACCCAG CCG AACCT
G GACAACTGCCCG TTCTGG
GTCGGCTTG GACCTG TTG A
CG GGCAAGCAT GACCAA CCG
CAT TTA AAA CGT AAA GCTGT
A CTG GTT GGCGTA AAT
DingTT GCA TTT CGATTCTGC
Ding CC Ding TCCAG ATCTACGCT GTT
CCGAGG ACG AGG AAG
GTCTAG ATG CGA CAA GGC
TGG CAG GGT ACC8TG AC
CCTG TCT To A TCCACCGTCC
CA TGG TACTGG GACAGA
TTT AGGACT TGCCAG GACG
CA Ding AATAG3゜TGA ACG GTCCT
G CGT ATT ATCAGCT 5゜GG-C
GTCGTGCACTGGATTTCGCAGTTGG
CGAATACAAAC8CCCAGCCGAACCDingG
GACAACTGCCCGTTCCATGACCAAC
C Another specific example of the synthetic gene of the present invention provided with the appropriate restriction enzyme recognition site is the following formula (5):
An example is the base sequence shown in

CCG CCG CGCCTG GGT GC
T GGCCCG ATG GACGGCGGC
GCG GACCAA CCA CCG GGCTAC
CTGGCG TCCGTA GAG GAA GAG
GGT GTT CGT CGTCGCAGG C
AT CTCCTT CTCCCA CAA
GCA GCAGCA CTG GAT TT
CGCA GTT GGCGAA TACAAC
CGT GACCTA AAG CGT CA
A CCG CTT ATG TTGAAA
CCT TCCAACGACATG TACCAC
TCCCGTTTTT CGA AGG TTG
CTG TACATG GTG AGG GC
AGCT CTG CAG GTA GTT CGCG
CT CGT AAA CAGCGA GACGTC
CAT CAA GCG CGA GCA
TTT GTCATCGTA GCA GGCG
TT AACTAC King TCCTG GACTAG
CAT CGT CCG CAA TTG ATG A
AG GACCTGGTCGAA CTG GGC
CCT ACT ACT TGCACT AAA
CAG CTT GACCCG GCA TGA TG
A ACG TGA TTT8CCCAG CCG
AACCTG GACAACTGCCCG TT
CTGG GTCGGCTTG GACCTG
TTG ACG GGCAAGCAT GACC
AA CCG C^T TTA AAA C
GT AAA GCTG Ding AC Ding G GTT
GGCGTA AAT TTT GCA TT
T CGATTCTGCTCCTTCCAG AT
CTACGCT GTT CCGAAG ACG
AGG AAG GTCTAG ATG
CGA CAA GGCTGG CAG G6
Ding ACCATG ACCCTG TC Ding AAA
CCACCGTCCCA TGG TACT
GG GACAGA Ding ■ Ding AGGACT Ding GCCAG GACGCA TAA TAG
3゜TGA ACG GTCCTG CGT
ATT ATCAGCT 5゜The gene of the present invention represented by the above formula (5) is cleavable with the restriction enzyme [si■] immediately before the nucleotide sequence encoding cystatin C contained in the gene of the present invention shown in the above formula (3). This nucleotide sequence is advantageous for constructing a cystatin C secretion expression vector, as described later.

The gene of the present invention as described above can be easily chemically synthesized according to the designed base sequence using a commercially available DNA synthesizer or the like according to a conventional method. The method includes, for example, the solid-phase phosphotriester method (Nature
, 310.105 (1984)), solid phase phosphoricidide method (S, L, BeaLICa(le.

and H, H, Caru extracts, Tetrahed
ron Letters, 22.

1859 (1981) )). The synthesized DNA fragments can be isolated and purified by conventional methods such as high-performance liquid chromatography, and each purified base sequence can be confirmed by secondary development using homochromatography (E, Jay, etc.). , R,A.

Bambara, R., Padmanbhan and
R, Wu, Nucleic Ac1dsRed, , Yu, 331 (1974)) and the Maxam-Gilbert method (^, H, Haxam and W, G11bert
, Proc., Natl.

Acad, Sci,, USA, 74.560 (19
77) :A,M.

Max and W, Gi 1bert, ) let
hods in Enzymol, 65.

499, Acad, Press (19130)
), etc., respectively.

Regarding the synthesis of the gene of the present invention, taking the base sequence of formula (4) as an example, this consists of the first half (subunit divided into two parts (subunit Y),
They can be advantageously synthesized by separately constructing them. This method will be explained in detail below.

In the above method, first, in order to synthesize subunit X, oligonucleotide fragment A having 37 to 46 bases is
-1 to A-5 and B-1 to B-5 are synthesized. In addition, for the synthesis of subunit Y, oligonucleotide fragments C-1 to C-4 and D-1 to D-4 each having 39 to 51 bases.
Synthesize. The number of bases and base sequences of each oligonucleotide fragment are shown in Table 1 below.

Table 1 Next, as shown in Figure 1, the oligonucleotide fragments are assembled and linked to create Block 1, Block 2, Block 3, and Block 4, and further assembled and linked to form the desired subunit. Obtain X and subunit Y.

The nucleotide sequences of each subunit thus obtained are as shown in the following formulas (6) and (7), respectively, and the RI and DAN restriction sites are present at both ends thereof.

<Subunit X><SubunitY> The above-mentioned subunit
It can be integrated into the RI and 3alI restriction enzyme cleavage sites. Specific examples thereof are shown in Examples below. Each of the plasmids thus obtained (plasmid pCYS-X and plasmid DCYS-Y) was subjected to, for example, the calcium method (E., Lederberq, S., Cohen,
J, Bacteriol,, 119°1072 (1
These can be transformed into E. coli according to conventional methods such as 974), and thereby can be stored and amplified.

