CN102234649A

CN102234649A - Efficient soluble expression method of active recombinant ribonuclease inhibitor

Info

Publication number: CN102234649A
Application number: CN201010162054XA
Authority: CN
Inventors: 王楠; 孙婧慧; 葛莹
Original assignee: WEIZHI LANSI MEDICAL TECHNOLOGY (BEIJING) CO LTD
Current assignee: WEIZHI LANSI MEDICAL TECHNOLOGY (BEIJING) CO LTD
Priority date: 2010-05-05
Filing date: 2010-05-05
Publication date: 2011-11-09

Abstract

The invention provides an efficient soluble expression method of an active recombinant ribonuclease inhibitor, belonging to the technical field of biological engineering. The invention discloses an effective method for obtaining an active recombinant ribonuclease inhibitor, which is simple and easy to implement and has low cost. A recombinant plasmid vector of a coding gene for expressing a mouse ribonuclease inhibitor (mRI) is converted into engineering bacteria with protease gene defect phenotypes, and soluble expression of higher-level active products can be obtained by appropriate induction.

Description

A kind of highly-soluble expression method of active RNAsin

Technical field:

The invention belongs to bioengineering field, particularly the prokaryotic expression of RNAsin.

Background technology:

Ribonuclease inhibitor (RNase Inhibitor RI) is the protein molecular that a kind of molecular weight is about 50KDa, can in conjunction with and suppress the multiple member of mammals pancreatic ribonuclease (RNase) superfamily.

RI is a kind of plasmosin, extensively exists in many mammalian tissues.It forms 1: 1 complex body by combining closely with multiple rnase, suppresses the activity of these rnases.Although some members' of RNase superfamily homology has very big-difference, the mixture that RI and they form, its dissociation constant is at level (the Lee FS of 0.5-200fM, Shapiro R, Vallee BL.Tight-binding inhibition ofangiogenin and ribonuclease A by placental ribonuclease inhibitor.Biochemistry, 1989,28:225-30; Shapiro R, Vallee BL.Interaction of human placental ribonucleasewith placental ribonuclease inhibitor.Biochemistry.1991; 30:2246-2255).The high-affinity of RI and these parts RNase, very rare at occurring in nature.

In some molecular biology researches, RI can be used for preventing that rnase from polluting the problem that may cause, as testing in the mRNA reverse transcription, acellular external protein translation experiment and other relate in the work of RNA research.

Natural ribonuclease inhibitor can (the Blackburn P.Ribonucleaseinhibitor from human placenta:rapid purification and assay.J Biol Chem.1979 of separation and purification from people's placenta; 254:12484-7), also can from some mammalian tissues, extract purifying (Gribnau AA, Schoenmakers JG, van Kraaikamp M, Bloemendal H.High purification of the RNaseinhibitor from rat liver by affinity chromatography.Biochem Biophys Res Commun.1970; 38:1064-8; Garcia MA, Klebe RJ.Affinity chromatography of RNaseInhibitor.Mol Biol Rep.1997; 24:231-3; Chatterjee J, Maiti TK, Dasgupta S.Isolationand partial characterization of ribonuclease inhibitor from goat liver.Protein PeptLett.2006; 13:779-83).RI research (Burton LE, FucciNP.Ribonuclease inhibitors from the livers of five mammalian species.Int J PeptProtein Res.1982 to ox, pig, sheep, mouse and rat source; Show that 19:372-9) they have character and the amino acid close with the RI in people's placenta source and form.

Along with the development of genetic engineering technique,, also success (United States Patent (USP) 5,552,302 have been obtained intestinal bacteria and other expressive host by dna recombinant expression people, rat and pig RI; United States Patent (USP) 5,965,399; United States Patent (USP) 5,932,440; Klink TA, Vicentini AM, Hofsteenge J, Raines RT.High-levelsoluble production and characterization of porcine ribonuclease inhibitor.ProteinExpr Purif.2001; 22:174-9; Park JH, Hwang IS, Kim KI, Lee JM, Park YM, Park CH, Chung IS.Functional expression of recombinant human ribonuclease/angiogenininhibitor in stably transformed Drosophila melanogaster S2 cells.Cytotechnology.2008; 57:93-9).

