JPH01124388A - High level manifestation in e. coli of soluble matured hil-1 beta and derivatives of different biological activities - Google Patents

Abstract

PURPOSE: To produce a specific soluble protein, comprising plasmid pUC8 and amino acid sequence 5-153 of mature hIL-1β, by forming a plasmid having a specific translation initiation codon.

CONSTITUTION: This plasmid enables high level expression in Escherichia coli of a soluble protein which comprises plasmid pUC8 and a DNA encoding amino acid sequence 5-153 of mature hIL-1β and contains mature hIL-1β amino acid sequence 5-153 having ATG/Met translation initiation codon at the downstream of pUC lac promoter isolated from TCASer codon at 5-position by a 4-18 amino acid codon. E. coli cell is transformed with the plasmid, and a transformant is cultured to express a protein, which is recovered and expressed.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides mature human interleukin 1β (hIL-1β
) and the expression of derivatives of mature hIL-1β (mutant proteins) with amino acid substitutions and insertions occurring at the N-terminus and with concomitant different biological activities. In particular, the present invention relates to high level expression of such proteins in B. coli using plasmid vectors containing the lac promoter.

Human interleukin-1β (hIL-1β) is Mr3
Q, 747, 269 amino acids. It is first produced by stimulating mononuclear cells as a tannoplasmic substance. This was proteolytically converted into 1 from position 117 to position 269 of Mr17,377.
53 Aminojo Cal? The bioactive form consists of a xyl terminal end. This 269 amino acid protein is called pro-hIL-1.
The 153 amino acid protein is called mature hIL-1β.
It is called.

European Patent Application Publication No. 0165654 (June 1985,
Immunex Corporation) is a recombinant hIL''-1β with a methionine residue in the first position.
(methIL-1β) expression is disclosed. hIL
The gene encoding -1β was amplified in E. coli but expressed in yeast. June 1985, Immu
Journals published by NEX researchers (March et al., r.
Nature J 315.641-647, 198
June 20, 2013), recombinant mature met hIL-1β using a vector with the λ phage PL promoter.
The findings in E. coli are described. This journal, pages 644-5, states that the cells exhibited high levels of IL-1 activity. There is no disclosure regarding protein purification. In 1986, researchers at In;munex began publishing other journals. Kronheim et al.
otechnology J 4.1078-1082
．． December 1986), 1080 negative, MarC
The "preliminary structure" described in the journal H. et al. did not produce high levels of recombinant hIL-1β, indicating that purified hIL-1β had previously been obtained only from human mononuclear cells.

Journal of Kronh61m et al. uses the expression system pLNIL1β which also contains the λ phage PL promoter.
describe high-level expression of hIL-1β in li and purification of the thallus.

European Patent Application No. 0161901 and AurOn et al. Proc, Natl, Acad, Sci,
USA J, 81.7907-7911 (1984) describes cloning vehicles (pcD415 and pc
D121B) and various fragments thereof. European Patent Application Publication No. 0i61901 indicates that the sequences can be fused to expression vectors and expressed in either eukaryotic or prokaryotic cells, but does not disclose a specific expression system. Dinarello et al. [J.

C11n, Invest, J 77.1734-1
759 (June 1986) is a protein E, col consisting of amino acids 71-269 of pro-hIL-1β, prefixed with 24 amino acids provided by an expression vector.
Proh by expression in i and subsequent enzymatic decay
It discloses the production of 7mino powders 112 to 269 with IL=1β. The expression vector in the journal is pc
It has been shown that it was assembled by inserting a fragment of D121B into an E. coli plasmid, but no further information is provided. r Proc, Natl, Acad, Sc by Rossenwssθr et al.
i, USA J, 83.5243-5246 (198
(July 1997) disclose the expression of pro-hIL-1β and various deletion forms in COS monkey cells using plasmids pcD415 and 1218, and demonstrate the expression of mature hIL-1β in cal-cells. Also included was one consisting of about 136 amino acids at the xy-terminus.

