NZ625595B2 - Method for highly expressing recombinant protein in recombinant bacteria and use thereof - Google Patents

Abstract

Disclosed is a method for highly expressing a recombinant protein in recombinant bacteria, wherein the recombinant protein comprises a hydrophilic and a hydrophobic end, wherein the hydrophilic end is a colicin polypeptide and wherein the hydrophobic end is a polypeptide target moiety which is capable of binding a target, the method comprising: (1) transfecting a recombinant plasmid expressing the recombinant protein into E.coli bacteria with pET system to obtain positive monoclonal colonies and produce recombinant E.coli bacteria, (2) producing seed bacteria solution from the positive monoclonal colonies, and inducing protein expression and enlargement culturing of the seed bacteria solution in enlargement culturing medium; wherein the supernatant of the enlargement cultured solution contains the expressed recombinant protein, (3) extracting and purifying the recombinant protein from the supernatant, wherein the recombinant E.coli bacteria with pET system is E.coli B834 (DE3), and wherein the enlargement culturing medium has water as solvent and comprises the following components: NaCl 6.0~6.7 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 0.6~2.0 g/L, Na2HPO4·7H2O 6.8~18.3 g/L, KH2PO4 3.0~4.3 g/L, NH4Cl 1.0~1.4 g/L, MgSO4 0.2~0.4 g/L, CaCl2 0.01 g/L, and methionine 0~40 mg/L. Also disclosed is a medium for recombinant E. Coli bacteria with pET system. wherein the medium has water as solvent and comprises the following components: NaCl 6.0~6.7 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 0.6~2.0 g/L, Na2HPO4·7H2O 6.8~18.3 g/L, KH2PO4 3.0~4.3 g/L, NH4Cl 1.0~1.4 g/L, MgSO4 0.2~0.4 g/L, CaCl2 0.01 g/L, and methionine 0~40 mg/L. le of binding a target, the method comprising: (1) transfecting a recombinant plasmid expressing the recombinant protein into E.coli bacteria with pET system to obtain positive monoclonal colonies and produce recombinant E.coli bacteria, (2) producing seed bacteria solution from the positive monoclonal colonies, and inducing protein expression and enlargement culturing of the seed bacteria solution in enlargement culturing medium; wherein the supernatant of the enlargement cultured solution contains the expressed recombinant protein, (3) extracting and purifying the recombinant protein from the supernatant, wherein the recombinant E.coli bacteria with pET system is E.coli B834 (DE3), and wherein the enlargement culturing medium has water as solvent and comprises the following components: NaCl 6.0~6.7 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 0.6~2.0 g/L, Na2HPO4·7H2O 6.8~18.3 g/L, KH2PO4 3.0~4.3 g/L, NH4Cl 1.0~1.4 g/L, MgSO4 0.2~0.4 g/L, CaCl2 0.01 g/L, and methionine 0~40 mg/L. Also disclosed is a medium for recombinant E. Coli bacteria with pET system. wherein the medium has water as solvent and comprises the following components: NaCl 6.0~6.7 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 0.6~2.0 g/L, Na2HPO4·7H2O 6.8~18.3 g/L, KH2PO4 3.0~4.3 g/L, NH4Cl 1.0~1.4 g/L, MgSO4 0.2~0.4 g/L, CaCl2 0.01 g/L, and methionine 0~40 mg/L.

Description

Specification METHOD FOR HIGHLY EXPRESSING RECOMBINANT PROTEIN IN RECOMBINANT BACTERIAAND USE THEREOF TECHNICAL FIELD The invention relates to biotechnology, especially to a method for highly expressing a recombinant protein in recombinant bacteria and the use f.

BACKGROUND OF INVENTION In earlier studies, the inventor conducted creative experiments and invented a series of new recombinant peptides with Colicin as attack point, which operationally connects a polypeptide (natural or cial design) with identification and binding ability to target cells. For example, the new antibiotic PMC—AMl disclosed in patent No. 10092128.4, named “Novel otic comprising an antibody mimetic, its preparation and uses thereof”, shows a broad—spectrum antibiotic property and has stronger antibacterial ty on Neisseria meningitidis, Mulridrug-resistance Pseudomonas aeruginosa, Vancomycin-resistant Enterococcus faecalis or Methicillin-resistant Staphyiococcus aureu compared to the known otics. Another ion by the inventor is titled “A novel antibiotic, its nucleotide sequence, methods of construction and uses thereof”, with CN patent No.2L2009101575645, and discloses a series of new anti—staphylococcus antibiotics, such as PMC-SAl, PMC—SAZ, PMC—SA3, PMC—SA4, PMC—SE as well as PMC—PA. In vivo and in vitro experiments, these antibiotics showed better targeting y and stronger antibacterial activity than current antibiotics, antifungal antibiotic and herapeutics drugs. onally ed with current antibiotics, these new antibiotics showed incomparable biological security and anti drug—~resistance teristic.

