JPS5993093A - Muteins with loss of biologically active protein cystine andmanufacture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

The present invention is in the general field of recombinant DNA technology.9 More specifically, the present invention relates to the production of parent analogs by rearrangement/deletion of one or more cysteine residues.
al'ogs) refers to a mutationally modified biologically active protein that has become W. Biologically active proteins produced microbiologically by recombinant DNA (rDNA) technology contain cysteine residues, which may not be essential for activity or may be intermolecular. or intramolecular. In the process of producing IFN-β using rDNA technology, one intestine containing 1"J1+'
,E. Microbiologically produced IFN-β timers and oligomers are formed in the Col+) extract.
Something was discovered. Because of this multimer formation, IF
The purification and separation of N-β becomes extremely laborious and time-consuming, and purification steps such as preserving the protein during purification and then diluting it into IIQ to return it to its original conformation are necessary. Some additional steps are required in the procedure, thereby increasing the possibility of incorrect insulfite bond formation. Furthermore, microbial It was also found that IFN-β, which is produced biologically, exhibits the lowest specific activity in the body. It does not adversely affect its activity or reduce its ability to form intermolecular cross-links or intramolecular bonds that would result in the protein adopting undesirable secondary structures (e.g. structures that reduce the activity of the protein). It would be desirable to modify the biologically active protein in a manner that eliminates it. (Such proteins are called "muteir+:").
1 Biography/Cytogenetics Terminology Dictionary” (Glossary o
f genetics and cytogenetics
cs) No. 41, 38jJ, Splinsher...
Hellag C+97 [Year i)). In this regard, the authors of Nature (1981 I'T') 29
4, pp. 563-565) describes a munoin that can be substituted with cysteine or tyrosine at position 1.41 of the aminobutyric sequence of IFN-β (human IF
N-, β has cysteines at positions 17.31 and 141. Gene (1980) Vol. 10 N-+5%, and Nature (1980) Vol. 285, 542-54.
7N-), this mutin has a portion 1'+' with a G-+A transition 1 at nucleotide 485 of the IFN-β gene.
was generated by bacterial expression of a hybrid gene constructed from the II F N-β cDNA clone. This l,tin lost the original biological activity of IFN-β, leading the authors to conclude that the substituted cysteine was ligneous in activity. Directed mutt W induction technology
Genesis) is well known, and Lather, R.F., Lecoq, J.P., Gene+, IC Engineering.
earing), Akatemi, Kupresun 1 (1983
(2011) Pages 31-50, was once examined. Oligonucleotide-directed mutagenesis has been described by M. Smith, M., and Gilam.
lam, S.), C., Network Engineering, Principles and Methods, Plenum Press (1981) 3.
Specifically discussed in Volume 1-32. One aspect of the invention is that biologically active proteins with at least one cysteine residue are free to form cisulfi F bonds and are nonessential for physical activity. It is a synthetic mucin, and is characterized in that at least one of the mutin or cysteine residues is deleted or replaced with another amino acid. Another aspect of the present invention is that a synthetic structural gene having a DNA sequence specifically designed to code for a synthetic mutin is a synthetic mutin. 1n1 is an expression containing such structural designer genes (futa, hosts transformed with such pectas) - cells or organisms, and such transformed organisms or their f'grandchildren. The method is to produce a combination of 1 and 1 and 1 and 1 and 1 and 1 and 1 by culturing 1 and 1 and collecting 1 and 1 and 1 and 1 and 1 and 1 and collecting 1 and 1 and 1 and 1 and 1 and 1 and 1 and 1 and 2. ! In the case of Uchijime I1 Ishikawa+1 also one no-tin type, the therapeutic composition containing Muchii/phase treatment I1 effect+H is another li'+i of the present invention. A further aspect of the invention is to treat white matter with one or more cysteine residues that are free to form undesired cisulfite bonds. This method is characterized by mutagenicly modifying the protein by deleting cysteine residues or replacing them with other amino acids. A further aspect of the invention provides that oligonucleotides 7 can be produced by oligonucleotide-directed mutagenesis characterized by the following steps:
．． This is a method for creating the synthetic structural gene described below. (a) Parent protein IIF? ``+ structure Kokaden r- no. 1
A single-stranded single-stranded DNA is hybridized with a mutant oligonucleotide primer (with this single-stranded cysteine cordon to be deleted or replaced, or in some cases with an antisense trinucleotide that forms a hybrid with this cordon). The Blend 1 primers are complementary: a codon for deletion of the coron I/n or other amino acids,
Or, as the case may be, this does not apply to antisense, 1 replay, 1, or any discrepancy. ), (b) Extend the primer with DNA polymerase to generate a mutagenic heterozygomer (
heteroduplex) into form gIi, and (c) replicating this mutant heteromer. The mutant oligonucleotide primers used in the [Human No. 1] method are another aspect of the invention. The present invention proposes (5I) that non-woody cysteine residues are responsible for the biological activity of biologically active proteins due to intermolecular cross-linking and
In order to eliminate the site of bond formation - mutins such as mutins that are internally deleted or replaced with other amino acids/acids, mutagenesis procedures that add mutins to lf6H5, wave This is the first stage of r′ that creates a mutin class like Hiko. Proteins that can be mutationally modified according to the present invention can be used in living organisms! i: cysteine plasticity of proteins active in rl'r;
The role played by cysteine residues and cysteine residues in terms of activity and secondary structure can be established from the available information. For proteins for which no such information is available in the literature, each of the cysteine residues of the protein can be systematically altered by the procedures described herein to improve the biological activity of the resulting mutins and their desired properties. This information can be determined by testing for the tendency to form intermolecular or intramolecular disulfide bonds. However, biologically active compounds containing functionally non-essential cysteine groups that make proteins susceptible to the formation of disulfide bonds are desirable. It will be appreciated that the following teachings apply to other proteins. "Sudden changes according to the present invention other than IL-2 such as IFN~β"
Examples of intraF white matter that are candidates for Cisse1 modification are lymphoxoxy〉' (tumor necrosis factor), colony-stimulating factor r-1, and 6 candidate proteins, IFN-alpha, which are usually found in odd-numbered cysteine molecules. It has residues. In the case of IFN-β, both glycosylated and terminally glycosylated IFNs show essentially the same specific activity; therefore, the glycosyl moiety does not participate or contribute to the biological activity of IFN-β. This was reported and reported. [2
Oak, non-glycosylated IF produced by bacteria
N-β is significantly lower than glycosylated native IFN-β. IFN-/3 is known to have cysteine residues at positions 17.31 and 141. cysteine l
4. It was demonstrated by Shepard et al. (1i1f) that 1 is essential for biological activity. 4
In IFN-α containing cysteine residues, there are two intermolecular -5-S- bonds;
ysl38, others between cysl and C1/S98. T based on homology +1 between IFN-β and IFN-α,
IFN-β(7) Cys141 and cys31 intramolecularly
Although it is a 5-8 bond, the remaining cysl7 can freely form intermolecular crosslinks. By deleting c y s i 7 or substituting it with another amino acid,
Whether cysl7 is biologically ligneous can also determine its role in -S-S-1I synthesis. If cysl7 is not essential for the protein's biological activity, the resulting cys17-deficient protein is cys1
The 7-substituted t-protein exhibits a specific activity close to that of native (7) IFN-β and would also likely facilitate single-sided purification of the protein. 1FN-β crushing cys17 I cordon (cod
on: Is this row 1- complementary to the area of l?