Pro-human interleukin-1α (pro-hIL-1α) is a protein consisting of 271 amino acids with approximately 27% homology to pro-hIL-1β. When this is proteolyzed, prohlL-1α cal? A biologically active protein of approximately 17 kD consisting of 159 amino acids at the xyl terminal is obtained. Gubler et al.
Mmmunology J, 136.2492-2497
(1986), using a plasmid with the λfursoPL promoter to develop a pro-hIL-1α vector. Discloses the expression of the xyl-terminal 154 amino acids in E, C011.

Plasmid vectors with lac promoters such as pUCB have been described (Vieira and Mess et al.
ing et al., Gene J, 19.259-268.
(1982)), Pharmacia (Piscat
away%NJ) and BsthesdaRese
archLaboratory (Bethesda,
It is commercially available from IMD. European patent application publication 01
No. 61901 and Rossenwasser et al. describe the structures of cloning and expression vectors as well as the above-mentioned pUCB, but only as a source of polylinker 7 fragment used in the assembly of plasmid pcD121B.

We inserted the DNA encoding mature hIL-1β or a derivative of mature engineered L-1β with amino acid substitutions into plasmid pUC8, or inserted the ATG/Met/initiation codon downstream of the lac promoter into codons 4 to 18. by insertion in one or more of positions 1 to 4 in such a way that they are separated from the TCA/Ser codon for position 5 of mature hIL-1β.
Alternatively, it has been discovered that a plasmid capable of high-level expression in E. coli of a soluble protein having the amino acid sequence of an hIL-1β derivative can be obtained.

Derivatives of hIL-1β with amino acid changes within the N-terminal sequence of mature hIL-1β were found to exhibit various degrees of biological activity. The specific biological activities of the assembled hIL-iβ variants varied from approximately 7-fold increase to approximately 700-fold decrease compared to mature hIL-1β. These results indicate that the structure of the N-terminal sequence of mature hIL-1β is important for the biological function of the molecule. The present results identify the N-terminal sequence of mature hIL-1β, particularly the N-terminal 4 amino acid residues, as a target for additional structural changes that result in increased or decreased biological activity of the molecule. That is, changes in the N-terminal sequence of mature hIL-1β
It has been shown that it can be used to modify the biological activity of -1β.

The IL-1β derivatives of the present invention having a biological activity greater than about 10 times that of native hIL-1β can be used to promote hematopoiesis (Oppenheim et al., r Immunology
TodayJ, 7.45-56 (1986): D
inerello, r Bull, In5t.

Pa5teur J, 85, 267-185 (1987
)). Approximately 1 of natural hlL-1β
The IL-1β derivatives of the present invention with low biological activity find use as research materials, for example as negative controls in assays for the biological activity of IL-1.

The proteins produced by the present invention are soluble. When E. coli cells expressing the protein are lysed by sonication and centrifuged, the membranes are found in the supernatant rather than in the sediment.

Therefore, Tan? can be easily purified to achieve homogeneity in functional structure.

The plasmid of the present invention of E. coli strain JM 101 was deposited with the ATCC in accordance with the Budapest Treaty on the Deposit of Microorganisms.
(Ameri an Type Culture Co1
1ection.

12301, Parklavrn Drive%Roc
Kvilla, MD, USA). The deposited plasmids and their ATCC accession numbers are shown below.

pDP506 67302 pDP516 67303 pDP516-22a 67304pDP516
-18 67305 Figures 5 to 8 show the DNA sequence of the above-mentioned plasmid and the amino acid sequence of the protein expressed by it. Figures 9 and 10 show two other plasmids of the invention, pDP516-23 and pDP51.
6-16 DNA sequence and the amino acid sequence of the protein expressed by it are shown. Numbers 1 and 117 in Figures 6 and 5-10 indicate position 1 at the amino terminus of mature hIL-1β, which corresponds to position 117 of pro-hIL-1β. Numbers 153 and 269 are mature hIL
Position 153 at the carboxy terminus of -1β is shown, which corresponds to position 269 of pro-hIL-1β.