The foresaid novel antibiotics as a whole are a kind of water-soluble proteins with 600 amino acid residues, but in which there is a hydrophobic domain with 40 amino acid residues near carboxyl terminal. Compared to preparation of other water-soluble ns with one fold structure, there is more difficult in assembling and expressing of the novel otics, which inevitably affects protein yield. It is necessary to improve current expression process to achieve high yield and priority of the novel antibiotics. It will make sense for bringing the novel antibiotics into actual clinical application and practice.

Summary of ion According to the peptide structure and characteiistics of the new antibiotics disclosed in the current patent application, the present disclosure provides for a method for highly expressing recombinant protein in recombinant bacteria.

In one aspect, the present disclosure provides for a method for highly expressing a recombinant protein in recombinant bacteria, wherein the recombinant protein comprises a hydrophilic and a hydrophobic end, n the hydrophilic end is a colicin polypeptide and wherein the hydrophobic end is a polypeptide target moiety which is capable of binding a target, the method comprising: (1) transfecting a recombinant plasmid expressing the recombinant protein into E. coli bacteria with pET system to obtain positive monoclonal colonies and produce recombinant E. coli ia, (2) producing seed bacteria solution from the positive monoclonal colonies, and inducing n expression and enlargement culturing of the seed bacteria on in enlargement culturing medium; 'wherein the supernatant of the enlargement cultured solution contains the expressed recombinant protein, (3) extracting and ing the recombinant n from the supernatant, wherein the recombinant E. coli bacteria with pET system is Ecoli B834 (DE3), and n the enlargement ing medium has water as solvent and comprises the following components: NaCl 6.0~6.7 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 0.6~2.0 g/L, NagHPO4'7H20 6.8~18.3 g/L, KHZPO4 3 g/L, NH4Cl 1.0~1.4 g/L, MgSO4 0.2~0.4 g/L, CaClz 0.01 g/L, and methionine 0~40 mg/L. 2O In a preferable exemplary embodiment, said enlargement culturing medium has water as solvent and comprises the following components: NaCl 6.0 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 2.0 g/L, NazHPO4-7H20 6.8 g/L, KH2P04 3.0 g/L, NH4C1 1.0 g/L, MgSO4 0.2 g/L, CaClz 0.01 g/L, and methionine 0~40 mg/L.

In some exemplary embodiments, said enlargement culturing of the seed bacteria solution comprises the following steps: adding the seed bacteria liquid into a container and ing for 2 to 3 hours at 30°C, when the OD value reaches 0.4—0.6 heat shocking the solution for 42°C for 30 minutes, and then cooling the solution to 37°C and culturing for a further 1.5 to 2 hours. In some exemplary embodiment, IPTG with a final concentration of 0.5 mmol/L is added when the on is at 42°C.

In some exemplary embodiments, n in conducting heat shocks the IPTG with the final y 0.5 rnmol/L was added into said enlargement-culturing medium.

In some exemplary embodiments, wherein said extracting and purifying of the recombinant protein from the supernatant comprises use of a CM ion exchange IS column, wherein the loading quantity of the supernatant s on the ratio value which is 2.5 mg/ml between the weight of the recombinant protein in the atant and the volume of Gel particles used in the CM ion exchange column.

In some exemplary embodiments, the eluent solution used for said extracting and purifying in CM ion exchange column is boric acid buffer solution with 0.2 mol/L NaCl.

In most exemplary embodiments, said recombinant plasmid expressing the recombinant protein is selected from the group consisting of Al, pBHC—SA2, pBHC-SA3, pBHC—SA4, pBHC—SE, pBHC—PA, and pBHC—P or In a further aspect, the present disclosure provides for the applications of any foresaid methods in preparing the recombinant peptides PMC— SAl, PMC-8A2, PMC—8A3, PMC—8A4, PMC -SE, PMC-PA or PMC—AM.