Synthesis containing changes in rl4-nu in multiple salts),
Oligonucleotide primer and 4 (to, oligonucleotide
The designer gene f- was created using the mutagenesis procedure directed in /10. cysl7 or any other person, use a dangerous amino acid/acid 1. Review the results obtained. When deleting +0 is desired, the A-ribonucleotide primer lacks the cys17 river codon. , cysl7 with glycine, valine,
This is the simplest method for converting it into urinary neutral amino acids such as alanine, loin, isoleucine, 1,000, phenylalanine, histidine, l-lip), serine, threonine, and methionine. Serine and threonine are the most preferred alternatives due to their chemical similarity to cysteine. When Cysti> is deleted, the adult A1 mutin is one amino acid shorter than the IFN-β produced by natural regulatory white matter or microorganisms. H)・IL-2 is a protein of 58.105 and 125
What if there is a cysteine residue at the i<1 position? It is outdated. , as in the case of IFN-β, IL-2 is in an aggregated oligomeric form when isolated from 411 blank bacterial cells, and in order to obtain a good yield 114 from bacterial extracts, , must be reduced with a reducing agent. In addition, purified and reduced IL-2 is unstable;
It is easily converted into an oligomeric inactive form during storage. The presence of three cysteines means that during l'j M formation, the protein has one of three possible intramolecular cisulfite bridges, whereas there is only one correct bridge as seen in natural molecules.
It is a pleasure to be able to randomly form individual pieces. Since the structure of the native IL-2 protein (disulfide) is unknown, we created mutations in codons 58.105 and 125 of IL-2 protein to determine which cysteine groups are required for activation. The present invention can be used to ascertain which is most likely also involved in the formation of natural disulfite bridges. Similarly, cysteine residues that are not required for activity can be altered to prevent the formation of spurious intermolecular disulfite crosslinks and to minimize the chance of intermolecular disulfite crosslinks by removal or replacement of cysteine residues. The size of oligonucleotide primers is surprising! A change I
It is determined by the conditions necessary for the safe formation of a primer to the genetic f region into which M should be introduced, as well as by the limitations of currently available oligonucleotides and synthesis methods. To design oligonucleotides I for use in oligonucleotide-directed mutagenesis! 1' Also, aging factors (eg, overall size, size of the portion that bypasses the mutation site) are described by M. Smith and Niss-Killam (previously + IA). generally,
The total length of the ori' nucleotides is such that it optimizes stable and unique hybridization at the mutation site;
The extensions from the mutation site to the 5' and 3' ends are sufficiently large to avoid editing of the mutation by the exonuclease activity of the DNA polymerase. Oligonucleotides used for mutagenesis according to the invention typically have about 12 to about 24 bases, preferably about 14 to about 20 bases, and more preferably about 14 to about 18 bases. salt7. Contains <. These usually contain at least about three salts of the codon, j, L3', which are altered or deleted. The method of producing modified IFN-β gene in one human body is as follows:
Genetic mutagenesis is performed to either eliminate codon 17 or change it so that it encodes a different amino acid. By changing cysteine to threonine and hybridizing the primer with antisense of the IFN-β gene r, the preferred nucleotide primer is GCAAT.
TTTCACTCAG (the underline indicates the modified cordon). When it is desirable to delete cysteine, the appropriate primer is A G CA AT T
TTCAGCAGAAGCTCCTG, which is C
The TGT code 1 for 'IS' is omitted. One temple that replaces cysteine with serine is the 17-nucleoti) pla fmar GCA that includes the AGT cordon of the serine river.
ATTTTCAGAGTCAG is the primer for Hikoju J (Hikoju J). By rearranging the TA of the -i group in cys17, a change from cysteine to serine occurs. When introducing a deletion, the expression of 1 in the desired protein is DNA for
It must be recognized that the sequence must have an appropriate decoding frame (I") 4!r l. The primer is It is recognized that the phage can carry either the sense strand or the antisense strand of the gene.The phage carries the 77-J--1=nce strand. Sometimes, the primers are identical to the region of the sense button containing the codon to be mutated, except for a coton deletion, or a mismatch with this codon that determines a triplet coded with another 7 my/' acid. When carrying the sense strand, it is complementary to the region of the sense strand containing the codon that is mutated, except for appropriate mismatches in the codon to be deleted and the triplet that pairs with the codon to be deleted.Hybrid The conditions used for formation are described by M. Smith and Gillam (ibid.). Temperatures are typically from about 0°C to 70°C and generally about io'
It is in the range of L50'c. After hybridization, the primers are combined with DNA polymerase 1. T+ DNA polymerase is extended on the phage DNA by reaction with DNA polymerase, transcriptase, or other suitable NA polymerase. The resulting dsDNA is cleaved into closed ring dsDNA by treatment with a NA ligase such as T4DNA fI-ase.
DN A containing 1 strand region that is converted to A
Min-f- can be destroyed by S1 Endonuk1/Aase treatment. Oligonuclease-directed mutagenesis is an IL
It can similarly be used to create a mutant IL-2 gene that has -2 activity but encodes a mutin with cys125 changed to serine 125. When the phage carries the sense button of the gene, the nasty oligonucleotide primer used to construct this mutant IL-2 gene is GATGATGCTTCTG AAAGG.
It is TAATC. This oligonucleotide (±, ■L
-Make a pair with the cordon 125 of 2-year-old I Yunko]・There is a change from C to G in the 18 groups in the middle of the reprent. The resulting mutant Q+l-tello star bodies are used to transform host 1': organisms or cells capable of infection. Replication of the heterodimer by the host) produces progeny from both strands. Following replication, isolate the mutant strain gene from the progeny of the mutagenized strand. The vector is inserted into a suitable vector and this vector is used to transform ili/i into a suitable host organism or cell. One preferred vector is the plasmid pER322pCR1 and its mutants, synthetic heptoplasts. Suitable host organisms are Escherichia coli (E, Co11), Shootmonus (
Pseudomonas), Bacillus subtilis
5ubtilis), /<Bacillus ◆Bacillus thuringie++5
is), various yeast nJ strains, D1 Bacillus
lus thermophilus), house mice, rats, and Chinese hamster eggs (CHO).
) such as animal cells, plant cells, animal and plant hosts, etc. It must be recognized that when transforming the chosen host with the vector, appropriate promoter-operator sequences are also introduced in order for the mutin to be found. Inn 1 Warrior It can be a prokaryote or a eukaryote (DN to Matsuchi cell)
The method for inserting A is the PC published on September 3, 1981.
T application number US81100239 and υ? US81100
240). E. coli and CHO cells are preferred hosts. The mutins obtained according to the present invention may be glycosylated or non-glycosylated depending on the cosylation and the host organism used for mutin preparation. If desired, Escherichia coli (E,
The terminally glycosylated mutins obtained when Bacillus and Bacillus are the host organisms may optionally be glycosylated in vitro by chemical, enzymatic and other modifications known in the art. In a preferred embodiment of the invention regarding IFN-β,! As shown in Figure JS 1, the cysteine residue at position 17 in the amino acid sequence of IFN-β is
Site-directed mutagenesis of codon 17 of the sense strand of the DNA sequence encoding N-β, in which q-IQg is changed to serine by T-4A rearrangement, results in synthetic 1
7-Nucleotide I Primer GCAATTTTCAG
A, using TCAG, B, and BJ, this primer was used to identify the sense button of IFN-β in the region of codon 17, except for the mismatch of the -J group in Gorton 17's mth, (in -17 nucleotides), arrays, and circuits. This mismatch is at nucleotide '12 of the primer. The genetic code is degenerate, and many amino acids have more than one 1-
shall be allowed to be encoded by the same coden. For example, the 1n base code for serine is TCT,
There are six types of degeneracy, as the codens TCG, TCC, TCA, AGT, and ACG all code for serine. Convenience V, AGT as a preferred embodiment
Cordon is sent and like l-0, threonine is ACT,
Any of the ACA, ACC, and ACG cordons is numbered 1lrf. 1 for specific amino acids
When specifying a codon, this indicates that the amino acid is II (
(intended to include all degenerate codons that occur).
hybrids are formed. Next, DNA polymerase I
Using a Klenow transducer, the oligonucleotide is extended on the DNA by 7 and 1 times. The dsDNA is converted to closed s=DNA with T4 licase. The resulting mutant heterogeneity is confirmed and screened by other methods, cloned from the DNA strand containing the mismatch, by replication of the polymer. When replacing cysteine with serine, a new H4nfI restriction position is created in the structural gene by the T-$A rearrangement shown in FIG. The mutant clones were blob 1. in the hybridization screen of the mutated 71-silak.