Plasmid pDP506 contains Hg1A, a DNA encoding the amino acid sequence 116 to 269 of prohIL-1β.
It was created by inserting I~Pstl into the PstI site of pUC8. The pDP516 plasmid series was created by digesting pDP506 with EcoRI and Sal I to generate linear DNA, digesting the linear DNA with Ba, 131 exonuclease, filling in the 3' ends by Klenow reaction, and making blunt ends. was created by ligating with T4 DNA IJ gauze and recircularizing the DNA. Other plasmids of the present invention were also prepared using pDP506 by the method described below.
Created from.

E. coli cells of the JM101 strain were transformed using the plasmid, and the cells were cultured to express the protein.

The bacterial cells were sonicated, the lysed cells were centrifuged, and the supernatant was subjected to anion exchange column chromatography to purify the protein to obtain homogeneous proteins.

Table I lists some of the plasmids of the present invention, and the amino acid sequences of the proteins they express are listed by LehnLnger.
r Biochemistry J, 2nd edition, p72,
Worth Publishers Inc. (19
The amino acid residues described in 75) are indicated using letter symbols indicating the amino acid residues.

Recombinant hIL-1β- phIL-1β hIL-1βpDP-516
DP-516 pDP-516-22a DP-516-22ap
DP-516-18DP-516-18pDP-516
-23DP-516-23pDP-516-16DP-
516-16pG1y-3Gly-5 pPro-3Pro-3 pLeu-ro Leu-3pIle-
311e-”+ pThr-3Thr-3 pGlu=4 Glu-4 pTyr-4Tyr-4 pLys-4Lys-4 115,111115116117118119120
121...2691 2345...153 A P V Ft 5-8
T N , A P V
R8・5TPVRS...S TMVRS...5 TINAPVRS...S TMGRS...S TMPFI S...S TMLRS...S TMI'R8...S T'MTR8...S Mature hIL- N-terminus of 1β and several hIL-β derivatives.

The end sequences are shown in Table 1. The hIL-1β derivative tongue/4' tail was produced using the plasmid expression vector shown in Table 1.

It was expressed in E.coli. E. The hIL-1β-derivative tanf expressed in E. coli has an N-terminal Met residue (not shown) in front of the sequence shown in Table 1.
This may be removed in E8 coli by the action of aminopeptidases. Amino acids present in hIL-1ββ derivatives that differ from the sequence of native mature hIL-1β are underlined in Table 1. Numbers in two horizontal columns are pro hIL-1β (top row) and mature hIL-1β (bottom row)
It shows the aroma of the amino acid residue. Dots indicate mature hIL-1
1 of prohIL-1β corresponding to positions 5 and 153 of β.
Linear amino acid residues between positions 21 and 269 are shown.

Assembly of pDP506 As shown in Figures 1 and 2, plasmid pDP50
The starting material used to create B is plasmid rpcDI218.
As mentioned above, this is the European Patent Application Publication No. 016
No. 1901 and AurOn et al. and Rossenwa
It is described in the literature of Sser et al. prohIL-1
1.6 kilobase pairs (Kb) containing the DNA encoding β
) fragment was excised from pcDI218 by BamHl digestion. FIG. 2 shows the restriction enzyme cleavage site within the 1.6 Kb fragment and the probe and mature hIL-1
β amino- and cal? 1, 34 corresponding to the xy terminus
Sites of 8/351 and 807 nucleotides are shown. The 1.6 Kb fragment was inserted into the Barn of plasmid pUC9 (commercially available from BRL and Pharmacia).
By subcloning into the H1 site, hybrid plasmid pU
C9-prohIL-1β was created.