By cutting out the subunits possessed by each of the above-mentioned plasmids and ligating them, a ligated product containing the cystatin C gene of the present invention represented by the base sequence of formula (3) can be obtained. By integrating this ligated product into a suitable plasmid vector, such as pBR322, a desired recombinant plasmid of the present invention, ie, pcYs-XY, which will be described in detail in Examples below, can be obtained. Like the plasmid pcYs-X and plasmid DCYS-Y, this plasmid can also be transformed into a suitable host cell such as E. coli, and can be stably stored and amplified within the microorganism.

Furthermore, the nucleotide sequence represented by the above formula (5), which is another preferred specific example of the synthetic gene of the present invention, is a plasmid pcYs- It is obtained by cutting out a portion containing the above base sequence from XY and ligating a predetermined synthetic oligonucleotide thereto, and the base sequence thus obtained is similarly inserted into an appropriate plasmid vector such as pBR322 to obtain the desired nucleotide sequence. The recombinant plasmid of the present invention, that is, pCYS, which will be detailed in the Examples below.
-XYA, which can also be transformed into a suitable host cell such as E. coli, and can be stably stored and amplified within the microorganism. In particular, the base sequence represented by this formula (5) has an N5iI restriction enzyme cleavage site immediately before the translation initiation codon of the cystatin C gene, and using this, for example, secretion expression can be performed immediately after the codon. There is an advantage that a base sequence encoding a signal peptide for use as a system vector can be linked. I will write more about the details.

The E. coli JM-103 strain harboring the above-mentioned plasmid recombinant pCYS-XYA is rEscherichia
coli, Jt4-103. It has been deposited with the designation pcYs-XYAJ as FERM P-9799> at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.

In constructing each of the above-mentioned plasmid vectors, usual operations, methods, etc. in genetic recombination technology can be employed. This involves processing DNA using various restriction enzymes, S1 nuclease, etc.
cleavage treatment, DNA ligation treatment using T4 DNA ligase, etc., DNA isolation and purification by agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc., DNA recovery and purification by phenol extraction method, etc. .

In addition, the presence of each plasmid vector in host cells can be confirmed using the alkaline-3DS extraction method (H, C, Birn
boim, and J. Doly, Nucleic
Ac1dsRes, 7.1513 (1979
)), etc., and then treating the plasmid DNA with various restriction enzymes and examining the presence or absence of secondary restriction enzyme recognition sites and the length of the resulting NA fragments.

Furthermore, for example, gene base sequences can be directly analyzed using the dideoxy method (F, Sanger, et al., Proc, Na.
tl, Acad, Sci.

USA, 74.5463 (1977)-) and the like.

The source vector used for the construction of the recombinant of the present invention is preferably pBR322 or various plasmid vectors derived therefrom, but is not particularly limited to these, and various conventionally known vectors may be used. , various viral vectors including bacteriophages and animal and plant viruses,
Various plasmids, cosmids, etc. may be used.

In order for the transformant obtained by introducing the gene to express the target cystatin C, the vector carrying the gene of the present invention must contain, in addition to the gene of the present invention, various regulatory factors necessary for its expression, such as a promoter. It is necessary to have factors for transcription such as a transcription termination signal, a poly A chain addition signal (when a eukaryotic cell is used as a host cell), and factors for translation such as a bosome binding site. Such regulatory factors are well known depending on the host cell, and examples of promoters include υ'-promoter, EnL promoter, Nihe promoter, λPL promoter, Mark L promoter, Nihe promoter, etc. in E. coli, and Bacillus subtilis promoter. 5PO1 promoter, 5PO2 promoter, concave L promoter, etc., and PH0 promoter in yeast and other eukaryotic cells.
Examples include the 5 promoter, the PGK promoter, the GAP promoter, the ADHI promoter, and the SV40-derived promoter. 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 them from a plasmid containing them according to a conventional method, or by chemically synthesizing them. By inserting them into an appropriate vector, each can be present in the vector of the present invention, and thus a desired cystatin C expression vector can be obtained.

The cystatin C expression vector (recombinant) thus obtained has cystatin C expression information, which is obtained by linking a promoter and a ribosome binding site upstream of the structural gene of the present invention, and a transcription termination signal downstream. By introducing it into a suitable host cell and transforming the cell, the cell can be caused to express (produce, accumulate) the desired cystatin C.

In particular, when Escherichia coli is used as a host cell, the expression system vectors include both a system in which the target cystatin C is directly expressed within the bacterial body and a system in which it is secreted and expressed in the periplasmic layer. According to the latter secretion expression system, the target cystatin C is expressed in Escherichia coli as a fusion protein with a signal peptide, and transferred to the inner membrane by the action of the signal peptide, where the signal peptide is enzymatically processed by a signal peptidase. In this way, only the desired cystatin C is selectively accumulated in the periplasmic layer of E. coli. Therefore, by using this secretion expression system, there is an advantage that the target protein can be stably stored in the periplasm layer while avoiding the risk of decomposition of the target protein by proteases etc. present in the E. coli body. The protein can be easily separated and purified from the periplasm layer, and the fusion protein transferred to the inner membrane is selectively cleaved by signal peptidase immediately after the signal peptide, that is, just before the target protein, eliminating any other unnecessary amino acid sequences. It has the advantage of being able to collect only the target protein that does not have the same amount.

Such a secretory expression system can be constructed using a base sequence in which a gene encoding a signal peptide is directly linked upstream of the 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 precursors such as various secreted proteins. The signal peptide acts to guide the protein out of the membrane, and is itself separated from the precursor, and thus can secrete and express secretory proteins etc. by the action of the signal peptide. The use of such signal peptides in gene recombination methods and the advantages thereof are known, and are described in detail in, for example, Japanese Patent Application Laid-Open No. 149089/1989.