The oligonucleotide primers are used if the oligonucleotide primer is used. As shown in Figure 2, the primers will have either 111 [1 mismatch when hybridized to the parent or 1[lambda] perfect match when hybridized to the mutated phage DNA. The oligonucleotide primer is mutated from the parent DNA lI'I! DNA can be used to create a type of H species (one type of cedar) that will lead to hybridization. The newly generated HinfI site also has IFN-β
Serves as a means of confirming monogenic mutations in genes
'itsu. M13 phage DN carrying mutated gene
A can be released Qi and integrated into a suitable expression vector, such as plasmid pTrp3, and this vector can be used to express human intestinal obstruction MM.
Transform the 294 strain. Suitable growth media for culturing transformants and their progeny are known to those skilled in the art. The discovered mutin of IFN-β is isolated and purified, and converted into a specific protein. The following examples are presented to aid in a further understanding of the invention and are presented for illustrative purposes only. They should not be construed as limiting the scope of the invention in any way. Examples 1-9 describe the preparation of IFN-β mutins. Examples 10-15 describe the preparation of IL-2 mutins. Example I Cloning of the IFN-β Gene into the M13 Vector The use of the M13 phage vector as a source of end-strand DNA templates F-Temple (
G, F. Temple) et al., Nature (198
2) was demonstrated in Vol. 296, pp. 53L-540. Plasmid pβit rp (Figure 3) containing the IFN-β gene under the control of the E. coli trp promoter was digested with restriction enzymes HindIII and Xho11. M
13mp8 (Jay Φ Mensing (1Messing)
, 3rd Creepland on giant fractions
Symposium 1, z Recombinant DNA Japan Society, Edited by Woichiton, Elsahia Press, p. 43-153 (1981
)] Replicative form (RF) [)NA (Fig. 4) was digested with restriction enzymes Hindm and BamHI, and HindI
P/3 which had been whitened with II and X h o II
1. mixed with trpDNA. Then add the mixture to T4DNA
The ligated DNA was ligated with licase, and the ligated DNA was transformed into E. coli JM103 strain capable of infection.
Nucleic Ac1ds Res (1
881) Vol. 9, pp. 309-321). Recombinant fγ-
Remove the plaque (white plaque) containing the bacteria,
A new sonic base (ench d paan poim) of JM103 and minipremb, an RF molecule made from infectious bacteria.
H,D. Birnboim) and J.Dol.
y), Nucleic Ac1d Res (197
9) Volume 7 + pages 511-1523). IF
To identify clones containing N-β inserts, RF molecules were digested with various restriction enzymes. The restriction map of one such clone (M13-β1) is shown in FIG. One unstranded phage DNA-A was generated from the M13-β1 clone and used as a template for site-specific mutagenesis using synthetic oligonucleotides. Example 2 Zyte-specific mutagenesis 0.1 m M adenosine orthophosphate (ATP), 50 m m
- HCl) pH 8,0, l (++nM
M g C12, 5mM dithiothreitol (D
DT) and T4 kinase 9-fold, 50 p
-1 in synthetic oligonucleotide GCAATTTTCA
40 pmol of GAGTCAG (primer) was treated with T4 Ginase for 1 hour at 37°C. 50mM NaCl, 1,0mM Tris-HCl, pH 8
, 0, I Om M M g Cl 2 and υlomM
501 L of mixture containing β-mercaptoethanol
Hybridization of this Chylase-treated primer (12 pmoles) to single-stranded (ss) ML3-βI D N A 5 TLg by heating at 67°C for 5 min and at 42°C for 25 min in I let it happen. The annealed mixture was then cooled in water and diluted with 0.5 m M each deoxynucleotide triphosphate (
dNTP), 80mM1.lis-
HCl, pH 7, 4, 8mM MgCI
2. 100mM NaCl, DNA polymerase I Klenow fragment 91″#, 0.5mM ATP
and reaction mixture 5 containing 2 units of T4 DNA licase.
0p1 and coalesced for 3 hours at 37°C and 2 hours at 025°C. The reaction was then stopped by phenol extraction and ethanol precipitation. DNA in 10mM Tris-HC
l, pH 8.0, IOm M ethylenediaminetetraacetic acid resistance (
EDTA), dissolved in 50% sucrose and 0.05% bromophenyl blue, containing 7 gg/mu of 1,000 sium bromide.
The samples were subjected to electrophoresis on a 0.8% acarose gel. The DNA band corresponding to the RF type of M13-β1 was detected using the Percollet-1 method [R.
, Q, []avis) 3, "Higher Bacterial Genetics"
(8 advanced Bacterial Genet
ics), Cold Curved Sprinkle Herba r%
, NY. 178-179 C1980)] was thus eluted from the gel slice. The eluted DNA was used to transform JM103 cells capable of infection, the bacteria were grown overnight, and 5sDNA was extracted from the clear solution. I'JfIL. Prime this 5sDNA
Phage plaques are obtained by using phage cells as a template and step 17 to transform the gel-purified RF+J DNA into JM103+WIII foam capable of infecting, plating on agar plates, and incubating overnight. Example ↓ The experiment of Example 2 on site-directed mutagenesis is repeated completely, except that the synthetic oriconucleotide primers used range from one encoding cysteine to one encoding threonine. 1.FN-β
G CA A T to change codon 17 of the gene
']" T '1' CA G A CT CA G. EXAMPLE 4 The experiment of Example 2 on wit-specific deletions is repeated, except that the synthetic oligonucleotide primer N:IIi'N- A G CA A TT to delete codon 17 of the β gene
T TCA G CΔGΔAGCTCCTG. Example 5 Sieving and Identification of Mutagenic Weathered Plaques Flulates containing mutated M13-β1 plaques (Example 1) as well as unmutated M13-β1
The two plates containing phage plaques were cooled to 4°C and the plaques from each plate were transferred onto two nonitrocellulose circular filters and, in the case of the first filter, a 50% dry filter onto the base plate. In the case of the second filter (I moved it by car for 15 minutes. Next, 0, 2
N NaOH, 1,5M NaC/! . Place the filters on two thick pieces of paper soaked in 02% l-IJton X-100 for 5 minutes, then 0.5M) Lith H
CL, pl+ 7.5, and 1,5 M NaC
The mixture was further neutralized by overlaying it on tVC-soaked P paper for 5 minutes. In a similar manner, the filters were washed twice with 2×SSC (standard citrate), dried, and dried in a vacuum drying oven at 80° C. for 2 hours. Duplicate filters and 10 ml per filter of I) NA hybridization buffer (5X SSC), pH 7.0, 4x Anne's solution (IP + J vinylpyrrolidone, Ficoll and bovine serum albumin, 1x = 0.0 each). 02%),
0. ．． 1% sodium dodeane sulfate (SDS), 5
0 mM @l'ff thorium buffer, PI-17
, Q and 100 μ'i/me ('l' salmon sperm D
1) The sample was incubated with NA for 4 hours at 55°C. 112 by treating the oligonucleotide Zolimer with ATP labeled with 3?1)
A probe labeled with 1) was prepared. Wash the filters 3 times in 5 ml of DNA hybridization buffer per filter.