With intestinal bacteria is host's protokaryon recombinant expression method, has outstanding advantages such as easy and simple to handle, with low cost.Yet the expression of specific gene depends on all multifactor, and genetic expression meeting sometimes causes host cell death.Also can occur inclusion body during escherichia coli expression and express, produce the inactive protein product, need external renaturation.People, rat and pig RI have close character and higher dna homolog, and its prokaryotic expression mainly faces the problem that the non-activity inclusion body is expressed.Clone's people RI gene expression product exists with the inclusion body form of non-activity, need external folding renaturation, and its renaturation yield is very low; At people RI gene fusion pilot signal dna encoding peptide, also can't obtain active result; And some expression strategy can directly cause the death (United States Patent (USP) 5,552,302) of host bacterium.Cloned rat RI gene, need with other gene order amalgamation and expressions of small pieces, could obtain expression product, the activated protein of a small amount of solubility and the inclusion body of non-activity also are stored in the host bacterium; Clone's pig RI Prokaryotic Expression can cause that host's bacteria growing suppresses or death (United States Patent (USP) 5,965,399).Generally speaking, the output that is obtained active result by the RI gene prokaryotic is very low, general level (Klink TA at 0.25mg/L bacterium liquid, Vicentini AM, Hofsteenge J, Raines RT.High-levelsoluble production and characterization of porcine ribonuclease inhibitor.ProteinExpr Purif.2001; 22:174-9).Output for the active result that improves the RI gene prokaryotic, Klink etc. adopt the expression system of Trp promotor in the document, the highly-soluble that obtains pig RI gene is expressed, output is in the level of 15mg/L bacterium liquid or 1mg/g wet thallus during high density fermentation, but need to change nutrient solution and induce trivial operations.

The RI protein molecular contains the free sulfhydryl groups about 30, and sulfhydryl oxidase can cause the loss of activity of RI, therefore needs to preserve in the presence of certain density reductive agent such as DTT and use.The sulfydryl of mouse RI is arranged with the protein molecular of other kind different; U.S. New England Biolabs (NEB) company sells the explanation indication of recombined small-mouse RI (M0314), and mouse RI has tangible anti-oxidant deactivation, can keep active when lower concentration reductive agent very.Although existing commercial recombined small-mouse RI provides, and the complete gene order of encoding murine RI is reported (NCBI sequence number: AF071546; But it is in the method for effective recombinant expressed active result of intestinal bacteria or other expressive hosts, does not still have report NM_145135).

Summary of the invention:

The present invention relates to a kind of RNAsin (RI).The invention provides the method that a kind of simple and easy to do and low-cost high-efficiency obtains active RNAsin.The present invention finds, will contain the recombinant plasmid vector of the encoding gene of mouse ribonuclease inhibitor (mRI), transforms the engineering bacteria with proteinase gene defective, through suitably inducing, can obtain the solubility expression of higher level active result.

Implement the present invention, need at first obtain the full length cDNA clone of encoding murine RI aminoacid sequence.According to known complete gene order (NCBI sequence number: AF071546; NM_145135) or the corresponding polypeptide sequence, can directly obtain full length cDNA clone with reverse transcription PCR.Full length cDNA clone also can pass through the method for cDNA library screening, or the method for synthetic obtains.The gene order of synthetic can be identical with natural cDNA sequence, also can be according to mouse RI aminoacid sequence, with the synthetic different degenerate code of known method of design.

CDNA clone with encoding murine RI is connected in the expression vector, is the biology field known method.The prokaryotic expression carrier of extensive stockization can provide selection.As pQE30 and pQE80L (Qiagen), can generate the target protein that His-6 (six Histidines) label polypeptide merges, this fusion rotein is kept Yeast Nucleic Acid enzyme inhibition activity completely, and can use the specific metal affinity chromatography method of His-6 single step purification.The fusion expression vector of other type, as contain the expression vector of coding TrxA (Trx), can generate the target protein that TrxA merges.The activated protein of solubility and the inclusion body of non-activity also are stored in the host bacterium.Usually, induce for a long time at a lower temperature, the expression amount of soluble proteins can obtain raising.Be that through conventional expression condition optimization, after recombined small-mouse RI (rmRI) solubility expression product in intestinal bacteria was purified, output was apparently higher than the report of general document for unexpected.The present invention is also unexpected to be found, when different host bacterium were expressed, except that the target protein output of solubility expression to some extent the difference, the activity of its product also had notable difference.At BL21 and the deutero-engineering bacteria such as BL21 (DE3) pLysS etc. of proteinase gene defective, mouse RI can obtain highly-soluble and express, and its RNaseA suppresses the quite active of active and people RI; When engineering bacterium expression such as TG1 or Top10, the RNaseA of purified product suppresses active obviously to be reduced.In small-scale (200-400ml shake-flask culture) abduction delivering of laboratory, the active rmRI output of purifying can be up to 4-8mg/L, or 1mg/g wet thallus, express (Klink TA with the high dissolubility of reported literature, Vicentini AM, Hofsteenge J, Raines RT.High-level soluble production and characterization ofporcine ribonuclease inhibitor.Protein Expr Purif.2001; 22:174-9) output is suitable, and simple to operate many.