Plasmid pUC9-prohIL-1β contains two Acc
It has 1 part. pUC9 from the Acc1 site of the 1.6 Kb pcD1218 derivative fragment (Figure 2)
(L5Kb non-coding region up to the Acc1 site of
pUC9-prohIL-1β was removed by digestion with cI and then ligated to generate the plasmid pUC9-prohIL-1βAAcc), which was digested with prohIL
DNA encoding L-1β was used to amplify in E. coli.

Figure 3 shows amino acids 112 to 269 of prohILlβ.
The nucleotide sequence encoding the prohIL position is shown.
P present within pUC9-prohIL-1βΔAccl approximately 150 nucleotides downstream from the -1β co-r sequence.
stl restriction site and the codon I (giA1 site) for positions 114 and 115. Horizontal and vertical lines in Figures 3-5 indicate sequences recognized and cleaved by the indicated restriction endonucleases. There is.

To assemble pDP50(S), a [L6Kb restriction fragment was created by digestion of pUC9-Biff hIL-1βCcl with Hg1Al and Pstl, and a 6Kb fragment containing the mature hIL-1β coding sequence was created by pU(:8 Figure 4 shows the PstI site and the upstream ATG/Met/initiation codon separated from the site by 11 amino acid codons.

Although the recognition sites for Hg1Al and Pstl are different, the 3' protruding ends produced by the enzymes are the same, so Hg1Al
The ends can be ligated to the Pst ends using T4 DNA IJ gauze, forming a sequence that is not recognized by either enzyme.

As shown in FIG.
It has the coding sequence for position ~269.

ATG/Met derived from pUC8, translation start codon is 16
Position 3 (Val) of hIL-1β by amino acid codon
separated from the codon for

pDP516, pDP516-16, -18, -22a
and -23 assembled pDP506, pDP516, pDP516-16, p
DP516-18, pDP516-22a and 1)D
P516-2! The assembly of 1 is schematically shown in FIG.

pDP506 was digested with EcoRI and 5all, and the linear DNA was subjected to Ba131 exonuclease digestion for approximately 10-15 seconds to remove the nucleotide P from both ends of both mirrors. At the end of this digestion, the deletion 3
' ends were filled in with a Klenow reaction, and the resulting blunt ends were then ligated with Ta DNA IJ gauze to complete the DNA.
A was recircularized. This formed a pool of plasmids that were subsequently analyzed by protein expression analysis and DNA sequencing to determine whether υpDP516, pDP516-16, -18, -
22A and 23 were found to be included.

As shown in FIG. 5, pDP506 has Smal and BamH1 sites in the region derived from pUC[3, which can also be used to obtain the plasmid of the invention. Additionally, other specific plasmids with single promoters, such as pUC12, can also be used to create the plasmids of the present invention.

All DNA manipulations described here are performed by Maniat.
is etc. r Molecular Cloning,
A Laboratory Manual J, Col.
Spring 11! Arbor Laborato
ry, NewYork (1982).

plasmid)'pDP506'st, phIL-
1β, pGly-3, pPro-5, pLeu-3, p
'rhr-3, plle-3, pGlu-4, pTyr
-4 and pLys-4 assembly plasmid rI)
DP506 has two Bindl11 sites.

One Bind11 site is within the fiIL-1β cod sequence. This site contains codon 133 of zolo hIL-1β, which corresponds to codon 16 of mature hIL-1β.

The other Bind1 site is located downstream (3') of the hIL-1β code sequence and is located within the pUC8-derived fragment of pDP506.
Adjacent to the stI site. pQc8 in pDP506
The derived H1ndI[1 site was removed by the method described below to form pDP506''St. Plasmid pDP
506 was digested with Pstl and Ba131 and ligated. Approximately 75 p.p. around the PstI site by exonuclease
base pairs were removed. Therefore, adjacent Bins at the same time
d[1 site was also removed. The obtained plasmid is pDP
It is shown as 506ΔPst. Plasmid pDP50aat
was digested with EcoRI and Eindlll, and the oligonucleotide from EcoRl to aindlll was converted into the coding strand: (AATTCCATA2 GGGTATTAC'A
'rGGCACCTGTAAGATCTC'I'G
AAC'I'GCACGCTCCGGGACTCACA
GCAAAAA) from EcoRI with atndI[
A plasmid phIL-1β encoding the mature form of hIL-1β was created by replacing the above with a 73 base pair oligonucleotide. The 73 base pair oligonucleotide contains the soma binding site (dashed and underlined), translation initiation codon ATG (underlined) and Bgl.
l[It has a restriction site (underlined), which is IL-1β
Although not naturally present in the co-P sequence, it was introduced without changing the amino acid composition at positions 4 and 5 of hIL-1β.