21) Primer labeled with 3.5×10'cprn/
mj was hybridized for 24 hours at 55°C. 0.1 Section S
I) Filters were washed at 55° C. for 30 minutes each in wash buffer containing SSC and decreasing amounts of SSC. Filters were first washed with a buffer containing 2XSSC,
Control filters containing unmutated M13-β1zolag were tested for the presence of radioactivity using a Geiger counter. Gradually lower the 5SCn degree,
Control filters containing unmutated M1.3-β1 flux were washed twice until no detectable radioactivity remained. 5SCO flight month] The lowest concentration is 0. There was an IX5SC. The filters were air-dried and sectioned into autotrautumegrans for 2-3 days at -70°C. Mutated Ml3-β] plaques/I80 and unmutated control plaques 1.
00 cells were sieved by kinased oligonucleotide fluorosol. In the control plaques, there were no blooms and foils [seeded], while five mutated M13-β1 plaques hybridized with the probe. 5 mutations M1.3-β1) 0 Lakh 1 (Ml
3-3Y2501) was taken up and inoculated with JM103 Doon Yaki-3. 5s DNA was prepared from the clear solution, and double-stranded (ds) DNA was prepared from the bacterial cells. 5sDNAtI! as a template for dideoxy sequencing of clones using M1, 3 universal primers!
Figure 6 shows the results of the sequencing analysis using 1.
J-0 This result is TGTcys:'-ton is AG'l
It is confirmed that it was converted to 'ser code)'. , Example 6 Mutagenesis in E. coli 11”N-β
Expression Ml, 3- FDN A from SY2501
f <fill 1Keda 11zosai, Ir1nd II
Digested with 52 (l bp of 1
111) (Fragment 1 agarose scale) 2 in a Kosei tube, 1), 11 pigeon 1 jrp 7' D motor-containing fluor smi l□'p'L"rp 3 (7th
Ml3-3Y2501 was purified using enzyme J (ind III and Bam IT I ('Port1').
DNA…I Kata and E 1. Then, in the presence of T4 DNA ligase, the ligated /ζ(l]!&Kichi1)N8 was transferred to Escherichia coli IVTM29/I strain. - Sieve/I'f Keb ζ in terms of sensitivity uI to Atraya 4 Clin. Plasmids from 5 ampinoline-resistant and tetrazycline-sensitive clones are screened for the presence of Ml, 3-3Y2501indu--1/KomeHi
Digested with nfI. Figure 8a shows one of the clones (
118Y2501), and the figure compares this with the peak nfl pattern of the original IFN-β clone, i.e., pβ1trp. As expected, the I) additional I in SY250] -(infl
There is Zayto, 1! 17 bp)JFN-β internal fragment 1 is cut into a 1.69 bp fragment and 281)■) (Fig. 8b). the law of nature

The restriction map of []-n psY250] is shown in FIG. Mutation 1 VAs J J.
The complete I)NA sequence of the ``N-β gene'' is shown in Figure 10 along with the predicted ft-7 amino acid sequence.
-71) The plasmid identified as SY2501 was sent to the Fermentation Laboratory, Northern Research Center, Education Bureau, U.S. Department of Agriculture, 6060/1, 1815 North Unipergidi Sl-IJ-1, Oria, Illinois. Age・I#+
It has been deposited in the L-t, Wolf, Power IL'ry-Koshiri-Dipun (NRRL). The designated deposit numbers are CMCC No. 1533 and NRRLB-1535,6. Cultures of psY2501 and pβ1 trp including progeny were grown at an optical density (0Dooo) i of 1.0. Creates an extract that does not contain bacterial cells and extracts 1 of IFN-β.
GM2767 virus activity was determined by micro-8 titration assay method.
1, the extract of clone pSY2501 showed 3-10 times higher activity than trJ:pβl trp (Table 1), clone pSY2501 showed IFN-β
Either more active proteins are being synthesized, or the protein produced is more expensive! Indicates whether the substance has specific activity. Table 1 Extract Antiviral activity (unit/ηre
) pSY2501 6X10'pβl tr
pI 'The gel was subjected to electrophoresis on acrylamide gel.
As shown in Figure 11, the protein was stained with 7-c to develop the protein. There was only one protein band corresponding to the 000 Dalton protein. This protein is about 20,000
It shows the migration pattern of a protein with a molecular weight of 18,000 Daltons, and pβ1 tr
By purifying this protein from extracts of p.
It was known in advance that i'N-β. Since there is less i2 of this protein in the psY2501 extract than in the pβ1trp extract, the clone psY
The specific activity of the protein in the 25011111 product was higher than that of clone pβILrp. Example 7 Purification of 11" N-βSer I 7 111
IFN-βSer 17'fz was recovered from Escherichia coli transformed to produce M-β8er I7f.
It was grown at an optical density of 11 and (dry Ichikawa 8.4?/l-). Component Concentration Nu, C
120rnM K2SO416, irnM KTI2PO478mM Na,, HPO41, 2-2mM MgSO4・7L0 3mMNa, Zytrate "2H201, 5mM Mn5O<" 4H203
0ttM ZnSO2φ7H2030/IM CuSO4" 5J (203trM L-1-Lyptophan 70mY/l FeSO4・7■-■20721tM Thiamine I-(Cl30 mg/l Glucose 40'
Harvest of transformed E. coli adjusted with PI + 9.97 (9,9K9)
to 20℃? 1, and the F liquid weight becomes 88 to 9. Harvest i-1, 10 kpa average pressure drop and 260 per minute
Steady P liquid flow of me - passed through a cross flow filter with a kite. , pour the condensate (approximately 1.1 cm) into the container, ]5'
Cooled to C. Concentrated liquid f: 5°G, ~69,000
The bacterial cells in the concentrate were disrupted by passing it through a Manton-Gorlin homogenizer at a pressure of 100 kpa. Phosphate buffered saline, P117°4. (PBS)1/=
The homogenizer f:: was washed and the washing solution was added to the crushed material to obtain a final volume of 2 tons. Total capacity with 50m/min
The solids were separated from the supernatant and resuspended in PB847 containing 1% SDS by weight. This suspension was incubated at room temperature for
After 5 minutes, there was no visible change. The m solution was then extracted with 2-butanol in a 1:1 volume ratio of two 2-zotanol solutions. 1σ in liquid-liquid phase separator
The extraction was carried out using a flow rate of 200 m12 min. Next, a1- The mechanical phase is separated and evaporated to dryness, resulting in protein 2
1.3! i'k happened. This was added to distilled water at a ratio of 1:10. The recovered product is the cytopathic agent of the virus (CP).
E) using a test based on protection against H) 11”
Assayed for N-β activity. This assay was performed in a microplate. A minimum of 50 pi of basic culture medium was placed in each container, 25 μL of the sample was placed in the first container, and dilutions of 1:3 volume were continuously performed in the next and subsequent containers. Virus (vesicle 1'1-Ll enditis). IFN-β is for me! It was 1100 units. The plate was then irradiated with UV light for 10 minutes. After irradiation, the cells were suspended 7f, (9 cells per me 1.2 x 10')
Add 100 ttL f to each container and fill the trays with 18-24
It was fed for 18 hours. [per cell] virus solution was added in plaque-forming units to each vessel except for the cell control plate. Virus control is 100
Trays were plated at dimensions showing %C1)E. This usually occurred 18-24 hours after adding the virus solution. Assay results were interpreted in terms of the location of the 50% CPE container of the standard IFN-β control. From this point, the interferon titer for all assays on the plate was determined. The specific activity of the recovered product was determined to be 5 X 107 U/m'j. Example 8 Shield preparation by acid precipitation and chromatography The procedure of Example 7 was repeated, except that after extraction, separation of the aqueous and organic phases and mixing of the organic phase with PBS in a volume ratio of 3:1, ice Mixture P11 was made low by the addition of acetic acid. The resulting precipitate was separated by centrifugation at 10000-1.7000x! for 215 minutes and the pellet was reduced to 10% W/V S.