The host bacterium of expressing as freeze-thaw method, sonioation method, other machinery or chemically fragmenting method, is collected the supernatant liquor that contains the solubility expression product through the breaking method of routine.Active rmRI product can carry out purifying by known protein purification technology, as salting-out process and various chromatography method.In the preferred scheme, active rmRI product can be by the affinity chromatography technology of label polypeptide, and as the specific metal affinity chromatography method of His-6, purifying obtains.

The rmRI of fusion tag polypeptide keeps Yeast Nucleic Acid enzyme inhibition activity wholly or in part usually.In the preferred scheme, the label polypeptide can remove with the proteolytic cleavage of specificity scinderin fusion sequence.

The active rmRI that the present invention obtains, has the Yeast Nucleic Acid enzyme inhibition activity of basically identical with recombinant expressed people RI, can be used for preventing that rnase from polluting may cause in some molecular biology researches of problem, or other are obtained in the external and body of certain biological effect in the application by suppressing ribonuclease activity.

Description of drawings

The transformation efficiency of Fig. 1 .mRI prokaryotic expression carrier in different host bacterium.With A: empty carrier pQE30; B: the recombinant plasmid pQE30-mRNH of expressing protein mRI; C: the recombinant plasmid pQE80L-mRNH of expressing protein mRI is transformed into respectively among four kinds of host bacterium TOP10, TG1, BL21 (DE3) pLysS, BL21 (DE3) Rosetta.The transformed clone of pQE30-mRNH obviously is less than the transformed clone of pQE80L-mRNH and empty carrier pQE30.

Fig. 2. the expression of the prokaryotic expression plasmid pQE80L-mRNH of target protein mRI in E.coli TOP10, TG1, three kinds of host bacterium of BL21 (DE3) pLysS.37 ℃, grow to bacterial concentration OD600 at 0.4 o'clock, 1mM IPTG induces 4h, collects full bacterium and carries out protein electrophoresis (SDS-PAGE).Being not blank with the corresponding host bacterium of IPTG inductive.Right side arrow is denoted as the target protein position.

Fig. 3. the abduction delivering of recombinant plasmid pQE80L-mRNH in E.coli BL21 (DE3) pLysS host bacterium.37 ℃, grow to bacterial concentration OD600 at 0.4 o'clock, 1mM IPTG induces 4h, collects full bacterium cracking, goes up cleer and peaceful precipitation and carries out protein electrophoresis (SDS-PAGE).Being not blank with IPTG inductive host bacterium.Right side arrow is denoted as the target protein position.

Fig. 4. the influence that expression condition is expressed in E.coli BL21 (DE3) pLysS recombinant plasmid pQE80L-mRNH.27 ℃ are cultured to bacterial concentration OD600 is 0.8 o'clock, uses 1mM and 0.1mM IPTG to induce respectively 4 hours or 16 hours, collects full bacterium and carries out protein electrophoresis (SDS-PAGE).Being not blank with IPTG inductive host bacterium.Right side arrow is denoted as the target protein position.

Solubility expression and the purifying of Fig. 5 .mRI recombinant plasmid pQE80L-mRNH in E.coli BL21 (DE3) pLysS bacterium.27 ℃ are cultured to bacterial concentration OD600 is 0.8 o'clock, induces 16 hours with 1mM IPTG, collects full bacterium.After cracking was centrifugal, supernatant liquor was through nickel affinity chromatography, and 5～50mM imidazoles solution cleans, 100～300mM imidazoles eluant solution.Protein electrophoresis (SDS-PAGE) separates, and right side arrow is depicted as the target protein position.

Fig. 6 .his-mRI molecular weight of albumen is identified.It is 51420 that flight mass spectrum (MALDI-TOF) is measured the his-mRI molecular weight of albumen.