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 these. Specific examples include Escherichia coli β-lactamase (bla), phosphate binding protein (p
hos), alkaline phosph 7turbi(1) hOA>
Examples include signal peptides of E. coli outer membrane proteins (OmpA, Sarasu-F, LPP, 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 nucleotide sequence encoding a colon signal peptide, more specifically, it has genetic information in which the tac promoter, SD sequence, and nucleotide sequence encoding a signal peptide are arranged in the same direction. Escherichia coli JM103 strain carrying pKTN is
ESCtlOriChiacoli, J)l-103
, DKTN-2-2J is displayed, and the number is 914.
6M (FERM P-9146).

As a preferable example of the secretory expression system of the present invention, the above-mentioned p
An example is the plasmid pCYS-XYATB, which is constructed from KTN and pCYS-XYA and will be described in detail in Examples below. The secretory expression vector pCY S -X Y
A T B Ha, especially Ni'ola-)'; A base sequence in which the first codon encoding the first amino acid of the cystatin C gene is directly linked after the base sequence encoding the It peptide, i.e., the above-mentioned signal peptide. and cystatin C
It has a nucleotide sequence that encodes a fusion protein with Cystatin C, and the fusion protein of the signal peptide and cystatin C is expressed in E. coli by the action of the tac promoter without fear of shifting the reading frame, and it passes through the inner thigh. This allows only the desired cystatin C to be secreted and accumulated in the periplasmic layer.

Escherichia coli JM103 strain carrying this plasmid pcYs-XYATB is [Escherichia co! i
.

JH-103, pcYs-XYATB j is displayed,
FERM P-9800>
It has been deposited as.

The cystatin C expression vector of the present invention obtained as described above can be introduced (transformed) into an appropriate host cell to impart the cystatin C-producing ability of the present invention to the host cell. The host cells used here are not particularly limited, and may be any of various known types, such as Gram-positive 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 these, H81 derived from 12 strains
01 strain (H, W, Boyer and D, Roull
and-Dussoix, J.) lol, Biol
,, 41.459 (1969)) and JMI03
(J.) Lessing et al., Nucl.
eic^cids Res,, 9.309 (198
1) ) 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 calcium chloride, and a vector is added to the solution (E, L
ederberg and S., Cohen.

J, Bacteriol, 119. 1072
(1974) ), etc. can be exemplified.

The present invention also provides host cell transformants transformed according to the above.

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 media such as L-broth medium, E medium, and M-9 medium can be used. Furthermore, various commonly known carbon sources, nitrogen sources, inorganic salts, vitamins, natural product extracts, physiologically active substances, etc. can also be added to these media.

Cultivation can be carried out by various methods employing conditions such as DH1 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 suitable, 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 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, for example, in E. coli transformed with the cystatin C secretion expression vector of the present invention, the fusion polypeptide is produced in the cytoplasm, and then secreted into the periplasm by the action of the signal peptide, and at the same time, the fusion polypeptide is secreted into the periplasm by the action of the signal peptidase. The signal peptide is separated from the polypeptide, and the desired cystatin C is secreted and accumulated in the periplasmic layer.

Cystatin C accumulated in the periplasmic layer can thus be separated and purified by conventional methods. This separation and purification operation can be carried out using, for example, gel filtration, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography, etc. alone or in an appropriate combination on the periplasm layer prepared by the osmotic shock method. Particularly, those secreted into the periplasm layer or into the culture supernatant have the advantage of being relatively easy to separate and purify.

Thus, according to the present invention, cystatin C can be produced by genetic recombination technology. The obtained cystatin C can be confirmed by, for example, high performance liquid chromatography (l
-1PLC), a single band by polyacrylamide gel electrophoresis (PAGE), etc. can be easily used as indicators. Furthermore, the target highly purified cystatin C can be identified using methods similar to those used for structural analysis of ordinary polypeptides or proteins, such as 5D
This can be carried out by molecular weight analysis by S-PAGE, isoelectric point measurement by isoelectric focusing, measurement of amino acid composition by an amino acid analyzer, amino acid sequence analysis by an amino acid sequencer, etc.

EXAMPLES Examples and reference examples are given below to explain the present invention in more detail. It should be noted that each method and operation used on each side 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. and Toyobo Co., Ltd., and the reaction solution used had the composition specified by each company. The reaction temperature was 37°C, and the mixture was left standing in a hot bath for 3 hours to react.

The standard amount of restriction enzyme used was 1 unit per 1 μq of DNA, and the final reaction volume was 100 μQ. A sterilized 1.5 mG 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, TE buffer saturated phenol [1mM
Add 10mM Tris-HCl containing EDTA (pH 8.0 > buffer saturated with phenol), stir thoroughly, and then centrifuge (12,000 rpm).
Then, the same amount of ether was added and the aqueous layer was collected by centrifugation. This operation was repeated 2 to 3 times. Add 0.1 volume of 3M sodium acetate buffer (pH 4,
8> and 2.5 times the volume of cold ethanol, stir once, leave at -80'C for 30 minutes or more, and centrifuge (12
000 revolutions/min) to precipitate and collect DNA.

3.81 Plant end of DNA by nuclease treatment Dissolve the DNA in an aqueous solution containing 30mM sodium acetate (pH 4,8), 50mM sodium chloride, and 1mM zinc sulfate, and add 8 m to 3 units per 1 μq of DNA.
1 nuclease (manufactured by BRL) and incubated at 37°C.
The reaction was allowed to proceed for 0 minutes. The final reaction solution volume was 100 μQ.

Then 2μQ of 0.5M EDTA and 1M Tris-HCl (p
The reaction was terminated by adding 1 μQ of H8,0), and then DNA was collected by the phenol extraction method described above.

4. Linking of DNA fragments using T4 DNA ligase 6'7mM Tris-HCl (pH 7,6), 6.7mM magnesium chloride, 10mM dithiothreitol and 1mM
Dissolve the DNA in an aqueous solution containing ATP, and
T4 DNA ligase in an amount of 1 unit for q (
(manufactured by Takara Shuzo Co., Ltd.) and incubate at 12℃ for 5 hours or more or 4 hours.
DNA was bound by reacting at °C overnight or more. The final reaction solution volume was 100 μQ. After the reaction was completed, DNA was collected using the phenol extraction method described above.