In another aspect, the present disclosure provides for a medium for recombinant E. c055 bacteria with pET system, wherein the medium has water as solvent and comprises the following components: NaCl 6.0~6.7 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 0.6~2.0 g/L, NazHPO4-7HZO 6.8~18.3 g/L, KH2P043.0~4.3 g/L, NH4C1 1.0~1.4 g/L, MgSO4 0.2~0.4 g/L, CaC120.01 g/L, and methionine 0~4O mg/L.

In some exemplary embodiments, said recombinant E. coli bacteria with pET system is E. COli B834 (DE3), and the medium has water as t and ses the following ents: NaCl 6.0 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 2.0 g/L, 4-7H20 6.8 g/L, KH2P04 3.0 g/L, NH4C1 1.0 g/L, MgSO4 0.2 g/L, CaClz 0.01 g/L, methionine 0~40 mg/L.

The pET expression system provided by Novagen Company is a common system for cloning and expressing recombinant proteins in Escherichia coli. In this invention, a series of BL-21 (DES) cells are transected with recombinant mutated plasmid disclosed in former patents and produced a higher n sion yield than the T61 cells does in this invention. By experimental data, we found that B834 (DE3), which is parent strain of BL21 (DE3), has a more ideal expression productivity than BL—Zl (DE3). The experimental data showed that the B834 (DE3) has increased protein expression productivity than the TG1 system does.

Medium is used for ing required carbon source, nitrogen source and inorganic salts for ium growth and multiplication. The invention also provides a medium with capability of improving the expression productivity of target protein, which has an optimum formula for inant bacteria tation. In this invention, the medium, named FB—M9 compound medium has an increased carbon source and nitrogen source and MgSO4, CaClz as well as some special amino acids that are required in growth of recombinant bacteria with pET system. The medium moderately improved recombinant bacteria reproduction speed and protein expression rate. Further, the material cost of the improved medium is relatively low, which es larger research space and higher development value for enlargement tion in the future.

According to guide of the product manual, the ng rate of CM ion gel particles used in purification system in this invention could not reach the ideal standard described in the t manual, which limits the recovery rate of target protein. In present invention, the recOvery rate has been significantly improved by the means of reducing loading quantity of sample while moderately increasing the gel volume, etc. The result also reflected that it is necessary to find or develop a kind of ion exchange gel with more efficient for large-scale rial production of the target protein. In addition, the recombinant proteins have fewer ties owing to eluent with optimized concentration used in the ion exchange steps of this invention.

In summary, this invention provides a variety of optional more optimized method of expressing E. coli inant bacteria recombinant proteins by the means of choosing recombinant strains, optimizing the composition of medium, improving the purification and recovery rate, etc. This also provides a possible research direction and technical route for finally finding an optimal procedure of high-efficiently expressing fusion protein needed. Compared with the original expressing system disclosed in former patents, the sing system developed by present invention has improved the expressing production of fusion protein dozens of times, and provided a beneficial basis of theory and practice for the uent large-scale rial production.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the conductance value of eluent in the protein elution s with different volume gel column. a. The elution process of protein with 150 ml CM gel column. b. the elution process of protein with 600 ml CM gel column.

The curve signified by two arrows in the figure represents the conductance value of the eluent. The area ted by the arrows is a conductance peak caused by the loss of the sample PMC—SA in loading process. The area of the conductance peak caused by the loss of the sample PMC-SA reduced by 70% after increasing the volume of gel.

Another curve: OD value of elutropic protein Fig. 2 shows GE Gel electrophoresis of the PMC—S From left to right in the order: a. 1. Marker, 2. PMC—SAl produced by TGl, 3. PMC-SAl ed by BL-Zl, 4. PMC—SAl produced by B834; b. l. Marker, 2. PMC—SAl eluted by boric acid buffer solution with 0.1 M NaCl, 3. PMC-SAl eluted by boric acid buffer solution with 0.2 M NaCl, 4.

PMC-SAl eluted by boric acid buffer solution with 0.3 M NaCl.

Fig. 3 shows the inhibition curve of the PMC-SA against MRSA (BAA42).

Y—axis represents light absorption value; X-axis represents bacterial growth time l: control group; Amp: ampicillin sodium; OXA: oxacillin; Ia—wt: wild type colicin Ia; PMC-SAl: anti—staphylococcus aureus polypeptide; PMC—AM: iplococcus meningitides polypeptide.

DETAILED DESCRIPTION OF THE INVENTION Following es are just used for explaining the invention rather than limiting the scope of the invention.

The experimental equipment and instruments used are as follows: 1. Bacterial strain E. coli TGl recombinant bacteria , K. lakes).