Fig. 7. recombinant plasmid pQE80L-mRNH is at E.coli BL21 (DE3) pLysS, and the solubility expression purification ratio is in three kinds of host bacterium of TG1 and TOP10.The pLysS that pQE80L-mRNH transforms, TG1, Top10,27 ℃ are cultured to bacterial concentration OD600 is 0.8 o'clock, induces 16 hours with 1mM IPTG, collects full bacterium.After cracking was centrifugal, supernatant liquor was through nickel affinity chromatography, and 5～50mM imidazoles solution cleans, 300mM imidazoles eluant solution.Expressed proteins has obvious degradation in the Top10 bacterium.Protein electrophoresis (SDS-PAGE) separates, and the right side arrow marker location is respectively target protein His-mRI and degraded band position.

Fig. 8. the solubility expression purifying of recombinant plasmid pRLTEV-mRNH in E.coli BL21 (DE3) pLysS bacterium.27 ℃ are cultured to bacterial concentration OD600 is 0.1 o'clock, induces 8 hours with 0.1mM IPTG, collects full bacterium.After cracking was centrifugal, supernatant liquor was through nickel affinity chromatography, behind the 300mM imidazoles PBS eluant solution, cut label with the TEV enzyme successively except that His-TrxA, heavy soluble protein sample is once more by nickel affinity chromatography behind the ammonium sulfate precipitation mRI, and effluent liquid is dialysed through 50% glycerine PBS solution, the protein concentrate sample.Each sample separates through protein electrophoresis (SDS-PAGE).

Fig. 9 .TrxA-mRI molecular weight of albumen is identified.It is 66298 that flight mass spectrum (MALDI-TOF) is measured the A:TrxA-mRI molecular weight of albumen; Behind the B:TEV excision TrxA, the mRI molecular weight of albumen is 50621.

Embodiment

The statement of following embodiment is for understanding principle of the present invention and application better, and does not limit use range of the present invention.

1. materials and methods:

Ox pancreas RNase A is available from Qinyuan, Beijing favour flag Bioisystech Co., Ltd; HRI (recombinant human RI) is Promega company product (trade(brand)name RNasin Ribonuclease Inhibitor), and yeast rRNA uses before through purification process available from Shanghai pattern ocean Bioisystech Co., Ltd.Forulic acid (FA) is available from Chemical Reagent Co., Ltd., Sinopharm Group.Ni-IDAAgarose is available from prestige lattice Lars Bioisystech Co., Ltd.

Purifying protein quantitatively: use 5 μ l respectively, 10 μ l, 20 μ l concentration be 0.2mg/ml bovine serum albumin (BSA) as standard control, together carry out the SDS-PAGE electrophoresis with purifying protein.Coomassie brilliant blue staining, QuantiScan software carries out gray scale scanning to gel, calculates target protein content according to the BSA typical curve.Mass spectroscopy: get 5 μ l protein samples through the centrifugal post desalination of Sephadex G-10, the sample that obtains mixes point sample with mass spectrometer matrix FA at 1: 1.(MALDI-TOF/TOF Analyzer 4800plus, Applied Biosystem) carries out qualitative analysis to sample protein through substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOFMS).

Ribonuclease activity and ribonuclease inhibitor determination of activity: determination of activity reference [the Umansky SR of rnase, Piker EG, Adler VV.Ribonuclease activity of rat thymus chromatinproteins.Experientia.1974; 30 (7): 734-6] the TCA precipitator method are with 25mM Tris-HCl (pH8.0), 1mM EDTA, 0.01%BSA is a damping fluid, an amount of RNase A and 8 μ g yeast rRNA, 37 ℃ of room temperature reaction 15min, place on ice, add the 10%TCA of equal-volume precooling, place 15min on ice.4 ℃ of centrifugal 10min of 12000rpm get supernatant, and OD280 detects optical density value.The ribonuclease inhibitor to be measured that adds different concns in the above-mentioned reaction mixes with 5 μ g RNase A, measures the restraining effect of ribonuclease inhibitor to RNase A.Suppress the RI activity of 5ng RNase A activity 50%, be defined as 1 activity unit (U).