5. Transformation method As the host cell, Escherichia coli HB-101 strain or JM-10
Three strains were used.

The host cell line was grown in L-broth medium (1% Batato tryptone, 0.5% Bat yeast extract, 0.5%
% sodium chloride) at 37°C until the absorbance at 600 nm reached 0.25. 10 m of this culture solution (2) was centrifuged (7000 rpm, 5 minutes) to collect bacterial cells and cooled on ice. To this was added 5 m of 0.1 M magnesium chloride to suspend and wash. The bacterial cells were collected by centrifugation (7000 rpm, 1 minute), and then added with ice-cooled 0, 1M calcium chloride and 0.
05M magnesium chloride mixed solution was added and suspended. After leaving this in water for more than 30 minutes, centrifugation (7000 rpm, 1 minute) was performed to collect the bacterial cells, and the same solution was added again. It was suspended in .5mQ.

To 0.2 ml of this suspension was added 20 μQ of a reaction composition of DNA ligated using -T4 DNA ligase, and 30
Cool on ice for a minute. Next, the mixture was heated in a 42.5°C hot bath for 30 seconds, 1.0 ml of L-broth medium was added, and this was left standing in a 37°C hot bath for 1 hour.

Thus, 1q transformed strains were selected using the following antibiotic resistance as an indicator. That is, L- containing 1.5% agar
A solution of the reaction composition solution obtained above was added to a plate medium prepared by adding 50 μg of ampicillin/Peng to a broth medium.
．． A 2 mQ portion was spread out and cultured overnight at 37° C., and growing colonies were isolated.

6. Plasmid isolation and purification The plasmid-carrying strain was treated with ampicillin 50μCI
/ill? L-broth medium 400TII supplemented with
Q and incubated at 37°C for 12-16 hours. This was centrifuged (6000 rpm, 10 minutes) to collect the bacterial cells, and this was added to solution I [50mM glucose, 10mM
EDTA, 25mM Tris-HCl (pH 8, O) and 2
mMmi2 'J'/Team, 14m for sterilized stool]
f2 was added and suspended, and the mixture was left in water for 30 minutes. Further, solution II [0,2N sodium hydroxide and 1%
28 mg of sodium dodecyl sulfate] was added, stirred, and left in water for 5 minutes.
pH 4.8>, 21 mg of sterilized stool] was added and left in water for 60 minutes or more. Centrifugation (8000 rpm,
10 min), collect the supernatant and incubate 2.5x m (150 IT
112> (7) Add cold ethanol and incubate at -80℃ for 30 minutes.
The precipitate was obtained by centrifugation (8000 rpm, 10 min). To this, 111 ml of solution IV [0.1M sodium acetate and 0.05M Tris-HCl (p108.0>) was added. Added and dissolved.Additionally 2.5 times the volume (2.
7.5 mQ) of cold ethanol was added and left at -80°C for 30 minutes. Another centrifugation (12,000 rpm,
15 minutes) and collected the precipitate.

Then, TE buffer [a solution of 10mM Tris-HCl (pH 7.5> and 1mM EDTA)] was added to the obtained precipitate for 4 hours.
Add mQ and dissolve it, and add cesium chloride 4.62C to this.
Add 1 and stir to dissolve, then add ethidium bromide 5
m(]#1G solution 0.42mf2 was 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). Upon 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 A-50 (Biogel
A-50.

Bio-Rad) column chromatography (2,
5cm x 15-20cm column size, elution solvent = T
E buffer + 0.5M sodium chloride solution, UV 25
Cesium chloride and contaminating RNA, etc., were removed using a 4-nm chromatography method (detection at 4 nm), and plasmid DNA was recovered using the phenol extraction method described above.

The amount of purified plasmid DNA obtained was determined at OD 260nm
0D26o=0.022 by measurement is 1μq/mQDN
Calculated in terms of AI.

7. Synthesis of oligonucleotides Chemical synthesis of NA is carried out by Applied By A Chemicals (
Model 381A DNA synthesizer (Nodel 381A) manufactured by Happlied Biochemicals
(type DNA5ynthesizer) [solid-phase β-cyanoedyl phosphoramidide method, using a 0.2 μM column]. Its operation followed the company's manual.

The oligonucleotide obtained by the synthesizer still has a column carrier resin attached to its 3' end, I% retention to each active group, and a dimethoxyl trityl group to its 5' end. , etc., and this operation also followed 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. Ia j force for this iron 7muha YHc
A-PACK AM-303003 (manufactured by Yamamura Chemical Research Institute, 4, 6 x 250mm, Waterstyp
e), and gradient elution was performed using 5% → 40% acetonitrile 10.1 M triethylammonium acetate aqueous solution (pH 7.2>) as the elution solvent.The system used was a pump CCPM manufactured by Tosoh Corporation; Ultraviolet-visible detector Uv-8000, controller CP.

8. Agarose gel electrophoresis Acarose gel concentration was 0.9% or 1.6%.

Agarose (manufactured by Isotope Kagaku Kenkyusho) was weighed out to the above concentration, TBE buffer (containing 0.089 M Tris-boric acid and 0.02 MEDTA) was added, and the mixture was dissolved by heating to form a gel. The electrophoresis was performed using a mini gel electrophoresis system Mupid 2 (Cosmo Bio Co., Ltd. 1) and a TBE buffer as the electrophoresis buffer. After electrophoresis, the gel was suspended in a 0.5 μq/mQ Ethidi 1 cum bromide solution, and DN, which emits fluorescence when irradiated with ultraviolet rays, was added.
Fragment A was confirmed. To elute DNA from the gel, cut out the desired band with a knife, etc., and insert it into a dialysis tube (350
The cells were placed in a 0 MW cut), filled with TE buffer, and electrophoresed for 30 minutes using the same MiniGlu electrophoresis device.

