CN1007907B - Purification of recombinant interleukin-2 - Google Patents

Abstract

The present invention relates to a method for preparing basically homogeneous recombinant mature IL-2 of a human body, which comprises: cell membrane ingredients are separated from a lysate of a converted microorganism which expresses and accumulates IL-2; then IL-2 is extracted from the cell membrane ingredients, and an extract is purified by a chromatography method.

Description

Purification of recombinant interleukin-2

Human leukocytes Jie element-2(IL-2), be called T cell growth factor (TCGF) in the past, it is a kind of soluble proteins, can be from being obtained the T cell of Sugar receptors or antigen activates, it has the function of regulating lymphocyte reaction, and helps the lymphocytic external long-term cultivation to the responsiveness T of antigen-specific.Know that also IL-2 has the thymocyte mitotic division of increasing and the lymphocytic reaction of inducing cytotoxic T.In view of the above, this lymphocyte instrumentality is verified and is strengthened body fluid and cellullar immunologic response and immune deficiency state returned to normal body fluid and cellular immunity is effective.The immunologic competence of the IL-2 that these have been identified indicates, and IL-2 treats immunological disease at medical immunology, comprise tumor disease, bacterium or virus infection, immunodeficient disease is useful (people such as Papermaster B during autoimmune disease or the like, the immunopharmacology progress, 507,1980).Recently, in one piece of review article on magazine the 249th the 2nd phase of volume of American Medical Association the 166th～171 page (January 14 nineteen eighty-three), various lymphokine has been discussed, particularly including the clinical application of IL-2 of human body.

From using rapid dynamical liquid chromatography (LC) (HPLC) method of multistep, be the uniformity material recently purifying by the IL-2 that obtains in the inductive human malignant cell.It is 1.4 * 10 that this homogeneous human body IL-2 has demonstrated a kind of every milligram ⁹The ratio work of unit (for example, referring to European Patent Application No. 83.109202.8, publication number 106179).

Ta Niguqi people such as (Taniguchi) is in three recombinant DNA (thymus nucleic acid) annual meeting of February 9 nineteen eighty-three in Philadelphia, Pennsyivania, report the clone of IL-2 gene and the arrangement of its order, and reported the expression (" nature " of the immature pattern of human body IL-2.302 phases 305～310 (1983)).IL-2 proteoplast aminoacid sequence is by the cDNA(complementary DNA), this cDNA is from by the mRNA(messenger RNA(mRNA) that obtains the con A inductive Jurkat cell) in prepare.Whole cDNA clone length is 800 base pairs, a kind of 153 amino acid whose protein of encoding.Equally referring to european patent application.Publication number 91539.

Other groups have also reported the cloning of IL-2 gene and the arrangement of its order.See De Wosi " molecular cloning of human body IL-2CDNA and the expression in intestinal bacteria E.coli thereof " that the people write such as (Devos)." nucleic acids research ".Nineteen eighty-threes 11 are rolled up 4307～4323 pages.Be also shown in european patent application, publication No. 118977.

Although these bibliographys have been described IL-2 in transformed host cells, the particularly expression in E.coli can not obtain purge process homogeneous or purified basically sophisticated recombinant human IL-2 but all disclose the professional who makes this technical field.

The present invention is the method for the sophisticated human body IL-2 that particularly recombinates of a kind of purification of Recombinant IL-2.The invention provides a kind of sophisticated human body IL-2 that purifies reorganization and make it to reach homogeneous method, this method has overcome the limitation of the method for purification that has earlier.This method comprises:

(1) cultivates a kind of organism of conversion of the dna sequence dna that contains the sophisticated human body IL-2 that to encode out.

(2) make the cultivation of the organism that transforms in a kind of (1) step to express and to accumulate sophisticated human body IL-2.

(3) culture of the organism that transformed of dissolving (2) step is to obtain the molten born of the same parents' of a kind of cell mixture.

(4) separating in the molten born of the same parents' mixture of the cell of cell membrane component from step (3).

(5) clean isolated cell membrane component to obtain containing the washing lotion of IL-2 with extracting solution.

(6) with the washing lotion of purification by chromatography (5) step to obtain the recombinant human IL-2 of purified basically or homogeneous.

The brief introduction of accompanying drawing of the present invention is as follows:

Fig. 1 has described the amino-acid sequence of dna nucleotide sequence and corresponding sophisticated human body IL-2 of pIL2-2B, and (arrow is represented the proteinic N-terminal of sophisticated IL-2.）

Fig. 2 has explained from the structure of the next pRC2 of pBR322 in the mode of scheming.

Fig. 3 illustrates the structure of the pRC23 that contains a pL promotor.

Fig. 4 has represented the formation for the structure gene of expressing Ser-IL-2.

Fig. 5 illustrates a not structure of the sophisticated IL-2 expression vector of tape starting subsystem (pRC201/IL-2).

Fig. 6 illustrates and contains a pL promoter systems (pRC233/IL-2).

Process of the present invention is to depend on the film fragment that the IL-2 protein of having found surprisingly by Institute of Micro-biology's expression tends to be accompanied by host microorganism, mainly is the inner membrance (bilayer lipid membrane is usually with hydrophobic protein) of microorganism.Thereby, in regaining the process of IL-2, these films are separated from transform microorganism, guaranteed that the last minute at whole purification process obtains the IL-2 of high yield and pure level.