DS, lOmM I)'1”T, 50mM
Redissolved in sodium acetate buffer, Pl-15,5,
Heat to 80'C for 5 minutes. Next, using the Beckman gradient system, Brownlee I is also P−
300,10/ZM, rAquaboa"Casino" was poured with the solution. Slow infusion solution A contains trifluoromeroacetic acid (TFA) in H20, and buffer 13 contains 0.0 in acetonitrile.
．． 1% i”FA. Detection was by UV absorbance at 280 nm. Solvent program was 0% i”FA for 3 hours.
Buffer B to IOO% buffer was a glandular gradient. The fractions containing the highest interferon activity were pooled, and the specific activity of this pooled interferon preparation against natural IFN-β was approximately 2 x 108 units per m1 of white matter. ,9.
0X107 to 3.8 X 1 081J/In! It was decided. Example 9 Biochemical characterization of IFN-βser 17 5, 7 N 1. (Ct, 20 of 0.1% phenol
Try putting 0μ inside F140 ttL f) 1 0
The amino acid composition was determined after hydrolysis at 8° C. Proline and cysteine were determined in the same manner after performic acid oxidation. In this case, phenol was omitted from the hydrolysis. Tryptophansi”,,57N) IC/! ．． , 1 0
Tests 4' and 14 in % mercaptoacetic acid (without phenol)
Analyzed after 24 hour hydrolysis of 00 μL. In addition, the analysis was performed using a Beckman 121. MB amino acid acid separation device 61 was carried out. Representative 24-, 48 of purified IFN-βSer 17
−. The amino acid composition resulting from 72 hours of acid hydrolysis is 1:39, in addition to lacking the N-terminal methionine.
The amino acid sequence of the first 58 residues from the single amino acid terminus of purified 'Ili'N+rJ, Sample 0.7mg
from 0.1 using a Beckman 890C sequence measuring device.
Determined using M Quadrol buffer. PTH amino acids were collected using the Altex Super Spherical ODS column (4.6
Reverse-phase high-pressure liquid chromatography (HILC) was performed at 45°C using 40% buffer B.
for 1.3 minutes, and for 8.4 minutes with 40-70 buffer solution 13.
It was decided that Here, buffer A is 0.0 1. 1.
5M acetic acid, 5M detrahydride, pH 51.1, and acetonitrile. Terminal amino acid sequence 1, except for the absence of the N-terminal methionine, T
) It matches the predicted sequence determined from the NA sequence. As shown in Figure 1, the IFN-βSer17Se side exhibits an activity that is very close to or even more extreme than the natural II ('N-β I antagonistic active water f).
r 17 has a sulfhydryl group in free flesh 11, but 3
One -S-8- bond is shown between cysteines remaining only at positions 1 and 141. Proteins are considered to be essentially in monomeric form, easily forming oligomers. The IFN-βser 17 obtained according to the invention is inert, non-commercial, non-allergenic and pharmaceutically available as a drop product or as a mixture of various types in a pharmacologically compatible combination medium. The product can be formulated into an injectable preparation for clinical use in cancer therapy or in conditions where interferon therapy is indicated. For the treatment of viral infections, 1 liter of such media can be used - distilled water, saline, link solution. This includes, but is not limited to, bank liquids and the like. Dextrose, H8A (hi) ■(1+clear albumin)
Other non-toxic stabilizing and solubilizing additives may also be included as appropriate, such as. The therapeutic formulation can be administered intravenously or parenterally, such as intravenously, intramuscularly, intraperitoneally and subcutaneously. The main advantage of the above-mentioned IFN-β mudeins is that the free sulfhydryl group (- 8l (group) is divided by 441, which results in VCcys 31 and c
The correct zosulfide bond is formed between ys 141 and the =I notation clearly required for full biological activity. Due to the increased specific activity of IFN-β5er17, lower doses can be used for treatment. Deletion of protein at position 17 resulted in free 1qfl-
8J (by excluding the group IFN-βser
17 The protein IFN-β, which is produced by microorganisms, is fragile and does not form dimers or mericomers. This facilitates purification of the protein and enhances its stability. Example 10 Human IL-216) Nucleic sequence of dDNA clone for darkening, procedure for creating IL-2' cDNA library, and procedure for sifting this into IL-2 V sieve were described by Taniguchi. ,T,)
, Nature (1983), Vol. 24, pp. 305 onwards. -1 powerful TI, -2 cDNA clones enriched in D
The NA library was constructed from the enriched mRNA fraction of II,-2 obtained from induced peripheral blood lymphocytes (PBL) and Duyakasoto cells by conventional procedures. IL-2 times (Ge rnRNA
Concentration (mRNA) was divided into four fractions, the fractions were injected into immature oocytes of Xenopus laevis, and the IL-2 activity of the oocyte lysate was assayed using IT-2 cells.
This was done by confirming the active fraction [Zuei Watso 7 (J, Watson) +
J, Exp-Med- (1979) Volume 150 1570
~1519 pages and Nis Gillis (S, G11lis)
et al., J-Immun. (]-978) Volume 120 202
Pages 7-2032]. Example 11 Screening and Identification of N,-2 NA Clones A colony hybridization procedure was used to screen an IL-2 cDNA library. Each microcondensation titration plate was copied onto duplicate nitrocellulose r paper (S&S Quizo BA-85) using the replica method, and anopicillin 50 tl'i
/mI! Grow for 14-1611.'7 on L agar containing f. Colonies were lysed and incubated with 500 mM Na, On, 1-5 M NaC
I) NA' (fixed on filter and treated with 5x standard citrate (SSC) for 5 min each)
Washed twice. The filter was air dried and oven dried at 80°C for 2 hours. Duplicate filter t filter per ')], Ome I) NA hybridization buffer (50% formamide, 5XS
S.C. pH 7.0, 5x Anhard solution (polyvinylpyrrolidone, Ficoll and bovine serum albumin; lx = 0.2% each
), 50 mM sodium phosphate buffer at pH 7.0, 0.2% SDS, 20 μfl/m7! Prehybridization was performed with polyU and 50,1lme denatured salmon sperm DNA for 6-8 hours at 42°C. A 32p-labeled 20-mer oligonucleotide ditozorope was created based on the IL-2 gene sequence reported by Taniguchi (supra). Princess G'l"GGCCTTCTTGGGCA
5 me of DNAI per filter containing 0, ``P'GI) NAzorope with TGTA! < 1 in species formation buffer, the sample was
A rough fief was formed at 24-36°C. Wash the filters twice at 500C for 30 minutes each with 2XSSC and 001% SDS, then 1X5SC and 0.1% SDS.