A series of 35 base pair sequences, 1) nucleotide)' (Bco
Rl to Bgln) was created with appropriate coding sequence changes, and EcoRI to BglI of phIL-1β was created with appropriate coding sequence changes.
A 35 base pair fragment of I was used to generate various derivatives of hIL-1β with amino acid substitutions at the N-terminus. Plasmid expression vector representation, hIL
-1β-derived tannolog and created hIL-
The N-terminal sequences of the 1β derivatives are shown in Table 1.

Transformation, Expression and Purification Ampicillin-sensitive JM1 [11 strains of E. coli cells were transformed with plasmid DNA. The bacterial cells were incubated with ampicillin (
100 μf/ynl) and IPTG (isopropanylthio-β-galactoside) were added and grown in 7'hL broth. The recombinant clone has a Klett value of 30 (
Klett-8ummerson photoelectric colorimeter, Klet
The cells were grown until they reached K (measured by tManufacturing Company), at which point IPTG was added to a final concentration of 1 mM. Bacteria were harvested at various time points and further characterized. Cells obtained from 500g of culture solution were treated with sonication buffer (50mM)! JspHaO1
1mM EDTA, 1mM DTT) 50
resuspended in m and sonicated for 7-10 seconds in a volume of 5rILl. Sonicated samples were centrifuged for 5 minutes at 4°C.

The supernatant and precipitate were stored separately. After filtering the sonicated lysate with a Millipore filter (0.45μ),
5ynchropak ion exchange column (2,1x 2
5 CIIL) (Synchrom, Inc.
, Linden, Indiana).

h in this step and the next chromatography step
The IL-1β-containing fraction was identified by Western blotting using a rabbit polyclonal antibody against mononuclear cell hIL-1β.

Fractions containing hIL-1β were pooled and AMICON
It was concentrated to 1 to 3 at by ultrap-filtration using a concentrator, and then transferred to LKB Instruments, Inc.
Gaithersburg.

ACA sinon chromatography on the back of MD (
2,4X100cR). The buffer used in the Ryoriki Ram was 50mM Tris pH 8, 1mM EDT.
A, 1 mM DTT. Natural hIL-1
β as Matsushima et al. Biochem J
, 25.3424-3429 (1986), from the bone marrow mononuclear T1 (P-1 cell line).

For gel analysis, samples were prepared in Laemmli sample buffer containing 1 mM DTT (Gubler et al., r J
, Immunol J, 136.2492-2497
) and boil for 3 minutes, then Pharmaci
a 5D8-PA()E vertical slurry containing 15 thiacrylamide or 8 to 25 thiacrylamide groups using the Phast system (BioR
ad and Hoeffer apparatus). The gel was stained with Coomassie blue and the molecular weight of recombinant hIL-1β was determined using a molecular weight standard (
Calculated by linear regression analysis using pre-stained BRL or Pharmacia molecular weight markers).

Western blot analysis was performed using Towbin et al. [Proc.
.

Natl, Acad, Sci, USA J, 76
．． 4350-4356. First, E. coli proteins were separated using 5DS-PAC) and subjected to nitrocellulose plotting on 45ml nitrocellulose paper. Nitrocellulose paper with bovine anti-hIL-1
Horseradish peroxidase (BioRad,
mond, CA) to reveal the recombinant HEC I, -1β band.