Though can use different known cytolytic methods in the process of the present invention, as enzymolysis or chemical dissolution, the most handy ultrasonic method for breaking.With known method such as centrifugal method the adventitia of cell and inner membrance are separated with other cellular constituents then.

In case cytolemma is separated from the dissolved mixture, just cleans it with extracting solution, tend to salt and washing agent, to obtain to contain the solution that is at least about 50 percent IL-2.In a reasonable embodiment, cytolemma divided for four steps cleaned with salt and washing agent.The most handy washed with saline solution cytolemma of the first step, the sodium chloride solution of the most handy 1M.In second step, clean cell membrane fragments, the most handy centesimal sharp logical (Triton) X-100 solution that goes with washing agent.The 3rd step, with another kind of washed with saline solution cell membrane fragments, the guanidine hydrochloride solution of the most handy 1.75M to 2M.Still a kind of salts solution of usefulness is cleaned in final step, the guanidine hydrochloride solution of the most handy about 4M to 7M.In the 4th step, promptly contain approximately five ten at least percent IL-2 in the washing lotion of final step gained.

The IL-2 washing lotion of final step is further purified with chromatography, and the most handy reversed phase high efficiency can liquid chromatography (LC).What this step obtained is almost to be the active IL-2 of absolutely purified or even character.It is also envisioned that the polyclone of IL-2 or the antibody affinity chromatography of monoclonal antibody, can use as the surrogate of high-effect liquid chromatography (LC).Other chromatography also can be used, as the dyestuff affinity column (as the red agarose (Procion red agarose) of Procion, be recorded in european patent application No. 83103582.9, October 26 nineteen eighty-three, publication number was in 92163 the communique), perhaps dextran polyacrylamide (Sephacryl) S200 post.Of the present invention another preferably among the embodiment, chromatography comprises a plurality of steps, promptly then uses dye affinity chromatography after high-effect liquid chromatography (LC).

According to the present invention, sublimed IL-2 noted earlier can be used for similar purpose as other known immune-regulating factor compounds, as a kind of method as processing immunosuppression condition.It can be used for medicinal acceptable oral, injection or local composition and the mode of using of.Dosage can parallel with the known immunomodulatory compounds that is used for clinical application now with dose rate, and being typically every day is 1～200 * 10 ⁶Unit.These medical compositions of the present invention are comprising said IL-2 and the medicinal acceptable carrier material compatible with it.The solid support material of any routine all can be used.Solid support material can be organic or inorganic, that be suitable for enteron aisle, via skin, or parenteral medication.Suitable carrier comprises water, gelatin, Sudan Gum-arabic, lactose, starch, Magnesium Stearate, talcum, vegetables oil, polyalkylene glycol, particularly polyoxyethylene glycol and Vaseline and analogue.Further, pharmaceutical preparation can comprise other medical active agent.Other additive as seasonings, tranquilizer, sanitas, emulsifying agent, buffer reagent and analogue thereof, can add according to the building-up process of the medicinal compound that can be used to put into practice.

Pharmaceutical preparation can include 1 and be used for oral solid form, photo agent, wafer, pill, pulvis, granule and analogue with any conventionally form; 2 are used for oral liquid form, as solution, and syrup, suspension, vina and analogue; The preparation that 3 non-enteron aisles are used, as thimerosal, suspension or emulsion, and the preparation of 4 topical application, as solution, suspension, ointment, emulsion, colloid, particulate pulvis, smoke substance and analogue.Pharmaceutical preparation can be disinfectant and (or) can contain adjuvant, as contain protective material, stablizer, wetting agent, emulsifying agent, be used to produce different osmotic salt and (or) buffer reagent.

Non-parenteral dosage forms can be the transfusion or injection liquid, this injection liquid can be used for intravenous injection or intramuscular injection.The medicinal activity material that can also comprise other in the preparation.Other additive as protective material, stablizer, emulsifying agent, buffer reagent and analogue, can add according to the receptible practical situation of pharmaceutical compound.

The structure of IL-2 expression vector is given more detailed description with the structure that can express the conversion product of IL-2 at this.

Fig. 1 has drawn the aminoacid sequence of sophisticated IL-2 proteoplast.Proteoplast can come out with its sophisticated formal representation, is ATG as the initiator codon of fruit gene, at N-terminal a methionine(Met) is just arranged so.After expressing, methionine(Met) can or cannot be removed by host cell.

The used expression vector of the present invention induces from pBR322, and this pBR322 contains isolated PL promotor from phage DNA.Select the PL promotor for use, be because it is a very strong promotor, and can be by the control conveniently and effectively of λ CI repressor, can be positioned at the gene on the microbial staining body, suitable carrier or the carrier identical with the carrier that contains the PL promotor can be controlled this promotor easily and effectively.The gene of coding repressor has cIts sudden change, and it makes repressor become responsive to temperature type.One can be used for of the present inventionly, and the carrier that contains cI sudden change is pRK248cIts, and it is a prior art, and Cann (Kahn) people of etc.ing once described it on " Enzymology method " rolled up 268 pages in 1979 the 68th.In the time of 30 ℃, the repressor function is normal, and during from 37～42 ℃, it is with regard to deactivation.Like this, the PL promotor is checked (being closed) in the time of 30 ℃, checks effect disappearance (being unlocked) in the time of 42 ℃.The ability of controlling the PL promotor makes people to make the culture growth and not give expression to gene product in the time of about 30～36 ℃, between the most in good time temperature is converted to 42 ℃ to produce desirable sophisticated human body IL-2 product from about 30 ℃.