The DNA eluted above was concentrated and solidified, a small amount of distilled water was added thereto, and the DNA was recovered according to the phenol extraction method described above.

9. Base sequence analysis The constructed base sequence of cystatin C was obtained using the M-13 dideoxy method (J. Hessing, Methods in
Enzymolo! IIV, 101, 20 (19
83) ). The operation is performed using Toyobo Co., Ltd.'s M-13 Sequencing Kit ()l-13se.
quenching kit) ([α-32P] dCTP
Amajam Co., Ltd.] was used according to the kit's manual. As a running gel, FUJIGENSORGEL 5IIEET (30
802, 8% concentration) was used.

Example 1 Construction of DCYS-XY ■ Synthesis of oligonucleotides In constructing the entire base sequence shown in formula (3) above containing the structural gene of cystatin C, first the same base sequence was converted to 37 to 5
18 oligonucleotide fragments of one chain length (A-1 to A-
5, B-1 to B-5, C-1 to C-4 and D-1 to D-
4), and each fragment was synthesized by the solid phase β-cyanoethylphosphoramidide method. Each fragment, its base chain length, and base sequence are as shown in Table 1 above.

(2) Linkage of synthetic oligonucleotides Construction of the entire base sequence shown in formula (3) above was carried out separately for subunit X and subunit Y. Subunit X consists of the first half of the entire base sequence, from the FCORI restriction enzyme recognition site to the restriction enzyme recognition site. Also subunit Y
is the second half 1 of the entire base sequence. A, Consists of an EC0RI restriction enzyme recognition site including an atl restriction enzyme recognition site to an alI restriction enzyme recognition site.

The base sequences of these are as shown in formulas (6) and (7) above.

In constructing each subunit, subunit X was divided into blocks 1 and 2 as described above, and subunit Y was divided into blocks 3 and 4, each of which was constructed separately.

The construction of each block was carried out as follows, and its outline is shown in FIG. In FIG. 1, the black arrow (dot) at the end of the solid line indicating each chemically synthesized oligonucleotide indicates the 5' terminal phosphate group.

Among the 18 chemically synthesized oligonucleotides A-1 to D-1 above, 1 excluding A-1, B-1, C-1 and D-1
For 4 pieces, 20 tI each of 1-3 μq/mQ solution such as
Take Q, add 5 units of T4 DNA polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.), 5 mG and 100 mM of reaction buffer (101811 condensate) specified by the company.
Add 1μQ of ATP and then add water to bring the final reaction volume to 5.
The reaction was carried out at 37°C for 1 hour and 30 minutes to introduce a phosphate group into the 5' end of each oligonucleotide.

Next, for each block to be constructed, take 2 μQ of DNA solution of each oligonucleotide as raw material (for example, in the case of block 1, oligonucleotides A-1, A-2, A-
3, B-3, B-4 and B-5), plus 100mM
Add 1μQ of ATP and 10μQ of ligase reaction buffer [660mM Tris-HCl (pl-17,6>, 66mM magnesium chloride, 100mM dithiothreitol solution]), make the final reaction volume 100μQ, and heat in a hot bath at 100'C for 2 minutes. After treatment, it was naturally cooled.Next, T4D
2.5 units of NA ligase (manufactured by Takara Shuzo Co., Ltd.) was added and reacted overnight at 4°C to ligate the two.

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 4 was constructed.

■ Preparation of pcYs-X and pCYS-Y Subunit X consisting of block 1 and block 2 constructed in (■) above.
Vector pcYs was integrated into plasmid pBR322.
-X and the vector pcYs in which the subunit Y consisting of block 3 and block 4 was integrated into the same pBR322.
-Y were produced by the following operations.

The outline is shown in Fig. 2. In the figure, Apr indicates ampicillin resistance and Tc indicates tetracycline resistance, and the restriction enzyme recognition sites in each base sequence are indicated by the following abbreviations. The same applies to each subsequent figure.

S...3al■ Further, Qri in the figure indicates the replication start point.

Preparation of (2)-1' DCYS-X First, pBR322 was treated with the restriction enzymes CORI and 1 (2) and subjected to 0.9% agarose gel electrophoresis to obtain a DNA fragment of about 3.7 kb.

This DNA fragment was mixed with block 1 and block 2 obtained previously, T4 DNA ligase, ATP, and a ligase reaction buffer, and reacted at 4°C overnight to ligate the DNA. Escherichia coli strain HB-101 was transformed with this reaction solution, and the resulting transformed strain was spread on a plate agar medium prepared by adding ampicillin to L-broth medium, and growing colonies were selected. Furthermore, one of these strains was cultured with shaking in a liquid medium containing 400 mQ of L-broth medium to which ampicillin was added, and plasmid DNA was isolated and purified.

The thus obtained plasmid DNA is treated with restriction enzymes and
2dan■, 1fall HI, goose■, M±l■,)1ind11.
It was treated with 5alI, HaelII, EC0RI, and pan, and the cleavage pattern was analyzed by polyacrylamide gel electrophoresis, which revealed that it contains subunit X,
Target plasmid pCYS- having the designed base sequence
It was confirmed that it was X.

■-Preparation of 2pCYS-Y Approximately 3.7k obtained in the above ■-1 in the same manner as in the above ■-1.
The DNA fragment of b was ligated to blocks 3 and 4, and the ligated product was transformed into Escherichia coli HB-101 strain, and plasmid DNA was collected from the - strain and purified.

This is the restriction enzyme Hindm, Dan alII, Dan R■, 1
As a result of treatment and analysis with HinfI, Hinf, H, H, H, H, and HinfI, it was confirmed to be a plasmid I) CYS-Y containing the desired subunit Y.