Colonies of cells transformed with pDP516 series plasmids were picked individually and grown overnight in L broth 1a supplemented with ampicillin and lac inducer IPT (). - After night culture, 0.5 a of each cell culture solution
centrifuge and resuspend in tamp lysis buffer;
It was subjected to 5DS-PAGE.

It has an apparent molecular weight approximately equal to that of pDP516, and hI
Transformants that showed high level expression of L-1β were selected. Individual clones identified were isolated and sequenced, and recombinant hIL-1β was purified as described above.

When cells carrying recombinant plasmids pDP506 and pDP516 series plasmids were grown in the presence of IP'Il'G, recombinant hIL-1β was found to be the main product of total cellular protein (15-30 weight%). When the soluble IL-1-derived protein was subjected to chromatography on a 5-synchropak anion-exchange column, a large amount of IL-1-derived protein was eluted in the effluent from the 5-synchropak anion-exchange column.

Fractions containing hIL-1β were pooled. Purity is 5DS-
As determined by PAGE and Western blot analysis9
It was 5 cents more expensive. The pooled fractions were further purified by ACA sizing column purification.

According to this step, the recombinant hIL-1β protein is 99
High purity was demonstrated. The protein endotoxin concentration was 6 n/lq lower than that of hIL-1β.

The identity of the recombinant plasmids was determined by DNA sequencing. Direct plasmid sequencing rDNAJ such as Chθn
, 4.165-170. Synthetic 16-mer orico9 nucleotide (GTGGAATTGT
GAGCG) was used as a primer. The identity of the recombinant protein 4 was confirmed by amino acid composition and sequencing of the amine-terminal 24 amino acids. Purified protein is Ee
Analysis was performed on a ckman spinning cup sequencer model 390M.

Plasmid pC)ly-3, pPro-3, pLeu-
3, pThr-5, plle-3, pGlu-4, p
E in a similar manner as described above using Tyr-4 and pLys-4.

E. coli was transformed, and the corresponding protein was expressed and purified.

Bioactive purified recombinant hIL-1β protein was purified by Lachman
etc. r Meth, in En, zymol, J
Standard murine thymocytes described in 116.467-479 (
C3HAeJ mouse) proliferation and standard gingival fibroblast prostaglandin E2 (PGE2 production assay) (PG
E2 RIA kit NEN research item, E, 1. d
u Font de Nemours and Comp
any, N, B111er a, made by M'A) υ
evaluated.

The biological activity of DP506 was determined by the natural h
It was about 10 times lower than that of IL-1β. DP516,D
The biological activities of P516-16 and DP516-23 were approximately equal to or better than that of native hIL-1β.

Table 2 shows hIL-1β measured by thymocyte proliferation assay;
DP516-22a, DP516-18 and Gl
The biological activity of u-4 in one test is shown. IL-
DP with alanine substitution for threonine at the N-terminus of 1
516-22a showed an approximately 4-fold increase in biological activity. Another mutant, DP516-18, in which the first two amino acids were substituted, showed approximately 7-fold increased activity.

a, Purified protein by thymic proliferation assay.

analyzed. One unit is equal to the amount of substance that provides half the maximum enhancement. Values are mean ± from five independent experiments.
Displayed by SEM and normalized to mature mononuclear hIL-1β.

b, Natural mature protein L-1β derived from mononuclear cell line THP-1
In, -1β was determined by examining the N-terminal sequence of hIL1-1β.
It was suggested that the protonated aldadine residue at the 4-position may be involved in salt bridge formation or hydrogen bonding, contributing to the stabilization of the active structure of the protein. Substitution of arginine by glutamic acid (()lu-4) results in DP516-
A single amino acid substitution for 18 resulted in an approximately 700- to 7000-fold decrease in specific biological activity.

This result indicates that the positively charged arginine residue at position 4 is responsible for hIL-1.
shown to be important for the biological activity of β.