Be used for carrier preferably of the present invention and equally also include an EcoRI restriction site (intestinal bacteria restriction enzyme RI point of contact), it is at SD sequence far-end (below 3 ' direction).Following narration is used to explain a kind of pRC23 of carrier preferably of preparation, and after this, the IL-2 gene is introduced in its top.Yet, also available other carrier.

According to Fig. 2,3 described processes, 20 microorganisms of pBR322 are used EcoRI restriction endonuclease enzymolysis, be used in then in two reactions, 1. with the SL nuclease handle with remove 5 ' dangle, 2. with big fragment (Klenow fragment) processing of archaeal dna polymerase I to fill up terminal.Two reactions all are to stop succeeded by ethanol sedimentation with phenol extraction.The DNA that obtains from each reaction is connected on a kind of synthetic Bgl II restriction endonuclease joint, with Bgl II and pst I restriction endonuclease enzymolysis, by the gel electrophoresis of 1% agarose.3600 base pairs in two reactions reclaim from gel electrophoresis with fragments 760 base pairs.In order to make pBC2, the fragment of 3600 base pairs that obtain from the Klenow reaction is connected on the fragment of 760 base pairs that obtain from the SL reaction.The mixture that connects is transferred on the intestinal bacteria RRL, and transformation substance is selected with every milliliter of substratum that contains 50 microgram penbritins.The conversion thing that contains desired plasmid structure promptly contains a BgL II point of contact on the plasmid, its position is to identify with the point of contact analytical method of isolating plasmid DNA near the EcoRL point of contact.The manufacturing of pRC23 is with the synthetic oligonucleotide that contains the ribosome bind site (RBS) that " agree with sudden change " or computer produce (people (Soherer) such as Xie Le, nucleic acids research, volume was 3895 pages in 1980 the 8th) be connected on 250 the base pair BgL II-Hae III fragment that contains λ PL promotor, and a product that connects is inserted on the pRC2.

In order to isolate the dna fragmentation of 250 base pairs that contain λ PL promotor, 1 microgram contains the BgL II-Hpa I dna fragmentation (from the #35260 base pair of lambda bacteriophage dna sequence to 35710 base pairs) of 450 base pairs, with Hae III phase enzymolysis, its product is separated with the gel electrophoresis of 5% polyacrylamide preparation.The fragment that contains 250 base pair BgL II-Hae III of about 200 nanogram(ng)s, 60 pmols with each synthetic oligonucleotide shown in Figure 3 are connected, RBS sequence that these synthetic oligonucleotides contain most " agreeing sudden change ", this is by being drawn as described Computer Analysis of people such as Xie Le.The molecule that connects comes enzymolysis (to eliminate oligomer) with BgL II and EcoRL restriction endonuclease, and uses the gel electrophoresis purifying.After this, the product that connects just is inserted among the pRC2, and this pRC2 be by the BgL II and by the EcoRL enzymolysis.Intestinal bacteria RRL(pRK248cIts) conversion of strain is to finish with the method for standard, and the material of conversion is to select in the time of 30 ℃ with the substratum that contains penbritin (every milliliter 50 microgram).Obtained 50 conversion products, from wherein 8, isolated DNA, and with Hinc II restriction endonuclease enzymolysis analysis they.6 among these 8 have demonstrated the pattern that desired restriction enzyme is cut, and have analyzed one of them with mark Sa Mu-gilbert (Maxam-Gilbert) nucleotide sequence analysis, have affirmed desired structure (being called pRC23).

Make a plasmid expression vector that is comprising the DNA of encoding mature human body IL-2.

1. the separation of the mRNA of coding human body IL-2

MRNA separates from H33HJ-JAJ cell (be stored in the U.S. culture model August 26 nineteen eighty-two and collect the center, store number be CRL-8163), and these cells are the clones of the Jurkat clone FHCRC after inducing with PHA and PMA.In the RPMI1640 tissue culture medium (TCM), grow 12000 milliliters H33HJ-JAI clone cell culture (10 ⁶Cells/ml), joining the fetal bovine serum of supporting adding 10% in the base simultaneously, the penicillin of 50 units per ml, the Streptomycin sulphate of 50 mcg/ml, left-handed-glutamine that the gentamicin of 50 mcg/ml and 300 mcg/ml are fresh.Collect these cells with centrifugal method, and they are suspended in 6 again go up in the nutrient solution of stating, serum not wherein, but and then add 1% the PHA and the PMA of 10 millimicro grams per milliliters.These cells have been divided into 6 liters of portions, and the glass of putting into the bacterium of having gone out shakes bottle, then, they are put into go (per minute 10 changes 37 ℃) in the mill that rolls again.