Wash at 50°C twice for 90 min each, air dry, -7
Autoradiographed 2-3 times at 0°C. Positive clones were identified and screened again with the probe 3. Clones of sufficient length were identified by restriction enzyme mapping and Tani! This was confirmed by comparison with the sequence of the IL-2'CDNA cone reported by ChiF-1 (cited above). Example 12. Cloning of the IL-2 gene into the M13 vector as described in Example 1 using the E. coli plasmid F pLWl (FIG. 2) containing the IL-2 gene in the regulatory domain of the E. coli trp promoter. IL-2 gene 1 as per
The clone was compared to M13rnp9. tE of p LW 1
, fl n 198: 3 August 4 [yo, 20852
D.Tuckville, Maryland, United States) and - Clone Drive 1.2301 sound location. It was entrusted to Ameri-ka-Tai 7', J Fulcher Koshi 7', and was designated ATCC No. 3!1.405. IL-
1 clone (M13-IL-
The restriction map of phage 3,1 (designated as 2) is shown in Figure 13.3. 3. Example 13 Oligonucleotide-Directed Mutagenesis As described above, jL-2 contains cysteine residues at amino acid positions 58°105 and 125. . ■ Based on the nucleotide sequence of the part containing codes for which three cysteine residues in the L-2 gene, three oriconucleotide primers are proposed 1 and used for these residues. was synthesized to mutate the codon into a se1non codon. These meligonucleotides have the following sequences: cys 58ii eru CT'l"CTAGAGAC
TGCAGATGT'rTC (1) M27) C:V S , + 05 f plle CATC8GC to TACTCAGΔC8TG ATG (1) M2S
) cyS125', (3 GATGATGCTCTG
AGA, A/HiroGGTAATC (I) Mg9) 0.1. mM ATI), 50
mM) Lis-HCl, P++
s, o. 10mM MgCl4, 5mM DTT and Q'T
In the presence of 9 units of 4-kinase, 40 pmol of each oligonucleotide was treated separately in 50 μL for 1 hour at 37°C. [kinase-treated)], each of the primers (1 mol of lO vinyl) was added at 100 mM
NaCt, 20mM to1nos-1-fct, pH
7-9, 20mM MgC1, and 20m1Vfβ-
Into 15μ of a mixture containing mercaptoethanol,
55M13-IL-2DNA 2.6 p'j% by heating at 67 °C for 5 min and 42 °C for 25 min.
(Seeded. Cool the annealed mixture on ice and then add 0.51 TIM each dNTP, 17 mM
) Lis-HCt (pH 7,!1), I7m
MMgc12.83 rnMNacl. 17mM β-mercaptoethanol, 1) 5 units of NA polymerase I Klenow fragment, 0.5mM
ATP, and T4DNA! The reaction mixture containing 2 units of J gauze was adjusted to a final volume of 25 μt and incubated at 37° C. for 5 hours. Stop the reaction by heating to 80°C,
The reaction'/J14 compound was used for t1 transformation of susceptible JM 103 cells, plated on agar plates, and cultured overnight to obtain phage plaques. Example 14 Screening and Identification of Mutagenic Thickened Fern Flowers Mutagenic Thickened IV113-IL-2 Flowers
and M1 without mutagenic thickening.
3- Cool the two plates containing IL-2 phage plaques to 4°C, transfer the phage plaques from each plate onto two nitrocellulose circular filters, and place the dry phage plaques on the platform of the first filter. Filter 0 rate for Japanese people
- for 5 minutes, and in the case of the second filter for 15 minutes. Then 0.2 N NaOH, 1,5 M NaCl
and 0.5]Vl! -Squirrel-L(-C/
(pH 7.5) and 1.5 M Na (J).
ii11≦Wash the filter 1-2 times soaked in 2XSSC, dry it, and then dry it in a vacuum drying oven at 80°C.
2)] Jj. Duplicate filters were added with 10me per filter I) NA hybridization buffer (5XS
SC), pH 7.0, 4x anno-de ig (7
1? Rivinylpyrrolidine, Ficoll and bovine serum albumin, ■×=0.02% each), 04% 5I)S
, 50 rnM sodium phosphate buffered 6 Da (pl(
7,0) and 1ootty/me denatured salmon sperm D
Prehybridization was performed with NA for 4 hours at 42°C. A 32p-labeled probe was created by kinase treatment of the oligonucleotide primer with fully labeled ATP. in 5 ml DNA hybridization buffer per filter.
Primer 0.32P labeled for 8 hours at 42°G.
IX 105cpη/m7! The filters were hybridized. 0.1 SI) in wash buffer containing S and 2X SSC twice at 500 G for 30 min each, then 0.
．． The filters were washed twice with 30 fl each of SDS and 0.2X SSC Te50'Cf. The filters were air dried and subjected to tradiogranometry at -70°C for 2-3 days. Oligonucleotide primer I) M28 and 0;0M2
9. Mutagenic frozen clones with new I)d
The force established to create the el limit position @f (first
Table 4), R1i'-DNA from several clones hybridized with these kinased primers was digested with the restriction enzyme 1)del. Primer DM2
The mutagenic frozen Ml3-H,2 gene generated with a new Ddel restriction position. Take one of the larries (Ml 3-LW44) and J
Inoculate M103 culture medium and extract 5sDNA from culture medium supernatant.
and make dsRF-DNA from the bacteria. Similarly, a plaque (Ml 3-LW46 ) hybridized with primer DM29 was removed and 5sJ)NA and Rli'-1)NA were made from it. Oligonucleotide primer 1) M27 is Dde 1
It is configured to create a new Pstl restriction position instead of the position. Therefore, this primer has 411
Specified plaques were screened for the presence of new Pst1 positions. To identify one such phage plaque M]:3-LW42), ss
DNA and RF-J) NA will be made from now on. Uncertainty that the target TGT cord for cysteine was converted into a TCT cord for serine 8. I) The sequences of NA obtained from all three clones were determined. Example 15 Recloning of mutagenized IL-2 genes for expression in E. coli Ml3-LW42, Ml3-LW44, and M1
．． 3-In'-DNAy from LW46 was digested with restriction enzymes Hindll and Ban II, respectively, and the insert was purified from 1% agar o-skool. Similarly, plasmid pTrp3 (Fig. 7) f Hind I
The large plasmid fragment containing the trp promoter was purified on agarose distillate using M13-LW/I2, M1.3-L.
W44 and M13-LW46 were isolated and ligated with each of the insert fragments. The ligated cilasmid was transformed into E. coli capable of infecting the 112MM294 strain. 3. The cilasmid DNA from these transformants was
was analyzed by restriction r81 genomic mapping to confirm the presence of plasmids pLW42, pLW44, and pi, W/16. FIG. 14 is a restriction map of pLW46. , % of these individual clones in the absence of Tosub1-fan to induce the trp-F0 promoter.
Grow z 2q and extract the bacteria-free extract with SDS-,
Three clones pl-, W42 were analyzed with IJ acrylamide cell. pLW4/1 and pLW46 are all 14. /IKd
114.5 similar to that found in pLW2, a positive net that has been demonstrated to synthesize II, -2 protein.
It has been shown that Kd protein can be synthesized. When these same extracts were assayed for IL-2 activity in mouse HT-2 cells, only clones pr, w2i (positive control) and pLW46 showed significant amounts of total IL-2 activity. (Table 2), cys 58 and cys 105 are important for biological activity, and changing them to serine (pLW42 and pI, W44, respectively) means that biological activity is lost. ing. On the other hand, cy
s125ij, this f ser 125 (pLW46
) It must be unnecessary for activity, since changing it to does not affect biological activity. 2nd expression IL-2 activity (tt/m
e) P I L 2-7 (($WfJI!(I)
1pLW21 (positive control)
113,000T) I-, W42 6
60 pLW44 1,990 pLW46 123.000 Figure 15a shows the nucleotide sequence of the coding strand of Ria-,/pIiW46. Compared to the coding strand of the natural human IL-2 gene, clone pLW46 contains nucleotide 3
74 has a single base change from G to C. Figure 151 shows: 1) the corresponding amino acid sequence of the IL-2 mutin encoded by LW46; This, Tin is HL-2
It is designated as Ser 125. Compared to natural IL-2,
Mutin is ranked 125th and has Selin in his ranking. −12MM294 to E. coli transformed with pLW46.