A at position 4 by Lya, as shown in Lys-4
Substitution of rg caused little change in the specific biological activity of hIL-1β-derived tongue and arm.

As shown by hIL-1β-derived Tyr-4, Tyr
Substitution of Arg at amino acid position 4 by a residue of approximately 1
A 0-fold decrease in specific biological activity occurred. These results are shown in Table 3, where N indicates the number of tests using the thymic proliferation assay.

Table 3 hIL-1β 62.79x10'α81x10727
9Tyr-431, 80X1060.17X10618
Gly-372, 87x1061.18X10629a
One unit is equal to the amount of substance that provides half the maximum enhancement.

b Mononuclear cell line TI (P-1 derived naturally mature hIL-1
A series of recombinant hIL-1β analogs with varying strengths of biological activity were generated by single site-directed mutagenesis for computational simplicity from specific activity relative to β. Manipulation of the amino-terminal sequence of hIL-1β has generated recombinant proteins with increased or decreased biological activity compared to native hIL-1β. In particular, hI
It was found that arginine at position 4 of L-1β is one of the key residues in the function of hIL-1β. mature h
It has been shown that specific substitutions within the N-terminal four amino acid residues of IL-1β alter the specific biological activity of the molecule.

When the identity of DP516 was confirmed by amino acid sequence,
Approximately 89 circles of the protein molecule start from the N-terminal threonine, probably due to incomplete removal of the starting amino acid methionine by E-coli aminopeptidase.
It was found that about 11 yen started from methionine. Mixed amine ends are a common phenomenon in prokaryotic gene expression. Figures 5 to 1 [ ] and Tables 1 and 2 are
Recombinant hIL-1β of the present invention! This shows that the reaction starts with a threonine, but some molecules can start with a methionine in front of the threonine.

It is expected that plasmid expression vectors with the RACK promoter can be used for high level expression in E. coli of hIL-1β substitutes and deletion mutants other than those specifically described herein. Such isolateral bodies are also included within the scope of the claims.

All documents mentioned herein are incorporated by reference in their entirety.

[Brief explanation of the drawing]

Figure 1 shows pDP506, pDP516, pDP516-
16. This is a schematic representation of the assembly of pDP516-18, pDP516-22a and pDP516-25. Figure 2 shows a 1.6 kilobase pair (Kb) fragment containing DNA encoding pro-hIL-iβ. Restriction enzyme cleavage sites within the probe and amino- and cal-? 1, 348/351 and 8 corresponding to the xy terminus
Figure 3 shows the amino acid positions 112 to 26 of prohIL-1β.
Figure 4 shows the PstI site and the upstream ATG/Met/initiation codon separated from the site by 11 amino acid codons; Figures 5-8 show the nucleotide sequence encoding position 9; is plasmid pDP506, plasmid pD
The respective DNA sequences of P516, plasmid pDP516-22a, and plasmid pDP516-18,
It shows the amino acid sequence of the protein it expresses, and Figures 9 and 10 show two other plasmids pD of the invention.
1 shows the DNA sequences of P516-23 and pDP516-16 and the amino acid sequences of the species in which they are expressed. Patent Applicant E.I. Dupont de Nemoirce & Conn. 7 Knee and 2 other persons Mimi Tennuma Litigation 88 Maki 88-he H-
Heto - Hetoi Vomit ← n88 Armor $8 - Gto - Heto Procedural Amendment (Method) % Formula % 1, Indication of the case 1988 Patent Application No. 14791 2, Name of the invention Soluble mature hlL-1β and High-level expression of derivatives with different biological activities in E. coli 3, relationship with the case of the person making the amendment Patent applicant Compagnie 4, attorney 5, notice of order for amendment March 31, 1988 (Date of dispatch: 1988) .4.26
) 7. Contents of the amendment: An engraving of the drawings originally attached to the application and an attachment (no changes to the content). that's all

Claims (1)