E. coii BL-21(DE3), B834(DE3), Nova B1ue(DE3) and 618 recombinant bacteria are all purchased from Novagen company.

Staphylococcus aureus ATCC BAA-42 is purchased from ATCC (American Type Culture Collection).

Plasmid: pBHC—SAl, pBHC—SAZ, pBHC—SA3, pBHC—SA4, pBHC—SE , pBHC—PA, orAl (these plasmids are recorded in patents ZL 2009100921284 and ZL 2009101575645, and preserved in the applicant’s laboratory. The applicant promised to offer them to the public for necessary verification tests). 2. Main reagents and medicine Yeast powder (OXIOD LP0021), peptone (OXIOD LP0042), as well as other chemical reagent are all analytical reagent; Dialysis bag Snake Skin Dialysis Tubing (Pierce, intercept molecular weight 1x104, Lot #KD32324); Streptomycin Sulfate for injection (NCPC) AMP ampicillin sodium for injection (Harbin pharmaceutical) Anion ge column gel (Pharmacia Biotech CM Sepharose Fast Flow Lot 016).

LB liquid medium: Sodium de 1 g, peptone 1 g, and yeast 0.5 g were added into a 250 ml flask with the on of 100 ml water, dissolved and autoclaved at 120°C for 8min.

LB solid medium: 100 ml LB solid medium containing sodium de 0.5-1.5 g, e 0.5—2 g, yeast 0.3—1 g and agar 0.8—3 g. The LB solid medium is used for plate culture of single colony after strain recovery. Reagents were added into a 250 ml flask with the addition of 100 ml water, dissolved and autoclaved at 120°C for 8min.

FB—M9 complex medium: NaCl 6.0~6.7 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 0.6~2.0 g/L, NaZHPO4-7H2O 6.8~18.3 g/L, KH2PO4 3.0~4.3 g/L, NH4Cl l.0~1.4 g/L, MgSO4 0.2~0.4 g/L, CaC120.01 g/L, methionine 0~40 mg/L.

Improved FB-M9 complex medium: NaCl 6.0 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 2.0 g/L, NazHPO4-7H20 6.8 g/L, KH2P04 3.0 g/L, NH4C1 1.0 g/L, .2 g/L, CaC120.01 g/L, nine 0~40 mg/L. The methionine is 40 mg/L in the process with Ecoli B834 (DE3) as recombinant bacteria. 3. Key Instruments Bio-Rad Protein chromatography purification system (BioLogic Duo Flow, BioLogic Maximizer, BioLogic QuadTec UV—Vis Detector, BioLogic Econo Pump); Ultrasonic Cell Disruptor (Soniprep ’150), protein purification ion exchange column with 5 cm diameter(Pharmacia Biotech XKSO), protein purification ion exchange column with 11 cm diameter (Shanghai Huamei); Centrifuge an Coulter Avanti J-I2OXP, Beckman Coulter Avanti J—25); Spectrophotometer ad Smart Spect Plus spectrophotometer); Automatic fermenter (Bioengineering AG LP351—42L); High pressure homogenizer (Italian NiroSoavi NSlOOlL2KSN 6564).

Statement: the ical materials adopted in this invention have been known before the application filing date and have been also preserved in this applicant’s lab. The ant promised to offer them to the public for necessary verification tests in the twenty years since application filing date.

Example 1. The option experiment of recombinant bacterial strains.

Classic plasmid carried the colicin Ia and its immune protein gene (GenBank ) are from laboratory of Dr. Finkelstein. (Qiu XQ et al. An engineered multi domain bactericidal peptide as a model for ed antibiotics against specific bacteria. Nat Biotechnol, 2003; 21(12): 1480—1485). The classic plasmid was modified into following seven kinds of restructuring mutation plasmids in former research: pBHC—SAl, pBHC-SA2, pBHC—SA3, A4, pBHC—SE, pBHC—PA, pBHC-PorAl.

Step 1. Transformation of competent cell 40 uL Novagen pET system recombinant bacteria BL-21(DE3), B834(DE3), Nova Blue(DE3), 618 were tively transformed with 100 ng recombinant mutant plasmids pBHC—SAI, and then ice-incubated for 5 minutes, heat-shocked at 42°C for 30 seconds, kept in ice for 2 minutes, added with 160 pl SOC medium and shake—cultivated at 220 rpm, 37°C for 1 hour and then coated (LB medium with 1% agar and SOug/ml ampicillin, and cultured overnight at 37°C). Single colonies are picked out and cultivated to obtain the seed strain, which is conserved at a low temperature.