(2) Preparation of DCYS-XY The outline of the preparation of this plasmid is shown in FIG. 2, and it was carried out as follows.

pCYS-X, which was obtained in ①-1 above, was digested with the restriction enzyme AatII.
and EC0RI, and a DNA fragment of about 0.21 kb was isolated and purified by 5% polyacrylamide gel electrophoresis.

Similarly, pcYs-Y obtained in the above ①-2 was treated with restriction enzymes and then twice in ③ to isolate and purify an approximately 0.18 kbm fragment.

Both of the above fragments and the approximately 3.7 kb DN shown in ①-1 above
A fragment was ligated with T4 DNA ligase, the reaction product was used to transform Cormorant cormorant HB-101 strain, ampicillin-resistant colonies were selected, and plasmid DNA was isolated and purified from the - strain. As a result of analysis of cleavage patterns with various restriction enzymes (e.g., Hae■, Danau3AI, l-1incII, etc.), the target plasmid p, which has a cystatin C structural gene with a total base number of 4.09 kb, was found.
It was confirmed that it was CYS-XY.

■ pCYS-X, pCYS-Y and pCYS-XY
Confirmation of the nucleotide sequence of pCYS-Y, an approximately 0.21 kb DNA fragment obtained by treating pCYS-X with restriction enzymes, L-RI and 1-inch.
Approximately 0.18 kb DA obtained by treating with the same restriction enzyme
The approximately 0.39 kb DNA fragment obtained by treating the fragment and pCYS-XY with the same restriction enzymes was converted into batataeriophage M-13rr+p18RF DNA and pCYS-XY.
r+p19RF DNA (M13 dideoxy method;
J, Messing, Methods in Enzy
mology, 1Q 1, 2Q (1983)
) was subcloned into the cleavage sites of the restriction enzymes CORI and CORI, and the base sequence was analyzed using the M-13 dideoxy method.

□As a result, it was confirmed that all of them had the designed base sequences.

Example 2 Construction of a secretory expression vector This example is an example of constructing a vector that secretes and expresses cystatin C into the periplasmic layer of E. coli.
A chemically synthesized cystatin C gene is inserted downstream of the c promoter and the bla signal peptide, and the structural gene is inserted so that the codon frame of the structural gene for cystatin C does not deviate along the flow of codons for the amino acid sequence of the above-mentioned signal peptide. were ligated to construct the desired cystatin C secretion expression vector.

The outline of its construction is as shown in FIG.

In the figure, the abbreviations at restriction enzyme recognition sites are the same as above or as follows.

Ns・N5iI Na-Nae ■ Also pt in the figure
ac indicates the tac promoter, XY indicates the cystatin C chemical synthesis gene, and the shaded area indicates the base sequence portion containing the bla=l mini signal peptide.

(2) Construction of pCYS-XYA A vector was constructed into which a restriction enzyme recognition site was introduced so that it could be cleaved immediately before the first codon of the amino acid at the amino acid position of cystatin C. First, use pCYS-XY as a restriction enzyme, βa
After cutting with ml-II and %maI, a DNA fragment of about 4.07 kb was obtained by 0.9% agarose gel electrophoresis. Using this and the chemically synthesized oligonucleotide shown below, a ligation reaction was performed in the presence of T4 DNA ligase, and the ligated product was used to transform E. coli strain JMI03 to obtain the target pcYs-XYA.

Synthetic oligonucleotide (linker): X-15°
GATCATGCATCTTCC3゜X-25'
CCGGGGAAGATGCAT 3' The vector obtained above is treated with the restriction enzyme Nsi■ and further treated with S1 nuclease to obtain a DNA that is cleaved immediately before the amino acid at the amino acid position of cystatin C.

The base sequence of the introduced portion in pcYs-XYA obtained above was confirmed by the M-13 dideoxy method.

■Preparation of pCYS-XYATB (7) pcYs-XYA (DNA: 2 μ
q) was cut with the restriction enzyme 1'4siI, and the single-stranded region of the resulting cut region was treated with 81 nuclease (manufactured by Takara Shuzo Co., Ltd., 5
The sticky ends were converted to blunt ends by digestion (at 37°C for 110 minutes).

Thereafter, it was digested with the restriction enzyme ECORI and subjected to 0.9% agarose gel electrophoresis, and a DNA fragment of approximately 4.08 kb containing the cystatin CMA-producing gene was recovered from the gel.

In addition, a plasmid vector pKdN (DNA:
6μq) was digested with the restriction enzymes N<1> and EC0RI,
After performing 5% polyacrylamide gel electrophoresis, approximately 0.46k containing the nucleotide sequence encoding the tac-promoter, SD sequence, and bla signal peptide was extracted from the gel.
The DNA fragment of b was recovered.

The two DNA fragments obtained above were ligated using T4 DNA ligase, and the ligated product was used to transform E. coli strain JM-103. Ampicillin-resistant colonies were selected, and plasmid DNA was isolated and purified from one of them.

As a result of analysis of the cleavage pattern of the thus obtained plasmid with various restriction enzymes (e.g., 3ma, HincII, HindI [I, etc.), it was confirmed that the plasmid was the target plasmid pCYS-XYATB with a total base number of 4.5 kb. .

In addition, the binding site between the bla signal peptide and the cystatin C structural gene contained in the obtained plasmid was confirmed by analysis using the M-13 dideoxy method.
It was confirmed that the amino acid at the first position of the protein was correctly linked without any codon frame shift.

Example 3 Expression and confirmation of cystatin C ■ Escherichia coli JM-1 harboring secretion expression vector pcYs-XYATB
Culturing of E. coli JM-103 strain carrying the vector pCYS-XYATB obtained in Example 2 was cultured with M of the composition shown in Table 2 below.
The cells were cultured in a shaking culture medium using -9-9 amino acid liquid medium as follows.

Table 2: However, marks 8 in the table indicate that the autoclave sterilization process (12
1° C. for 15 minutes) was used. If necessary, add filter-sterilized ampicillin to a final concentration of 5.
It was added and used so as to be 0 μcr/mQ.