[Claims] 1) Essentially plasmid pUC8 and mature hIL
It is composed of DNA encoding the amino acid sequence 5 to 153 of -1β, with T at position 5 due to amino acid codons 4 to 18.
has an ATG/Met translation initiation codon downstream of the pUC8 rack promoter separated from the CASer codon;
A soluble protein containing the mature hIL-1β amino acid sequence 5-153. A plasmid capable of high-level expression in coli. 2) The plasmid according to claim 1, wherein a DNA fragment encoding the amino acid sequence 5 to 153 of mature hIL-1β is inserted into the Pst I site of pUC8. 3) A plasmid according to claim 2, wherein the ATG/Met translation initiation codon is separated from the TCASer codon by 4 to 8 codons. 4) Translation start codon of ATG/Met is sequence Thr A
4. The plasmid of claim 3, separated from the TCA/Ser codon by 6 codons encoding snAla Pro Val Arg. 5) The plasmid according to claim 4, which is pDP516 with ATCC accession number 67303. 6) ATGMet/start codon is sequence Thr Pro
TCA/Se by 4 codons encoding ValArg
4. A plasmid according to claim 3, which is separated from the r codon. 7) pDP516-22 with ATCC accession number 67304
The plasmid according to claim 6, which is A. 8) ATGMet/start codon has the sequence Thr Met
TCA/Se by 4 codons encoding ValArg
4. A plasmid according to claim 3, which is separated from the r codon. 9) pDP516-18 with ATCC accession number 67305
The plasmid according to claim 8. 10) ATG/Met translation initiation codon has the sequence Thr M
etIle Thr Asn Ser Arg Gly
Ser Val Asp LeuRis Asp A
Claim 2 separated from the TCA/Ser codon by 18 codons encoding la Pro Val Arg.
Plasmids described in. 11) The plasmid according to claim 9, which is pDP506 with ATCC accession number 67302. 12) A plasmid according to claim 1, wherein the ATG/Met translation initiation codon is separated from the TCA/Ser codon by 4 codons. 13) The plasmid according to claim 12, wherein the 4 codons encode Thr Met Gly Arg. 14) The plasmid according to claim 12, wherein the 4 codons encode Thr Met Pro Arg. 15) The plasmid according to claim 12, wherein the 4 codons encode Thr Met Leu Arg. 16) The plasmid according to claim 12, wherein the 4 codons encode Thr Met Thr Arg. 17) The plasmid according to claim 12, wherein the 4 codons encode Thr Met Val Glu. 18) The plasmid according to claim 12, wherein the 4 codons encode Thr Met Val Tyr. 19) The plasmid according to claim 12, wherein the 4 codons encode Thr Met Val Lys. 20) The plasmid according to claim 12, wherein the 4 codons encode Ala Pro Val Arg. 21) E. coli transformed with the plasmid of claim 1. co
li cells. 22)E. 22. The cell according to claim 21, wherein the E. coli strain is JM101. 23) Following steps: (1) Claim 1, 7, 9, 12, 15, 19 or 20
E. using the hybrid plasmid described in any one of the above. c.
(2) culturing the cells to express the protein; and (3) purifying the protein to provide homogeneity. A method for expressing soluble proteins at high levels in E. coli. 24) Derivatives of mature hIL-1β with amino acid substitutions in one or more of positions 1-4. 25) The derivative according to claim 24, which has an amino acid substitution at only one or both of the 1st and 2nd positions. 26) The derivative according to claim 25, which has one amino acid substitution at position 1. 27) Claim 2 wherein Thr is substituted for Ala.
6. The derivative according to 6. 28) A derivative according to claim 25 with two amino acid substitutions at the 1 and 2 positions. 29) Thr and Met are respectively Ala and Pr
29. A derivative according to claim 28, substituted for o. 30) Claim 24 with three amino acid substitutions at positions 1 to 3
Derivatives described in. 31) Thr, Met and Glu are respectively Ala,
Claim 30 substituted for Pro and Arg
Derivatives described in.