After 8 hours, use the centrifuging collecting cell, extract mRNA with the phenol chloroform extraction method of standard.After the phenol chloroform extraction, with ethanol sedimentation it, it is coccoid with high speed centrifugation RNA to be become, and makes it to be suspended in the salts solution of 0.5M again, the mRNA of the polymerization A tail end in whole RNA colony is with the method collection that makes this material by low (dT) cellulose column.The mRNA resuspending of ethanol sedimentation is in water, and its concentration is 500 mcg/ml.The RNA trace of 30 nanogram(ng)s is injected in the ovocyte of African toad (XenoPus).In aseptic Bath liquid, after the hatching 24 hours, 4 ovum are transferred in the 1.5 milliliters aseptic Eppenderf centrifuge tube, again the fresh sterile Bath solution of 500～1500 microlitres in addition.After 48 hours, the substratum that is suitable for the ovum condition of 200 microlitres has just been gathered in the crops, and with the ctll cell of standard ³H-Tdr mixes test (people (GilliS) such as lucky think of.2027 pages of Journal of Immunology 1978 120 volumes) measure the activity of IL-2.MRNA preparation with ovocyte translation is the remarkable activity that produces IL-2, and these mRNA preparations are mixed, and marks their size with the sucrose density gradient centrifugation technology of standard, and with ethanol sedimentation to set up the cDNA library.

The synthesizing 2cDNA(complementary DNA (cDNA))

Synthetic double-stranded complementary DNA (ds cDNA), usefulness be that the mRNA(that 3.5 micrograms have been purified approximately is the 10S of saccharose gradient), adopt following method (Ge Bule and Huffman (Gubler and Heffman).Gene.25，263-269〔1983〕）。

(a) article one cDNA chain is synthetic

MRNA be suspended in 17.5 milliliters contain 50mM Tutofusin tris-hydrochloric acid (Tris-HCl), pH8.3, the magnesium chloride of 10mM, 10mM dithiothreitol (DTT), 4mM trisodium phosphate, 1.25mM dATP, 1.25mM deoxidation vernine triphosphoric acid, 1.25mM triphosphoric acid deoxythymidine, 0.5mM triphosphoric acid deoxycytidine, the few deoxythymidine 12～18 of 100 mcg/ml, and 10 Curie ³²P-dCTP(AMoShaMu (Amersham) 3000 Curie/mmole).Hatched 5 minutes at 43 ℃, add 3000 AMV of unit and reverse transcriptase/milliliter (life science company), this mixture was hatched 30 minutes at 43 ℃.This is reflected at when adding ethylenediamine tetraacetic acid (EDTA) (EDTA) to 20mM and stops, and extracts with phenol-cresols, concentrates it with ethanol precipitation again.Article one, the result of synthetic chain measures its radioactivity with insoluble Tricholroacetic Acid, and calculating output is 58.6 nanogram(ng)s (1.7%).

(b) the second chain is synthetic

The cDNA-mRNA hybrid is re-suspended in the water of 5.8 microlitres, the second chain synthetic mixture that in this solution, adds 7.7 microlitres, this solution contains Tutofusin tris-hydrochloric acid of 20mM, pH7.5, the magnesium chloride of 5mM, the sulfuric acid amine of 10mM, the Repone K of 100mM, 0.15mM β-shop enzyme 1, the bovine serum albumin(BSA) of 50 mcg/ml, the deoxy-ribonucleoside triphosphate of 40mM (dNTps), 8.5 the H RNA enzyme that obtains in the intestinal bacteria of units per ml (Bei Xisida (Bethesda) research department), the archaeal dna polymerase 1(Bai Linge-mannheim company (Boehringer Mannheim) of 230 units per ml), the dna ligase that from intestinal bacteria, obtains of 10 units per ml (New England biological products company (NewEngland Biolabs)).This mixture was hatched 60 minutes under 12 ℃ of conditions, hatched 60 minutes under 22 ℃ of conditions again.Stop when being reflected at adding ethylenediamine tetraacetic acid (EDTA) (EDTA), and extract with phenol-cresols to 20mM.CDNA separates with the free nucleosides mutually by the very thin dextran G-50 chromatography column of particle of the trolamine that contains 10mM (TEAB) damping fluid.The output of this reaction is the double-stranded complementary DNA (cDNA) (ds cDNA) (91%) of 107 nanogram(ng)s.

(c) renaturation and conversion

Method with standard adds the double-stranded complementary DNA (cDNA) acquisition tail end that deoxidation vernine triphosphoric acid (dGTP) makes 64 nanogram(ng)s.PBR322DNA with ECORV(New England biological products company) the phase enzyme connects, and makes its tail endization with triphosphoric acid deoxycytidine (dCTp), prepares carrier with such method.The CDNA of the tail endization of the pBR322DNA of the tail endization of 100 nanogram(ng)s and 1.25 nanogram(ng)s is inserted body (ratio is 80: 1) be placed on 250 microlitre 0.01M Tutofusin triss (Tris), pH7.5,1mM ethylenediamine tetraacetic acid (EDTA) (EDTA), 0.15M in the solution of sodium-chlor, under 58 ℃ of conditions, cultivate and made their renaturation in 90 minutes, and be transformed in the activated intestinal bacteria RRI cell.The cell that has transformed is placed on the LB plate that contains 100 mcg/ml penbritins (Bristol laboratory (Bristol labs)), hatches 12 hours under 37 ℃ of conditions.Whole 3200 bacterium colonies are used for producing IL-2 complementary DNA (cDNA) library.