One preculture solution was added to a Sakaro flask containing 100 mG of the same medium, and cultured with reciprocal shaking at 37°C.

Approximately 3 hours after the start of culture (at OD6oo=about 6oo=, I
PTG (isopropyl-β-D-thiogalactoside, manufactured by Sigma) was added to a final concentration of 0.25 mM, and the culture was continued.

(2) Extraction of target product from bacterial cells Culture was carried out under the conditions (2) above, the culture was stopped approximately 4 hours after the addition of IPTG, and both portions were 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,
Add an equal amount of 0>-20% sucrose buffer to the culture solution, suspend, add an amount of EDTA aqueous solution to a final concentration of 0.01M, and shake at 180°C at 24°C on a rotary shaker.
Shake at rev/min for 10 minutes, then centrifuge (60
00 revolutions/minute for 10 minutes). The supernatant thus obtained is used as a sucrose buffer fraction.

Furthermore, the same amount of water-cooled water as the culture solution was added to the sediment after the centrifugation to resuspend it, and the mixture was allowed to stand still for 15 minutes in water, stirred occasionally, and then centrifuged (10,000 rpm between rotating valves). The supernatant liquid and the sediment were separated. The supernatant thus obtained is designated as the periplasm fraction.

Also, add the same amount of ice-cold water to the above sediment to suspend it,
The supernatant was obtained by ultrasonication (100 W, twice for 50 seconds each) and then centrifugation (15,000 rpm, 10 minutes). This supernatant is used as the intracellular fraction, and the residue is used as the cell-insoluble fraction.

(2) Measurement of cystatin C activity The activity of cystatin C was measured by the degree of inhibition of papain (EC3, 4, 22, 2) activity, which is one of the thiol proteases.

Measurement of papain activity was carried out by Barrett, J.
Method of Enzymolo (I
V, 201, 535 (198B)), the synthetic substrate α-N-benzoyl-arginine-β-
Using naphthylamide (hereinafter abbreviated as rBANAJ),
β-nanothylamine liberated by papain is subjected to a coupling reaction with fast garnet GBC salt (manufactured by Sigma), which is a diazonium salt, and the coupling product 520
This was done by measuring the increase in absorption in nm.

Details of this measurement method are shown below.

Papain (crystallized twice, manufactured by Hanui Chemical Co., Ltd.),
50μQ solution with a concentration of 4μa/mQ obtained by diluting with 0.1% Brizi-35 aqueous solution (nonionic activator), 2mM
EDT, A-2Na and 4mM cysteine.
Measurement sample 0 diluted with 1M phosphate buffer (pH 6,8>0.5i112 and 0.1% Brisi-35 aqueous solution)
．． After 5 ml of each was placed in a test tube and incubated at 40'C for 5 minutes, 25 μQ of BANA (manufactured by Hanui Chemical Co., Ltd.) dissolved in dimethyl sulfoxide at a concentration of 40 mg/mQ was added and stirred vigorously.

After 30 min incubation at 40″C, 5mM
2% Brizy-3 containing Marυlic acid (Sigma'IA)
0.01% fast garnet GB dissolved in 5 aqueous solution
The reaction was stopped by adding 1 mQ of C salt, and after being left for 10 minutes or more, the absorbance at 520 nm was measured.

The papain inhibitory activity of cystatin C according to the above is 4 ng
The sample concentration that inhibited papain activity by 50% was defined as 1 unit.

■ Western blotting Cystatin C in the bacterial cells 1q obtained in (■) above was determined by Western blotting using a cystatin Cvf antibody (
H, Towbin, T, 5taehelin and
Gordon, J.

Proc, Natl, Acad, Sci, USA,
76, 4350 (1979)). The details are shown below.

A specific antiserum for human cystatin C was prepared by immunizing a rabbit with cystatin C purified from human urine as an antigen. This is lyophilized human cystatin C1mc
+ in 1 mQ of distilled water, emulsified by adding 1 mQ of Freund's complete adjuvant, and injected this into the backs of 3 domestic rabbits.Then, every 2 weeks, the emulsified cystatin C was injected into the backs of 3 domestic rabbits. The animals were immunized a total of 4 times by subcutaneous injection into the back, whole blood was collected 10 days after the final immunization, and the serum was prepared by separating it. The 1q antiserum thus obtained was converted to cystatin C.
was used for the detection of

Samples for polyacrylamide gel electrophoresis in the presence of sulfur dodecyl sulfate (helium) (hereinafter referred to as rSDS-PAGEJ) are, for example, bacterial cells obtained from a 1 mQ culture solution mixed with 2% SDS.
and 2% dithiothreitol in 10mM Tris-HCl buffer (pH 8, 0>1 mQ).
The supernatant was prepared by heating at ℃ for 5 minutes and then centrifuging (12,000 rpm, 10 minutes) to obtain a supernatant.

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

Laemmli, Nature, 227, 58Q.
(1970) ), 15% containing 0.1% SDS.
Using polyacrylamide gel, the sample prepared by the above method was electrophoresed at 2 mA/cm for about 3 hours. Detection of cystatin C is carried out by electrophoretically transferring the protein in the gel after electrophoresis onto a nitrocellulose membrane (manufactured by Bio-Rat), and specifically staining cystatin C from the protein using a specific antibody. did.

For the above staining, first, the nitrocellulose membrane after transfer was dyed with 0.1
Tris-HCl buffer containing 5M sodium chloride (p
Bovine serum albumin (hereinafter referred to as f'BsAJ) dissolved in H7,5 (hereinafter referred to as rTBsJ) to a 10% concentration.
After shaking for 1 hour at room temperature, 0.1% B
The above human cystatin C antiserum diluted 2000 times with SA-TBS was reacted at room temperature for 3 hours, and after the reaction, T
Peroxidase-conjugated anti-rabbit IC washed 4 times with BS and further diluted 2000 times with 0.1% BSA-TBS.
After treatment with IG (manufactured by Kappel) for 60 minutes and washing again,
0.03% hydrogen peroxide and 0.5mM mQ 4-chloro-
0.02M citrate buffer containing 1-naphthol'
(I) 86.5).