2. the clone of mouse RI cDNA:

Get the Kunming mouse lung tissue, extract total RNA by the explanation method with total RNA extraction reagent RNAVzol (prestige lattice Lars Bioisystech Co., Ltd).The total RNA of 5 μ g adds in the 20 μ l reverse transcription systems, this system contains 10 * RT-buffer, 2 μ l, random primer 200ng, dNTPs 0.5mM, DTT 10mM, RNasin (Promega) 10u and ThermoScript II M-MLV H-(prestige lattice Lars Bioisystech Co., Ltd) 1 μ l is hatched synthetic cDNA first chain of 50min for 42 ℃, hatch 10min deactivation M-MLV for 70 ℃ ,-20 ℃ of preservations.Design and synthetic PCR upstream primer 5 '-ataagatctatgagtcttgacatccagtgtgagc-3, downstream primer 5 '-atagtcgactcaggaaatgatcctcagggaagg-3 ' (Invitrogen, Shanghai), contain BglII and SalI restriction enzyme site respectively.Contain 10 * PCR-buffer, 10 μ l in the 100 μ l PCR reaction systems, cDNA 2 μ l, dNTPs (2.5mM) 4 μ l, each 1 μ l of upstream and downstream primer (40 μ M), LA Taq 2.5u.PCR reaction is through 94 ℃ of pre-sex change 3min, 35 comprise sex change (94 ℃/30s), annealing (55 ℃/30s), extend (72 ℃/50s) circulation and last extension (72 ℃/7min) step is finished.Reaction is got 5 μ l pcr amplification products after finishing, and 0.8% agarose gel electrophoresis detects, and obtains the PCR product of about 1.4kb.

The PCR product is connected with the pBSK-T carrier, transforms the Top10 competent cell.Obtain to contain and insert pulsating positive colony.Extract plasmid, the mRNH/pBSK of acquisition cuts the insertion segment that confirms to contain 1.4kb through BglII and SalI enzyme.MRNH/pBSK through two-way sequencing (Invitrogen, Shanghai), the full-length cDNA that obtains mouse RI contains 1371 bases, measures encoding sequence and known mouse gene order (NCBI sequence number: AF071546; NM_145135) compare, the consistence of its nucleotide sequence reaches more than 99%.The difference of gene order may derive from the hereditary difference of different mouse kinds system, but artificial false drop can not get rid of genetic manipulation or sequencing the time.

The protein sequence of this nucleic acid sequence encoding and known mouse RI protein sequence (NCBI sequence number: AF071546; NM_145135) compare, the consistence of protein sequence is respectively 98% and 99%.Compare with known RI protein sequences such as people, rat, pigs, its homology is respectively 87%, 94% and 87%.

3. the expression vector establishment of mouse RI cDNA:

The mRNH/pBSK plasmid DNA is through BglII and SalI double digestion, the dna segment of agarose gel electrophoresis separation and purification 1.4kb, the pQE30 that handles with BamHI and SalI respectively, pQE80L carrier (Qiagen), and BamHI is connected with the pRLTEV carrier (prestige lattice Lars Bioisystech Co., Ltd) that XhoI handles.Connect product and transform the Top10 competent cell.Obtain to contain and insert pulsating positive colony.Among the expression vector pQE30-mRNH and pQE80L-mRNH that obtains, 5 of mouse RI cDNA ' end merges has coding additionally to comprise the vector gene sequence of 12 amino-acid residues of His-6 (six Histidines), the theoretical molecular that they express target protein (His6-mRI) is 51212Da, among the expression vector pRLTEV-mRNH, 5 of mouse RIcDNA ' end merges the vector gene sequence that has coding additionally to comprise TrxA (Trx) and TEV protease cutting site (ENLYFQ), and the theoretical molecular that it expresses target protein (TrxA-mRI) is 65789Da; After the TEV protease treatment was removed TrxA, its proteic theoretical molecular was 50256Da.

4.His6-mRI expression and purification:

The plasmid DNA of expression vector pQE30-mRNH and pQE80L-mRNH transforms Top10, TG1, BL21 (DE3) pLysS and BL21 (DE3) Rosetta competent cell respectively.Compare with TG1 with Top10, pQE30-mRNH obviously reduces the transformation efficiency of BL21 (DE3) pLysS and BL21 (DE3) Rosetta, and pQE80L-mRNH is to the transformation efficiency and the empty carrier suitable (Fig. 1) of various competent cells.Because the pQE80L carrier contains the gene framework of the extra LacI that encodes, can suppress the basal expression of goal gene, infer that pQE30-mRNH has certain basis to express at BL21 (DE3) pLysS and BL21 (DE3) Rosetta, thereby to this bacterial classification toxigenicity.After IPTG induced, Top10, the TG1 and BL21 (DE3) the pLysS host bacterium that contain pQE30-mRNH and pQE80L-mRNH plasmid all had extra about 50kD protein expression (Fig. 2).Contain BL21 (DE3) pLysS of pQE30-mRNH and pQE80L-mRNH plasmid and BL21 (DE3) Rosetta host bacterium after IPTG induces, growth obviously delays.SDS-PAGE shows that target protein is based in the inclusion body precipitation (Fig. 3) after the host bacterium cracking of expression.