Strain samples were obtained September 268, 1983, United States. 910-N, Rockville, Maryland 20852
12301 Drive, American Type Culture Collection
lture Co11ection), and A.
TCC3! l. No. 452 was designated as Bu ζ. IL-2 muteins in which cysteine at position 125 is deleted or replaced with another amino acid, such as Mutia IL-2ser ] 25, retain H,-2 activity. Therefore, they can be formulated and used in the same manner as natural IL-2. Therefore, such mutins can be used in the diagnosis and treatment of bacterial, viral, parasitic, protozoan, and fungal infections, lymphagenesis, or manifestations of immunodeficiency, as well as in normal immune function in older humans and animals. For reconstitution, enzyme amplification,
Radioactive label. The development of diagnostic assays, such as radioimaging and other methods known in the art to monitor IL-2 levels in pathological conditions, and therapeutics and diagnostics that inhibit lymphonotwin receptor location. For the promotion of T cell growth in vitro,
It is useful for various treatments, diagnosis, and other applications. Regarding the various therapeutic and diagnostic applications of human IL-2, please refer to Niss A. Rosenberg (S.A. Rosenberg).
berg), E-1-Ikyo Grimm (E, A-Gr
A. im) et al., A. Mazumder et al., and E. A. Grimm and N. A. Rosenberg have reported on research. H,-
2 in 1, can be used by itself or in combination with other immunologically relevant B or ']' alveolar or other therapeutic agents. For therapeutic or diagnostic purposes, distilled water, Linker's solution, banks They can be formulated in non-toxic, non-allergenic, physiologically compatible carrier media, such as liquids, saline, etc. Administration of IL-2 muteins to humans or animals is determined by the physician as appropriate. Depending on the location, it may be oral, or intraperitoneal or intramuscular or subcutaneous. Examples of related cells id B
or T cells, natural killer cells, etc. Examples of therapeutic reagents that can be used in combination with the polypeptides of the present invention are various I:/ter7-erons, especially nigamma interferon. B cell growth factor, IL-1, etc.

[Brief explanation of drawings]

FIG. 1 is an amino acid sequence diagram of IFN-β. Figure 2 is a schematic diagram showing the preparation of the mutant IFN-β gene by oligonuclease-directed mutagenesis.
FIG. Figure 4 shows the cloning vector M 13 m p 8
FIG. 3 is a diagram of a phage. FIG. 5 shows the restriction map of clone M13-β1. Figure 6 shows mutant strain I showing a single base change in the coding region.
Ii'N-βser 17 gene sequence order determination key ξ turn is shown. FIG. 7 is a diagram of the expression plasmid pTrp3. FIG. 8a shows the Hinfl restriction pattern of Rion pSY2501, and FIG. 8b shows the resulting two 169
bp and 28 bp fragments are shown. FIG. 9 is a restriction map of clone I) SY2501. Figure 1 shows the N A sequence encoding Mutin IFN-β5er1.7 and the corresponding amino acid sequence. Figure 11 shows clones psY2501 and pβ1trp
A single 18.000 Dalton protein ζ protein corresponding to 7.J for IFN-βser 17 is shown in the extract. FIG. 12 is a diagram of plasmid pLW1 containing the human interleukin-2 (IL-2) gene under the control of the E. coli trp promoter. FIG. 13 shows the restriction of phage clone M13-IL2 "1!" ! , Figure 1. FIG. 14 is a restriction diagram of plasmid pLW46. Figures 15a and 15b show the dark side of clone pLW46.
Nucleotide sequence of Kbutton and IL-2ser 12
The corresponding amino acid sequences of IL-2 muteins designated 5 and 5 are shown, respectively. Applicant Cetus Corporation Agent
Patent Attorney Kato Asa Michi FIG, 1 F#win↓Human intestine Φ form support East f5 Hama different bed FIG, 2 FIG, 4 FIo 7 GATC FIG, 8a = FIG, 8b FIG, 9 // IFN-β 1 of 1 It's amino fitting
Song to Natsume 61 CTGTGGC 88TTG 8 to GGG to GCTTG
8 ATATT CCTCAAGG 8 CAGGA GA 8 CTTTG CATCTeu trp gln leu
asn gly arg leu gTu tyr
cys leu lys asp arg met a
sn phe asp ile21 CCTGAGG8GATTA to GC8GC to GC to GC
8 GTTCC to GAAGG 8 GG 8 CG to CG ATT
GACCATCTAT pro gTu gTu ile
Tys gin leu qln gln phe
g]n lys glu asp ala aha T
eu thr l)e tyr81 G to GATGCTCCAGA CATCT TGCT to TTTCAGAC to G^TC to TCTAGCAC
TGGCTGGMTglu met Ieu gin
asn jle phe ala jle phe a
rg gin asp ser ser ser th
r gay trp asn41 G to G CTATTGTTGAGAACCTCCTGG
CTAATGTCTATCA to C to GAT to CC to CTGAAGAC glu thr ile val
gTu asn 1eu Teu ala asn
valtyr his gin jle asn hj
s TeuS thr01 GTCCTGGAAGAAAAAAACTGGAGAAAG
AAGATTTCACCAGGGGAAA8CTCAT
GAGCAGTCTGval leu glu glu
lys leu glu to lys gTu asp
phe thr arg gly Tys Teu m
et ser ser Ieu61 CACCTGAAA8GATATTA GGGAGG8TTCTGCA TACCTGAAAGGCCAAGGA
GTACAGTCAChis leu Tys arg
tyr tyr gay arg jle leu
his tyr 1ea Tys aTa Tys g
lu tyr ser his21 TGTGCCTGGACC8TAGTCAG8GTG
Hehe ATCC AAGGA/LCTT Ding TACT Ding CA
To TTlΔCAGACTTcys ala trp t
hr jle vaT arg valgTu ITe
Teu arg asn phe tyr phe
ile asn arg Ieu81 FIG, IQ FIG, f2 FIG, 13 FIG, 15a C12R1/19) 676
0 priority claim @April 15, 1983■United States (US)
■486162 0 Inventor Leo Nis Lin USA 94539 California Fremont Los Pinos 40880 0 Inventor Sida Yule USA 94610 California #109 Oakland
366 Euclid Avenue

Claims (1)

[Scope of Claims] (1) A synthetic mutin of a biologically active protein: Q, which has at least one syntin residue that is free to form cisulfide bonds and is not ligneous for biological activity. Synthetic mutins are defined as having at least one cysteine residue deleted or replaced with another amino acid. (2) Synthetic mutins described in 1iii. Synthetic mutin according to claim 1, in which the presence of only one group is defined as 41i mold. iso-isin, histidine, tyrosine, phenylalanine,
Synthetic mutin according to claim 1, characterized in that it is substituted with Trizo)fan or methionine. (4) Synthesis 1 of the small Y described in Claim 1, wherein the 4N characteristic is that the cys-nacystine residue in +iii is replaced with threonine. (5) The synthetic whip according to claim 1, wherein the mutin is not glyconated.
in. (6) The synthetic whip-in according to claim 1, wherein the protein is IFN-β, IL-2, lymphokinin, colony stimulating factor-1, or IFN-01. (7) The protein is IFN-β, the cysteine residue is at position 17 of IFN-β, and the cysteine residue is replaced with serine (on the 17th position). Synthesis according to scope 1, totiine. (8) r+: The protein j or IFN-β, the cysteine residue is at the 17th position of IFN-β, and the cysteine residue is replaced with a serine residue. The synthetic mute according to claim 1, wherein the protein is IL-2, and the protein is not glycosylated. (9) The protein is IL-2; Ink 5 (the group is at the 125 position of IL-2, and the cysteine sequence) 1 (the '4+i' character is that the cysteine sequence is replaced with serine) (10) The protein is LL-2, the cysteine residue 1 (is located at the 125th position of IL-2, the cysteine residue is replaced with serine, and l-tin is glycosylated. Synthetic mutin according to claim 1, characterized in that: (11) at least one cysteine group that is free to form disulfide 1ζ bonds and is not ligneous for biological activity H; It is a synthetic mutin that is the most biologically active protein.