Step 2. Strain Recovery 1. Preparing recovered bacteria solution The ved strain was thawed at 4°C; 1.5 ml of the strain is transferred into 10 ml LB medium (containing 50 ug/ml of AMP) and cultivated at 220 rpm, 37°C for 5—8 hours. 2. Inoculation of single colony The recovered bacteria on was diluted 104 or 105 times; and 10 ul of the diluted bacteria solution was transferred on to LB solid medium plate (containing 50 ug/ml of AMP) and coated on the plate. The plate was placed in a humidity box and cultivated in incubator at 37°C for 10—12 hours till round single colonies have grown out on the surface of the medium.

Step 3. Enlargement culturing the single colonies ( 1) Single colonies with regular round shape and smooth edge were picked up from the plate and respectively added into 1.5 ml LB medium, and cultivated at 220 rpm, 37°C for —8hours. (2) Each 15 ml LB ia solution was transferred into a 100 ml LB medium, and cultivated at 220 rpm, 37°C for 5—8 hours. (3) Primary stage of ement culturing: the 100 ml of ia solution from the last step was added into 700 ml of improved FB-M9 complex medium and cultivated at 220 rpm, 37°C for 5—8 hours. (4) Secondary stage of enlargement culturing: 700 ml of bacteria on from the primary stage is added into 6x700 ml of the improved FB-M9 complex medium and cultivated at 220 rpm, 37°C for 5-8 hours. (5) Third stage of enlargement culturing: 6x700 ml of bacteria solution from the ary stage was added into 20 L of the improved FB—M9 complex medium and cultivated in a fermenter with stirring rate of 220 rpm and maximum oxygen flow volume, 37°C for 3-5 hours. (6) Fermentation of engineered ia and induced expression of protein: 20 L of bacteria solution from the third stage of enlargement culturing was added into 200 L of improved FB—M9 complex medium and cultivated in a ter for induced expression of protein with stirring rate of 220 rpm and maximum oxygen flow volume, at 30°C for 2~4 hours; 42°C for 0.5 hours; and 37°C for l~2 hours, note that IPTG is added at 42°C with a final concentration of 0.5 Step 4. Collecting bacteria by centrifugation 6000 g fermentation liquor obtained from step 3 was centrifuged at 4°C for 20 min. The precipitate was ted and added into 50 mM boric acid buffer ) for resuspension of the bacteria. Note: the boric acid buffer has 2 mM PMSF (Phenylmethylsulfonyl fluoride serine protease inhibitor). All consequent steps after bacteria resuspension was conducted at 4°C.

Step5. Cells ntation After suspension in pH 9.0 boric acid buffer completely, the bacteria cells was fragmented by a High Pressure Homogenizer at 500~600 bar for 7 times, with intervals of 3~5 minutes.

Step 6. itation of the bacteria DNA The fragmented bacteria solution was centrifuged at 55,000 g, 4°C for 40 min.

The supernatant was added with streptomycin sulfate (16 bottles of 1 million unit streptomycin sulfate were added into every 200 ml liquid supernatant), and d for 1 h with a magnetic stirrer.

Step 7. is The bacteria solution from the step 6 was centrifuged at 55.000g, 4°C for 20 min. The atant was placed into a dialysis bag and dialyzed for 8~12 hours in boric acid buffer, which was changed once every 4 hours.

Step 8. ing the protein medicine and obtaining antibacterial—engineered polypeptide The dialyzed bacteria solution was centrifuged at 55,000 g, 4°Cfor 20 min. The supernatant was measured the protein concentration in unit volume and placed into a Bunsen beaker for conducting protein purification by ion exchange method. The supernatant with known protein concentration was uploaded onto a CM ion exchange column. The sample loading and its ratio with the CM iron gel particular are according to the Product Manuals of CM ion exchange column.

After being washed completely the CM ion exchange column was eluted with 50 mM boric acid buffer containing 0.3 M NaCl to obtain the novel antibacterial—engineered polypeptide.

The results are shown as table 1, the expressing efficiency of PMC—SA by Ecoli B834 (DE3) is the highest.