(2) Measurement of cystatin C expression level Escherichia coli strain JM-103 carrying the secretory expression vector pcYs-XYATB was cultured and fractionated according to (1) and (2) above, and then measured according to (2) above to determine the expression level of cystatin C.

The results are shown in Table 3.

Table 3 In the table, N and D indicate not detected.

From Table 3 above, most of cystatin C was detected in the sucrose buffer fraction (approximately 5 mM
It appeared at the 200 position.

From the above results, cystatin C detected by the present invention
It was determined that after being translated within the bacterial cell, cystatin C passes through the intracellular membrane due to the action of a signal peptide, undergoes processing, and the signal peptide is cleaved, and cystatin C, which is the same as the natural type, is secreted.

Example 4 Production and identification of recombinant cystatin C ■ Extraction from bacterial cells Culture solution 1.6 obtained by the culture method shown in ■ in Example 3
9 was centrifuged (5,000 rpm, 10 minutes) to collect bacterial cells, which were treated according to the osmotic shock method described in Example 3 (2) to extract the sucrose buffer fraction.

The activity of the above fraction was measured by cystatin C activity assay, and the total papain inhibitory activity was 45,000 units.

■ Purification of cystatin C The majority of proteins produced by E. coli are acidic proteins, and human cystatin C is an alkaline protein with an isoelectric point of 9.2. For this reason, separation methods such as electrophoresis, chromatofocusing chromatography, and ion exchange chromatography that utilize differences in isoelectric points are preferred. As a result of various studies, separation and purification using hydrophobic chromatography and cation exchange chromatography was found to be effective. The outline is shown below.

First, ammonium sulfate was added to 400 times of the above sucrose buffer fraction so that the concentration was 0.6 Mm.
The mixture was passed through a butyl Toyopearl column (manufactured by Tosoh Corporation, 2.5×10 Cm) equilibrated with ammonium sulfate.

After washing the column with 200 mQ of 0.6M ammonium sulfate, the cystatin C-like papain inhibitor was eluted with 100 mQ of distilled water. .

Then, the obtained elution fraction was added to 25mM phosphate buffer (
After dialysis against pH 6.5>, a 0M Toyopearl column (manufactured by Tosoh Corporation, 2
．． 5×10 Cm) to adsorb the target substance. After thoroughly washing the column with the same buffer, 25mM phosphate buffer containing 0.1M sodium chloride (pl
-16,5>.

Furthermore, the eluted active fraction was applied to a Sephadex G-25 column (
After desalting using 2.5×25 Cm (manufactured by Pharmacia), the final purified product was obtained by freeze-drying.

■ 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, the same molecule 1 as cystatin C obtained from urine
A single band was shown at the 13200 position.

(2)-2 Amino Acid Analysis 6N hydrochloric acid was added to the purified sample and hydrolysis was carried out at 110°C for 24 hours, followed by amino acid analysis by the ninhydrin method using a Hitachi Model 835 amino acid automatic analyzer.

The results are shown in Table 4 below. Table 4 also shows the same results for cystatin C derived from human urine for comparison.

Table 4 ■-3 Analysis of Amino Acid Sequence Analysis of the amine-terminal amino acid sequence of the same purified sample was performed using the method of Hewick et al.
,,J.

Biol, Chem, 256.7990 (19
81)).

As a result, the amino terminal amino acid in the purified sample was identified to be serine (Ser), which is the same as human cystatin C.

The recovery rate of recombinant cystatin C obtained according to the above method was 61.3%, and the amount recovered was 3.751 Kl based on amino acid analysis of the final purified sample.

However, the specific activity of papain inhibition is 7400 units/mg, and that of human cystatin C is 7200 units/mg.
g, it had the same activity as human cystatin C.

From the above results, it was confirmed that cystatin C obtained by the method of the present invention is the same substance as that obtained from human urine.

[Brief explanation of the drawing]

FIG. 1 shows a schematic diagram of constructing blocks 1 to 4 from synthetic oligonucleotides and constructing subunits X and Y from these. FIG. 2 shows a schematic diagram of constructing DCYS-X and pc'y's-y using the subunit X and subunit Y, and constructing pCYS-XY from them. Figure 3 is a schematic diagram of constructing pcYs-XYA from the above pCYS-XY and synthetic oligonucleotides, and the pC
YS-XYA and I) K T N 7')' et pcYs
- Shows a schematic diagram of constructing XYATB. (and above) “p

Claims (1)

[Scope of Claims] [1] A chemically synthesized gene comprising a base sequence encoding cystatin C having the following amino acid sequence. [There is a gene sequence. [2] The chemically synthesized gene according to claim [1], wherein the nucleotide sequence encoding cystatin C is as follows. [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. [4] The chemically synthesized gene according to claim 1, having the following base sequence. [There is a gene sequence. [5] A recombinant plasmid obtained by inserting the chemically synthesized gene according to claim [1] into a plasmid vector. [6] The recombinant according to claim [5], which comprises a promoter and a ribosome binding site upstream of the chemically synthesized gene according to claim 1. [7] The recombinant according to claim [5], wherein the promoter is tac. [8] The recombinant according to claim [5], wherein the plasmid vector is pBR322. [9] A transformant transformed with the recombinant according to claim [5]. [10] The transformant according to claim [9], wherein the host cell is E. coli. [11] A method for producing cystatin C, which comprises culturing the transformant according to claim 9 and collecting and purifying cystatin C expressed. [12] The production method according to claim [11], wherein the purification is performed by hydrophobic chromatography and cation chromatography.