200～400ml is through the host bacterium of abduction delivering, and the centrifugal thalline of collecting is resuspended among the PBS that contains 1%Triton-X100.Through freeze thawing and ultrasonication, the centrifugal supernatant liquor that contains the solubility target protein of collecting.Supernatant liquor is by the absorption of Ni-IDA agarose affinity matrix, and PBS cleans and contain the PBS gradient elution of 20-300mM imidazoles, can obtain the His-mRI of purifying.0.1～1mM IPTG induced 16 hours for 27 ℃, the target protein expression amount increases (Fig. 4), and the amount of collecting of soluble proteins obviously improves.The solubility expression product identifies through mass spectrum that through nickel affinity chromatography purifying (Fig. 5) simple spike shows His-mRI molecular weight about 51.4kD (Fig. 6), with the theoretical molecular basically identical, meets expection.His6-mRI output BL21 (DE3) pLysS host bacterium is 4～8mg/L bacterium liquid, or the 1mg/g wet thallus; Output quite or lower slightly in Top10, the TG1 host bacterium.Compare with the activity of recombinant human RI, the His6-mRI activity of BL21 (DE3) pLysS host bacterium is approaching, but the RNaseA of Top10, TG1 host bacterium purified product suppresses active obviously reduce (table 1).In expression experiment repeatedly, run into the situation (Fig. 7) that degraded appears in expression and purified product sometimes, infer that the activity of the expression product of Top10, TG1 host bacterium is hanged down relevant with product degradation; And BL21 (DE3) pLysS host bacterium is owing to exist proteolytic enzyme defective phenotype, and possible expression product is difficult for degrading, and can keep greater activity.Top10 host bacterium is still kept than low activity (table 1) based on the expression product of obvious degradation segment, also may be not relevant with the activity of residual a small amount of degraded product.

4.TrxA-mRI expression and purification:

The plasmid DNA of expression vector pRLTEV-mRNH transforms BL21 (DE3) pLysS competent cell respectively.After 0.1mM IPTG induces, extra about 66kD protein expression is arranged, after the target protein of solubility expression carries out the nickel affinity chromatography purifying, 300mM imidazoles PBS eluant solution, after the TrxA label was removed in the effect of cutting of TEV enzyme, saturated ammonium sulphate mRI albumen used the protein sample after the PBS solution weight is dissolved, by nickel affinity chromatography, effluent liquid is through 50% glycerine PBS solution dialysis protein concentrate sample (Fig. 8) once more.The protein product optimization expression that TrxA merges, purified after the amount of collecting be about 4～5mg/L, identify that through mass spectrum molecular weight is about 66.3kD, with theoretical molecular basically identical (Fig. 9 A), its specific activity is about 16kunits/mg; The TEV enzyme cuts except that behind the TrxA, and the amount of collecting of the mRI of purifying is about 1～2mg/L, and the mass spectroscopy molecular weight of albumen is 50.6kD (Fig. 9 B), and specific activity is about 39kunits/mg, and is suitable with the activity of recombinant human RI.

Table 1:pQE80L-mRNH transforms His-mRI solubility expression of protein, purifying and the activity identification of different host bacterium.

Annotate: the activity unit of 1 ribonuclease inhibitor is for suppressing the content of 5ng ribonuclease A activity 50%.

Claims

One kind in the host bacterium in intestinal bacteria sources, by transforming the prokaryotic expression carrier of RNAsin encoding gene, through inducing under the certain condition, obtain the method for the highly-soluble expression of active RNAsin, be characterised in that:

(1) described ribonuclease inhibitor encoding gene is a mouse ribonuclease inhibitor encoding gene (cDNA);

(2) described host bacterium is the engineering bacteria with proteinase gene defective;

(3) described inductive condition is to be lower than abduction delivering under 37 ℃ of culture temperature.
2. claim 1 is described has a proteinase gene defective host bacterium, is BL21 or its deutero-engineering bacteria.
3. the described culture temperature of claim 1 is at room temperature to carry out abduction delivering.