Table l. Expressing ency of different bacterial strain (Average unit productionzGross production of extracted I/ volume of bacterial liquid) Recombinant strain TG1 BL—21 618 NavaBlue B834 Average unit production (mg/L) 0.8 10 5.8 8.1 24.4 The same operation was conducted on the other six restructuring mutation plasmids, the results appeared similar trend as the result listed in Table 1, namely, in contrast to other recombinant bacteria, E. coli B834 (DE3) showed the highest expressing efficiency on all seven restructuring mutation plasmids.

The operation of heat shock as following adopted to inducing sion of protein in this embodiment was different from that in prior arts: After erring the seed bacteria liquid into the tank, cultured the bacteria at an l temperature 30°C for 2 hours, when OD value had reached 0.4—0.6, ted the heat shock at 42°C for 30 minutes, then when the temperature low down to 37°C, cultured the bacteria again for 1.5 to 2 hours again. At this stage the OD value of bacteria liquid can reach to 1-3 or even more, and can be conducted collection. During this process, 0.5 mM IPTG was added to induce expression of pET recombinant bacteria.

Before proposing present method, the usual process for preparing the recombinant peptides was as following: 100 ng of the mutant plasmids was ice—incubated with 40 ul competent cell of BL-Zl engineered bacteria for 5 minutes, heat—shocked at 42°C for 30 seconds, ice-incubated for 2 minutes, added with 160 pl of SOC medium, shake-cultivated at 220 rpm, 37°C for 1 hour and then coated plate (LB medium with 1% agar and 50 ug/ml ampicillin, and cultured overnight at 37°C).

Single colonies were picked out for enlargement culturing. ement culturing: 8—10L FB medium, 250 rpm, at 37°C for 3—4 hours; was added with IPTG, 250 rpm, at 28 °C grew for 4 hours again; ted centrifugation to precipitate bacteria at 4 °C, 6000 g, 20 minutes. The precipitated bacteria was added with 80—100 ml 50 mM boric acid buffer (pH 9.0, 2 mM EDTA) kept at 4°C to suspend, then added with 50 ug PMSF and broken by ultra sonication (4°C, 400 w, 1 minutes, repeat 4 to 5 times with intermittent 2-3 s for keeping the ature of the liquid). Then the broken bacteria was conducted high-speed fugation (4°C, 75000 g, 90 s), the supernatant was added with 5 million units streptomycin sulfate to precipitate DNA (4°C stirred for 1 hour), and 10,000 g, 4°C, for 10 minutes centrifugation. The supernatant was put into dialysis bag with the molecular weight 15,000 on 4°C, and dialyzed by 10 L 50 mM boric acid buffer overnight, then conducted centrifugation at 4°C, 10000 g, for 10 minutes once again. The supernatant was loaded on CM ion exchange column, after being flushed completely, eluted by 0.3 M NaCl + 50 mM boric acid buffer, the new antibiotics can be obtained.

Example 2 Improving medium The classic FB medium for colicin la ation (Qiu XQ et al. An engineered multi domain bactericidal e as a model for targeted otics against specific bacteria. Nat hnol, 2003; 21(12): 1480-1485 IKaren lakes, Charles Abrams, Alan Finkelstein, et a1. Alteration of the pH-dependent Ion Selectivity of the Colicin El Channel by Site-directed nesis. JBC, 1990; 265(12): 991) has components as follows: peptone 25.0 g/L, yeast powder 7.5 g/L, NaCl 6.0 g/L and glucose 1.0 g/L.

In this invention, we adopted FB medium without glucose, the components of which as follows: peptone 25.0 g/L, yeast powder 7.5 g/L and NaCl 6.0 g/L.

And the FB medium without glucose was configured with M9 medium at a special volume proportion to obtain the FB-M9 compound medium.

The mother liquor of M9 medium is 5><M9 and has components as follows: NaZHPO4-7HZO 64.0 g/L, KH2P04 15.0 g/L, NH4C1 5.0 g/L, NaCl 2.5 g/L, MgSO4 1.5 g/L, CaC12 0.05 g/L, and 2% glucose.

A preliminary attempt of the compound medium: FB-M9: volume ratio between FB2M9 was 7:10, the components as follows: NaCl 6.7 g/L, e 25.0 g/L, yeast powder 7.5 g/L, NazHPO4~7H20183 g/L, KHZPO44.3 g/L, NH4C1 1.4 g/L, MgSO40.4 g/L, CaC120.01 g/L and glucose 0.6 g/L.

This invention adopted this formula for bacteria fermentation. The process was as step 3 in Example 1. The result shows in Table 2, wet bacteria weight got from per liter culture solution is significantly higher than that done through FB . The collected protein production is significantly improved with average production up to 30 mg/L.