1. A structural gene characterized by having in the gene a DNA sequence in which at least one of the following amino acids is deleted or has been replaced with another amino acid. (l 2 ) A structural gene of nil, characterized in that the synthetic totiine has a cisnascystine residue. (] 3) + The above cysteine residue 1 (serine,) l/onine, glycine, alanine, valine, leucine, isoleucine, histidine, tyrosine, phenylalanine, tri-11-fan or hamethi-tenine was substituted for the cysteine residue of the mutin described in JF. It is characterized by Structural gene of 41 fraud A+i request; written in Chima No. 11. Cl 4 ') + iii 1, the structural genetic r of claim 11, 4, characterized in that the cysteine residue in iii) of tin is replaced with threonine instead of serinso. (15) rii + 1, non-glycosylation of tin is an 'IN sign, 1 to amended claim 11
Structure 11 gene described in section. (l Ili) riii protein is IFN-β, I
L-2, Lymph size, Colony J'1JIlfi
1. The structural gene according to claim 1, which is characterized in that it is IF N-1 or IF N-01. (17) The protein is IFN-β, the cysteine residue is located at position 17 of IFN-β, and the cysteine residue is replaced with a serine residue. Structural gene according to scope 11, item 1. (18) The protein is IFN-β, and the cis-in residue is replaced with a serine residue in the 17 (Vn; old) of IFN-β, and Special 81 range of interest characterized by the fact that the mutin is not glycosylated
;I blame Inomiko. (18) The quality of the song is IL, -2, and the above-mentioned Soste
It is characterized in that the in residue is located at the 125 I☆ position of IL-2, and the outside of the cysteine is replaced with (or serine). (20) The protein III is II-2, which is located at the 125th position of cysteine 9 (or IL-2).
In addition, the cysteine residue is replaced with a serif,
The structural genetic material according to claim 11, characterized in that the mutin is not glycosylated. (21) Synthesis of biologically active proteins that freely form disulfide bonds 7 and have at least one cysteine residue that is not essential for biological activity +1. Well, outside of that Mutin or Sistine,
(i
: An expression hector characterized by containing (, -\lid- or an expression at a position containing 51 volumes. (22) Disulfite!1111 can be freely formed,
Biological i: (at least one cysteine sequence not essential for J activity), (1 synthetic protein which is biologically active and has (at least one cysteine sequence not essential for activity), one of which is a cysteine sequence) :).The vector can express a structural gene that has a DNA sequence in the gene that implies that at least one of (23) The host cell or host organism is transformed by an expression vector or its progeny containing an ancient locus.
, Co11) according to claim 22. (24) A synthetic mutin of a biologically active protein having at least one cysteine residue that is free to form a '/sulfide bond and is not woody for biological activity, the mutin comprising: Structural genes that have a DNA sequence in the gene with the number u9 indicating that at least one cysteine residue X is deleted or replaced with another amino acid.
? Synthesis 1. Synthesis 1. Method for producing mutin (25) ) A protein with at least one cysteine residue such that a disulfide bond is free to form can be suddenly modified by deleting the cysteine residue, 15, or by replacing a silent cysteine residue with another amino acid. May it be mutatedly modified! A method of preventing proteins from forming disulfide bonds. (2B) It is assumed that the protein is biologically active and that the cysteine is not woody in this biological activity.
ν, of) J horn. (27) +iii Cystine remaining or serine remaining 1±
4+, characterized by being hit by Threonino
! "The method according to claim 25 or 26 of Ai. (28) Forming a cisulfite bond in 1-11, I7,
A synthetic mutin of a biologically active protein having at least one cysteine residue that is not ligneous for biological activity I, the mutin having at least one cysteine residue that is not ligneous for biological activity I.
1 (at least one of the groups is missing, or nu has been replaced with another ami-'d).
A method for producing a structural gene containing an NA sequence in the gene, characterized by C containing the following three points: (a> 1 of the structural gene encoding the protein) Hybridize the 1% DNA with the mutant oligonucleotide primer (this 1 codon for the cysteine residue of the wood button, or optionally an antisense triplet complement that makes one pair with this codon). (!-j L, IIA of the missing amino acid in the codon or other amino acids replaced)
(The codonne that adds the trible for -5 conversion to jjL is the antisense l 7' I, and this does not apply to the matching with the nt.) (b) Extending the pla rmer with DNA polymerase (c) causing the mutant heterodimer to form, and (c) replicating the mutant heterodimer. (29) The method according to claim 28, characterized in that the triblet defining the non-current is an 11-coded version of threonine. (30) The above-mentioned 1 wooden button DNA is 1 unchained Fuji that includes the 1 wooden button, and the mutagenic heterodimid of the above (b) is converted into a closed cyclic heterodimeric field body. The method according to claim 28, characterized in that: (31) The mutant heterozygous φ form is a closed ring heterozygous form.
29. The method according to claim 28, wherein the replication in step (C) is carried out by feeding a transformed susceptible bacterial host. (32) +iii- [After the replication of process (c), the progeny of the mutant strain of Sarani was separated, and the DN was extracted from the progeny.
Separate A, and add the gene r- from ii4 D N A to I'l
Claim 28JJ' characterized in that
The method described in f. (33) the protein is HiI-IFN-β,
I'iii Sistine is in the 17th position, dj
33. The method according to claims 28 to 32, wherein the in code that defines the j inconsistency encodes serine. (34) Claim 33, wherein the first tree strand is an antisense button of IFN-β, and the mutant stomach strain oligonucleotide 1' primer is GcAATTTTCAGAGTCAG-c. The method described in section. (35) the protein is human IL-2, 1iii
33. The method according to claims 28 to 32, characterized in that the cysteine residue is located at position 125 and is based on a codon containing a mismatched serine at position 118. (36) Except for codon deletions for cysteine residues or mismatches in the codons that defined the l-represent encoding other amino acids, the codon for cysteine residues in the structural gene 1 tree chain, or in some cases this codon Oligonucleonate-directed mutagenesis characterized by having an antisense reference triblend nucleotide sequence that pairs with at least one cysteine residue that is non-woody for biological activity. Synthetic oligonucleotides for creating structural genes with encoded DNA sequences in which groups are deleted or replaced with other amino acids. C37) 17 (IFN- with cysteine residue at η position
A therapeutic composition comprising a therapeutically effective amount of a synthetic mutin, with or without glycosylation, in which the cysteine residue of β is replaced by a serine residue, and a pharmaceutically acceptable carrier. (3B) 1.25 (a therapeutically effective amount of a synthetic mutin with or without glycosylation in which the cysteine residue of IL-2 with a cysteine residue in the V position is replaced by a serine residue) (39) Plasmid pSY2501. (40) A bacterium whose 4N sign is that plasmid psY2501 is transformed by its progeny. (41) The bacterium is Escherichia coli (E. 40.C42) IFN-β ser17. (43) Plasmid pLW46. <44) A bacterium characterized in that the AIlli shield is transformed by plasmid pLW46 and its progeny. (45) Special Al (bacteria according to claim 44), characterized in that the bacterium 11j is Escherichia coli. (4B) IL-7 se r 1 2 5.