3. for obtaining IL-2 similar gene separating complementary DNA (cDNA) library

Total length for the copy cDNA that measures the IL-2 gene, with a synthetic deoxy-oligonucleotide probe, this probe is the 45th～65 Nucleotide of corresponding human body IL-2 complementary DNA sequence, as Ta Niguqi (Taniguchi) and (nature 302 305～310 1983) that other people deliver.This probe (ACAATGTAC AGGATGGAACTC) is with solid phase phosphodiester method synthetic, and purifies it with high performance liquid chromatography (HPLC), uses again ³²The P Triphosaden ( ³²P-ATP) (ICN medicament .7000 Curie/mmole) and polynueleotide kinase mark it (New England biological products company).Represent the bacterium colony in complementary DNA (cDNA) library, transfer to (Enzymology method 68379 (1979)) in the nitrocotton filter with Ge Lusitan (Grustein) and Hogness (Hogness) method.Through after 30 ℃ 16 hours hybridization, under 45 ℃ of conditions, washed filter 45 minutes, drying, radioautograph with 4XSSC.At this moment found a positive bacterium colony, and found that it contains whole IL-2 encoding sequence.This bacterium colony is denoted as pIL-2B, and prepares a kind of thymus nucleic acid with it, this thymus nucleic acid be used for nucleotide sequence analysis and be used in expression in escherichia coli.Nucleotide sequence analysis shows, it equates with people's reported sequence such as Ta Niguqi, just have 2 difference: pIL-2B to contain one and insert body, it originates in the 17 Nucleotide of the sequence that Ta Niguqi names: and the replacement of a G is arranged on the 503rd.

4. make the e. coli plasmid vector of the thymus nucleic acid that contains the Ser-IL-2 that encodes.

A colibacillary expression vector that is used for IL-2, be the (see figure 4) with 3 sections manufacturings: (1) has the carrier pRC23 of λ p promotor, (2) synthetic connectors and (3) Hgi AI-Aha III fragments of separating from the IL-2 complementary DNA (cDNA).The carrier of preparation thymus nucleic acid is the pRC23 thymus nucleic acid of 50 nanogram(ng)s and EcoRI and EcoRV enzymolysis mutually, then with phenol extraction with use ethanol sedimentation.Synthetic connector is involutory two complementary synthetic deoxy-oligonucleotide sequence A and B and obtains;

（A）5′AATTCAATTATGAGTGCA3′

（B）3′GTTAATACTC5′

This double-stranded connector can be in its 5 ' terminal and EcoRI site and involutory with the HgiAI site at its 3 ' end.The insertion body of complementary DNA (cDNA) is with pIL2-2B thymus nucleic acid and BamRI restriction enzyme (Bei Xisida (Bethesda) research department) enzymolysis and obtaining mutually, thereby inserts body by producing one the 1 right IL-2 of kilobase in this bacterium colony.BamHI fragment gel-purified, so with HgiAI(New England biological products company) and Aha III (New England biological products company) enzymolysis mutually, then use phenol extraction, use ethanol sedimentation.HgiAI cuts between the L-Ala of the section start of the IL-2 of encoding mature sequence and proline(Pro).The bacterium colony scheme of expressing is the EcoRI site with contiguous PL promotor in the pRC23 carrier.Synthetic connector molecule can add between this EcoRI site of the HgiAI site that begins to locate of complementary DNA (cDNA) IL-2 sequence and carrier.This connector is to be designated as to produce the methionine(Met) codon begin to translate.The codon of Serine and L-Ala, i.e. first amino acid of sophisticated IL-2, at connector and complementary DNA (cDNA) when HgiAI site enzyme connects and produce.It is that the blunt end enzyme in the EcoRV site by Aha III site and carrier thymus nucleic acid connects and finishes that complementary DNA (cDNA) is added on the pRC23.These three fragments are placed on one 10 microlitre volume, contain carrier, synthetic connector, each 0.05pM of complementary DNA (cDNA) insertion body thymus nucleic acid, the pH value is 7.6 ethylenediamine tetraacetic acid (EDTA), the magnesium chloride of 10mM, 0.5mM Triphosaden and the liquid of the beta-mercaptoethanol of 15mM in, the enzyme of finishing them connects.The mixture that enzyme connects is cooled to 4 ℃ with 65 ℃ of heating 5 minutes, adds the T of 200 units then ₄Deoxyribonucleic acid ligase (New England biological products company) was hatched 16 hours under 4 ℃ of conditions then.When being heated to 65 ℃ 5 minutes the time, T ₄Deoxyribonucleic acid ligase loses activity.Then, the DNA sequence that connects is with EcoRV restriction endonuclease enzymolysis, removes the thymus nucleic acid carrier that those can not enzymolysis.Then, with the RRI strain (pRK248cIts) of this mixture transformed into escherichia coli, these cells are placed on make its growth 15 hours under 30 ℃ of conditions again.

According to the described method in front colony lift in the nitrocotton filter, the baking, use again ³²The radioactivity probe hydridization of the synthetic connector A of P mark it.A male is named and is pRC23/IL-2, and the bacterium colony of #4-1 is selected does analysis.This bacterium colony is found when inducing λ PL promotor to be used to express when at 42 ℃, has the ability of synthesizing greater than the IL-2 of 200,000 units per ml.Its IL-2 activity is measured with bioassay method, and used ctll cell is a kind of indicating clone for IL-2 (described at Journal of Immunology 120 2027 (1978) as lucky this people such as (Gillis)).