Table 2 contrast of target n production from test medium (PMC - SAl / BL — 21 recombinant bacteria).

Fermenting in BF medium Fermenting in FB-M9(7: iurn Bacteria1 weight (g). Protein Bacterial Protein contents ts (mg) weight (g) (mg) 1 255.07 280.8 847.82 L 2 246.3 519.94 343.47 643.71 3 302.28 461.965 366 779.3 4 276.67 465.179 388.44 946.34 AV 270.8 431.971 361.9325 804.2925 The final improved FB—M9 medium was obtained by r research and repeated comparison in this invention. The production rate of the target protein can reach 34 mg/L as Table 3 shows, in the same fermentation conditions as example 1.

The ents of the improved FB-M9 medium as follows: NaCl 6.0 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 2.0 g/L, 4-7HZO 6.8 g/L, KHZPO43.0 g/L, NH4Cl 1.0 g/L, MgSO4 0.2 g/L and CaClg 0.01 g/L. As the methionine was required in the growth of B834 recombinant bacterium, in the process of B834 as recombinant ia the methionine (40 mg/L) is added into the final improved FB-M9 medium.

Table 3 comparison of ed FB — M9 medium with other medium on productivity BL—21 B834 Recombinant strains and FB FB—M9 Improved FB FB—M9 Improved the medium (7: 10) (7: 10) FB-M9 Average unit production(mg/L) Example 3. Optimizing conditions for ing protein The basic structure of recombinant polypeptide (PMC- SA1, PMC-SAZ, PMC —SA3, PMC ~SA4, PMC —SE, PMC -PA, PMC-AM) prepared in this invention is Colicin Ia. The isoelectric point of colicin Ia is about 9.15, therefore the classic purification adopted is Ion Exchange Chromatography (Qiu XQ et al. An engineered multidomain bactericidal peptide as a model for targeted antibiotics t specific bacteria. Nat Biotechnol, 2003; 21(12): 1480-1485).

The principle is: In pH 9.0 boric acid buffer system, the majority of PMC-SA molecules exist as positive charge ions. When the CM gel particles with negative charge go through the chromatographic column, the recombinant protein molecules with positive charge was hung on the CM gel particles due to the electric charges attraction, while the other miscellaneous n was rushed out of the gel column.

In this example, the other steps were as that in example 1, but after the laneous protein was rushed out completely, using boric acid buffer of 0.1 to 0.3 M NaCl gradient to elute the gel column.

Owning to Na+ ions having stronger positive ty than the recombinant protein molecules, the inant protein was replaced from CM gel particles by Na+ ion. There are two variables to be manipulated in the process of ion exchange and purification for a better protein yield: 1. NaCl with different concentration within 0.05—1 M can be chosen respectively to elute protein molecules with different positive charge mounted on CM gel particles; 2. The amount of CM gel particles adopted can be optimized: In the environment with certain ionic th, the amount of protein carried by every CM gel particles is relatively nt. The volume of gel column is indispensable to be enlarged in order to increase the amount of protein carried by gel column.

CM Sepharose Fast Flow is anion exchange column gel produced by GE y. According to the manual, every 100 ml gel can combine with 9 mM cation. The actual usable combination capacity varies with the nature of sample in the s of dynamic combination, and molecular weight is inversely proportional with combined capacity. Its standard sample that has equivalent molecular weight with the recombinant peptides manufactured in this invention is Bovine COHb—(Mr69kD), which has theoretically dynamic combined capacity 30 mg/ml. Namely, with 100 ml CM Sepharose Fast Flow glue to retrieve recombinant protein molecules, the tically t recovery rate is about 300 mg (0.004 mM). But according to its manual operation, the actual dynamic combination capacity of recombinant protein molecules to CM gel particles reached only 3 mg/ml, just reached 10% of theoretical combined capacity.

In the ment, we found that in the latter half process of washing out the miscellaneous protein, conductance curve will raise a small peak (as shown in figure 1 a). According to this enon, we speculate that when there is a large amount of recombinant protein in the sample, due to the limited capacity of CM gel les with target protein, only a little part of the recombinant protein molecules can be recovered. The recombinant protein without being mounted on the CM gel particles has to be flushed out gel column together with miscellaneous protein. As the recombinant protein is positively charged, a short rising peak s in the conductance curve.