5. be used to produce the manufacturing (first amino acid of its amino terminal is L-Ala) of the coli expression carrier of sophisticated IL-2

For at the sophisticated IL-2 of expression in escherichia coli, carry out following step (as shown in Figure 5).A point of contact of HgiAI restriction endonuclease is selected in Serine-L-Ala junction, promptly is selected in that fracture place for the signal peptide that removes IL-2 people (Robbetal) pNAS 80 5990-5994 (1983) such as () spreading out.Then after the HgiAI enzymolysis, use T ₄Deoxyribonucleic acid polymerase is handled, and makes its terminal become blunt end.The blunt end of the molecule that is produced just is connected with the Bgl II-Hae II fragment of 108 base pairs, this fragment is separated from the lambda particles phage thymus nucleic acid, and with Bgl II and Xba I restriction endonuclease enzymolysis, be inserted into carrier pRC23/IL-2, between the Bgl II site of #4-1 and the Xba I site.The step that this clone's intermediate forms comprises guaranteeing T ₄The processing of archaeal dna polymerase will be according to desired such generation, this can from the Hae III terminal with the leveling of HgiA I end combine and produce the Stu I site that makes new advances and obtain sure.With Stu I enzymolysis once more, making initiating terminal easily is that the blunt end of CCT proline(Pro) codon is in this site regeneration.The plasmid structure of this intermediate is designated as pRC201/IL-2.Designed 2 synthetic deoxy-oligonucleotides, they are to mix mutually with an ATG-translation initiation codon, recover the L-Ala codon and make an EcoR I end.These oligomer are connected on the EcoR I end of expression vector pRC23, with Pst I enzymolysis, and the fragment gel-purified of resulting 1025 base pairs.The fragment of 1025 base pairs (from the Pst I to blunt end) is inserted between the emerging Stu I site and Pst I site of PRC201/IL-2.The conversion product that contains needed structure is to confirm with the inscribe analysis of isolating plasmid deoxyribonucleic acid.The plasmid structure of being confirmed is designated as pRC233/IL-2(and sees Fig. 6).When transforming with a carrier that contains the gene that relates to IL-2 of the present invention, reasonable host's organism comprises bacterium, as each strain of intestinal bacteria, and Bacillaceae, as subtilis and similarly.Yeast is one group and better is used for microorganism transformed.One good especially, is e. coli k-12 strain 294 as the microorganism of acceptor in the conversion process, is described among the English Patent publication No. 2055382A, is stored in U.S. culture sample collection center, and it stores number is on October 28th, 31446,1978.But other known intestinal bacteria strains are arranged also, as strain RRI, U.S.'s culture sample collection central storage numbers 31343, perhaps other microorganism, wherein major part is stored in the microorganism storage center of admitting and can obtains from this, this class center such as U.S. culture sample collection center etc., these microorganisms can be used as host living beings.Realizing when of the present invention that the culture that the intestinal bacteria that transformed are spent the night is placed in 30 ℃ the LB liquid nutrient medium their are grown.One liter of culture that spends the night preferably is diluted in 10 liters the solution of a spot of M-9 substratum that contains casamino acids.At logarithmic phase, culture is moved under 42 ℃ of conditions to induce generation IL-2 by 30 ℃.Then under 42 ℃ of conditions, cultivated 2-3 hour, collect bacterium with centrifuging.Whole fermentation and process thereof are that the recombinant DNA guide of abideing by National Institutes of Health carries out.The detailed step of purifying is described in following example.

Example 1

1 gram is produced its NH of IL-2( ₂End has Serine) the intestinal bacteria transformant be suspended in and contain the 30mM tris-HCI buffer, pH8.0, the ethylenediamine tetraacetic acid (EDTA) of 5mM in the solution of 1mM phenyl methyl sulfonyl fluorochemical, and makes its dissolving with supersound process.Dissolved mixture centrifugal 15 minutes with 14000g.Film component in molten born of the same parents' thing after centrifugal taken out and be separated with other compositions in molten born of the same parents' thing.Film component extracts with flushing of the step of four shown in the table 1 or IL-2, and the washing lotion in each step is used the amount of 5 milliliters in every gram cell at least.Most IL-2 actives is in the end to extract in the flushing that goes on foot, and sees table 1 for details.Contain most IL-2 active substance in the liquid of final step flushing gained, and detect its purity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-pAGH) and be at least 50%, then this liquid is passed through RPHPLC (reversed-phase high-performance liquid chromatography) (Rp-CS).Be almost 100% IL-2(at NH with regard to being recovered to 40～60% purity like this ₂End has Serine).The activity of measuring IL-2 is at least 1 * 10 ⁶Unit/milligram.

Example 2

This example is to be to tend to combine with the film component of the e. coli host cell that has transformed in order to proof IL-2.

In order to prove this point, will produce its NH of IL-2( ₂End is for Serine) intestinal bacteria transformant 1 gram be suspended in that to contain 10 ml concns be 20% sucrose, in the solution of 30mM tris-HCI buffer pH8.0, carry out molten born of the same parents to isolate protein with N,O-Diacetylmuramidase-ethylenediamine tetraacetic acid (EDTA).These protein concentrate in the gap of periplasm, and remaining molten born of the same parents' mixture is again through supersound process.Cytolemma part (inner membrance and adventitia) is separated with sucrose gradient centrifugation and other cellular constituents again, and inner membrance and adventitia also are separated simultaneously.Table 2 demonstrates most IL-2 active substance and all concentrates in the cell inner membrance.

Example 3

Intestinal bacteria transformant 1 gram that produces ripe IL-2 is suspended in contains 30mM tris-HCI buffer pH8.0, the 5mM ethylenediamine tetraacetic acid (EDTA) in the solution of 1mM phenyl methyl sulfonyl fluorochemical, and makes its molten born of the same parents with supersound process.To dissolved mixture centrifugal 15 minutes with 14000g.The membrane portions of the molten born of the same parents' thing of centrifugal and other molten born of the same parents' compositions are separated.Film component extracts with flushing of the step of four shown in the table 3 or IL-2 extraction step.The amount that each goes on foot used washing lotion is at least 5 milliliters in every gram cell.Most IL-2 active substance is to extract in the final step shown in the table 3.Contain most IL-2 active substance in the washing lotion that final step obtains, measure its purity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and be at least 50%, this liquid is passed through RPHPLC (reversed-phase high-performance liquid chromatography) (Rp-C again ₈).Being recovered to about 60% purity is 100% ripe IL-2 substantially, measures the IL-2 activity and is approximately 4 * 10 ⁸Unit/milligram.

Example 4

Following this example has provided with adding the method for red this step of agarose chromatography of Procion (procion) with purification ripe IL-2 from the intestinal bacteria paste that has transformed outside the high performance liquid chromatography.

Cytolemma extracts

Freezing Bacillus coli cells (containing the plasmid as human body IL-2) is melted, restrain buffer A (the 0.03M tris-HCI buffer that this material is added to 5 milliliters to 1, pH8.0, the 0.005M ethylenediamine tetraacetic acid (EDTA)) in.After mixing 10 minutes, it is centrifugal to put into Sorval ss-34 rotary head, thereby these cellular segregation is come out (per minute 10.000 left the heart 10 minutes).Supernatant liquor is removed, again cell is suspended in 5 milliliters the buffer A again.The cell of these suspensions was with a gloomy supersonic cell pulverizer 350(6 of Blanc * 30 seconds) fragmentation.Broken cell carries out centrifugal (per minute 10.000 left the heart 10 minutes), and the supernatant liquor that contains soluble protein is thrown away.Wash throw outs once with 5 milliliters buffer A, and centrifugal (per minute 10.000 left the heart 10 minutes).The throw out that contains the film fragment is suspended in buffer B (1M sodium-chlor, 0.03M Tutofusin tris-hydrochloric acid, FH8.0,0.005M ethylenediamine tetraacetic acid (EDTA)), put into a Hui Dun-Du Ensi (Wheaton Dounce) tissue homogenizer, mix after 10 minutes, (per minute 15000 changes with Sorval ss-34 rotary head, 10 minutes) centrifugal, the film chip separation is come out.Resistates is put into 5 milliliters damping fluid C(1% and is removed sharp logical X-100,0.03M Tutofusin tris-hydrochloric acid, pH8.0) in, carry out homogenate, mix and centrifugal (per minute 15000 changes 10 minutes).Resistates resuspending after centrifugal carries out homogenate in the guanidine hydrochloride solution of 5 milliliters of 1.75M, mix and centrifugal (per minute 15000 changes 10 minutes).Resistates washes once with 5 milliliters of buffer A and is centrifugal.Extract film fragment (resistates) with 5 milliliters 7M Guanidinium hydrochlorides.After centrifugal, the extract that contains IL-2 is preserved, and the 7M Guanidinium hydrochloride of resistates with 5 milliliters extracted once more.Current extract is same to be preserved.

The red agarose chromatography of Procion:

A post is set at ambient temperature, walks before the post for the first time, wash this post with the 7M guanidine hydrochloride solution of twice column volume.When being 4 ℃, temperature make this post reach balance (level pad is 0.01M Tutofusin tris-hydrochloric acid, pH7.9,0.035M sodium-chlor) with level pad.At least will be with the red agarose of 1 milliliter Procion to desired per 100 microgram IL-2.Flow velocity is 2 column volumes per hour.The 7M Guanidinium hydrochloride extracting solution that contains IL-2 dilutes 40 times with level pad, and removes throw out with centrifuging.Then supernatant liquor is packed in the post.Level pad with two bed volumes washes this post, and behind the elution buffer wash-out with 1.5～2 column volumes, the IL-2 of wash-out reaches a peak value (elution buffer is 0.01M Tutofusin tris-hydrochloric acid, pH7.9,1.035M sodium-chlor).After IL-2 is extracted, with the flushing of the guanidine hydrochloride solution of 6M to remove irrelevant material.

At RP-P(C-18) chromatography on (reversed phase high performance liquid chromatography) post:

The pH value of the red elutriant of Procion adjusts to 7 with the acetic acid that slowly adds 1.0M.In the front and back that the pH value is adjusted, get 0.1 milliliter at every turn and be used for measuring.Neutral elutriant (60 milliliters) pumps into the speed of 1 milliliter of per minute in one 0.31 * 25 centimetre the RP-18 post, and uses the level pad that is mixed and made into by 5% high performance liquid chromatography damping fluid II and 95% high performance liquid chromatography damping fluid I to make this column equilibration.The liquid that flows through pillar is collected to measure protein content and IL-2 biological activity.At ambient temperature, according to this post of the described gradient elution scheme of table 4 wash-out.Effluent is located monitoring at 220 millimicrons.Each component that wash-out is collected is 1.5 milliliters, analyzes its protein content and IL-2 activity then.The active peak-peak of IL-2 is approximately appearance in 74% o'clock in high performance liquid chromatography damping fluid gradient.Each component is kept at 4～8 ℃, and according to mixing than living.

The institute of chromatography all at room temperature finishes in steps.

Table 1 extracts IL-2 from the Bacillus coli cells film

The total IL-2 specific activity of gross protein is lived

Extracting solution (milligram) (unit) (unit/milligram)

1M sodium-chlor washing lotion 0.4mg 0.03 * 10 ⁶0.1 * 10 ⁶

（IMNacl）

1% removes sharp logical washing lotion 2mg 0.75 * 10 ⁶0.4 * 10 ⁶

（1%Triton）

1.75M Guanidinium hydrochloride washing lotion 1mg 1.5 * 10 ⁶1.5 * 10 ⁶

（1.75Mguanidine-HCl）

7M Guanidinium hydrochloride washing lotion 4.7mg 250 * 10 ⁶53 * 10 ⁶

（7Mguanidine-HOL） （99%）

252.3×10 ⁶

The distribution of table 2 IL-2 in Bacillus coli cells

The active IL-2 rate of recovery of the total IL-2 of gross protein

(milligram) (unit) percentage ratio (%) partly

Soluble part 37mg 2 * 10 ⁶0.3

Periplasm part 2mg 10 * 10 ⁶1.5

Adventitia 6mg 8 * 10 ⁶1.2

Inner membrance 15mg 6.6 * 10 ⁶97

Table 3 extracts sophisticated IL-2 from the Bacillus coli cells film

The total IL-2 specific activity of gross protein is lived

Extracting solution (milligram) (unit) unit/milligram

1M sodium-chlor 0.5 0.3 * 10 ⁶0.6 * 10 ⁶

（IMNacl）

1% goes sharp logical 3.3 5.5 * 10 ⁶1.7 * 10 ⁶

（1%Triton）

1.75M Guanidinium hydrochloride 2.5 10.0 * 10 ⁶4.0 * 10 ⁶

7 mole hydrochloride guanidines 10.1 779 * 10 ⁶77.1 * 10 ⁶

98%

794.8×10 ⁶

Table 4 carries out the condition of chromatography to a red eluate of typical Procion on the RP-18 post

Add sample size: 60 milliliters

The area of post: 0.41 * 25 centimetre of flow velocity: 1 milliliter/per minute

HpLC damping fluid I: 0.01M phosphoric acid, 0.05M lithium chloride

HPLC damping fluid II: 0.01M phosphoric acid, 0.05M lithium chloride

80% acetonitrile

The %HPLC damping fluid II time length (minute)

Balance 5 20

Dress sample-60

Wash 5 15

The first step, gradient 5～35 15

Second step, gradient 35～85 50

The 3rd step, constant density 85 5

The 4th step, constant density 55

Claims (11)

1, produce the ripe recombinant human interleukin--2's (IL-2) of basic homogeneous method from the cracking culture that transforms microorganism, this microorganism contains encoding mature human IL-2's dna sequence dna, it is characterized in that:

A. isolated cell film component from the cracking culture;

B. handle cell membrane component with the extracting solution that contains the 4M-7M Guanidinium hydrochloride of having an appointment, produce the washing fluid that contains IL-2;

C. pass through the washing fluid of affinity chromatography or highly pressurised liquid chromatography purification step (b), to produce recombinant human il-2 pure basically or homogeneous.

2, method according to claim 1, the culture ultrasonic method fragmentation of the microorganism that has wherein transformed.

3, method according to claim 1, cell membrane component wherein are to separate from the molten born of the same parents' culture of cell with centrifuging.

4, method according to claim 1, the cell membrane component of wherein separating washed with salt and washing agent before with the extracting solution flushing.

5, method according to claim 4, the salt wherein and the flushing of washing agent be divided in order three the step finish.

6, method according to claim 5, what wherein the first step of cell membrane composition flushing was used is sodium chloride solution, the second step usefulness be washing agent, the 3rd step usefulness be the guanidine hydrochloride solution of volumetric molar concentration 1.75～2.0.

7, method according to claim 1, wherein extracting solution is actual is the Guanidinium hydrochloride of 7M.

8, method according to claim 1, wherein the chromatography purification is to use the high-performance liquid chromatography (LC).

9, method according to claim 1, wherein the chromatography purification is to use affinity chromatography.

10, method according to claim 9, wherein chromatography purification is to use dye affinity chromatography.

11, method according to claim 1, wherein the chromatography purification is the rapid process of multistep of using high-effect liquid chromatography (LC) and affinity chromatography.

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