In an optimized example of this invention: in order to reduce the loss of recombinant proteins, we reduced loading amount of sample to 1/3 of the manual regulation, and sed the volume of gel from 150 ml to 600 ml, namely the protein amount in the atant fluid: gel particle volume = 2.5 mg/ml. The loss of the recombinant protein decreased in the process of elution.

The experimental data showed that the ry rate of recombinant proteins was increased 3.5 times; the results shown in figure 1b.

In addition, we set the gradient concentration of NaCl as O.l-O.2-0.3 M in the boric acid buffer used in elution, and 0.2 M showed the highest eluting ency and protein purity, as shown in figure 2b.

Example 4. Detecting protein purity and activity Step 1. SDS—PAGE electrophoresis The fusion protein samples obtained by optimized conditions of example 4 were conducted SDS-PAGE electrophoresis and silver nitrate dyeing. As shown in figure 2, there is a clear protein—imprinting stripe at the point of about 70kD relative lar weight, namely PMC — SAl manufactured in this invention in the electrophoresis map a the map b shows the protein has eliminated mixed zone through the improved gradient elution in example 4 and the purity is improved. The rest six kinds of recombinant ns manufactured through the zed method of this invention have also showed similar improved purification.

Step 2. Detecting the antibacterial activity With the inant protein PMC — 8A1 and PMC—AM that produced by the improved manufacturing method in the e 1, 2 and 3 we conduct the following antibacterial activity test.

The Merhicillin—resistant staphylococcus aureus (MRSA, ATCC BAA-42) bacteria liquid 10 pl (105 cfu/ml) was inoculate into th ml BM medium and added with antimicrobial agents. According to the antimicrobial agents we set six parallel groups: ampicillin sodium 2 ug/ml, oxazocilline 4 pig/ml, wild type colicin Ia, PMC-SA and Ph—NM (4 , and blank control group. Culturing at 37°C, 210 rpm, and testing optical density value per hour (595 nm), drawing the bacteriostasis curve, as shown in figure 3.

The bacteriostatic curve shows that the recombinant proteins produced by improved methods of this ion have good cterial activity

Claims (10)

1. A method for highly expressing a recombinant n in recombinant ia, wherein the recombinant protein comprises a hilic and a hydrophobic end, wherein the hydrophilic end is a n polypeptide and wherein the hydrophobic end is a polypeptide target moiety which is capable of binding a target, the method comprising: (1) transfecting a inant plasmid expressing the recombinant protein into E. coli bacteria with pET system to obtain positive monoclonal colonies and 10 produce recombinant E. coli bacteria, (2) producing seed bacteria solution from the positive monoclonal colonies, and inducing protein expression and enlargement culturing of the seed bacteria on in enlargement culturing medium; n the supernatant of the enlargement cultured solution contains the expressed recombinant protein, 15 (3) extracting and purifying the recombinant protein from the supernatant, wherein the recombinant E. coli bacteria with pET system is E.coli B834 (DE3), and wherein the enlargement culturing medium has water as solvent and comprises the following components: NaCl 6.0~6.7 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 0.6~2.0 g/L, NazHPO4-7H20 6.8~18.3 g/L, KH2P04 20 3.0~4.3 g/L, NH4C1 4 g/L, MgSO4 0.2~O.4 g/L, CaC120.Ol g/L, and methionine O~4O mg/L.

2. The method according to claim 1, wherein the enlargement culturing medium has water as solvent and comprises the folloWing components: NaCl 6.0 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 2.0 g/L, NazHPO4-7HZO 6.8 g/L, KH2P04 3.0 g/L, NH4C1 1.0 g/L, MgSO4 0.2 g/L, .Ol g/L, and methionine 0~40 mg/L.

3. The method according to claim 1 or claim 2, wherein said enlargement culturing of the seed bacteria solution comprises the following steps: adding the seed bacteria liquid into a ner and culturing for 2 to 3 hours at 30°C, when the OD value reaches 0.4—0.6 heat shocking the solution for 42°C for 30 s, and then cooling the solution to 37°C and culturing for a further 1.5 to 10 2 hours.

4. The method according to claim 3, characterized in that, IPTG with a final tration of 0.5 mmol/L is added when the solution is at 42°C.

5. The method according to any one of claims 1 to 4, n said extracting and purifying of the recombinant protein from the supernatant ses use of 15 a CM ion exchange column, wherein the loading quantity of the supernatant samples depends on the ratio value which is 2.5mg/ml between the weight of the recombinant protein in the supernatant and the volume of Gel particles used in the CM ion exchange column.

6. The method according to claim 5, wherein the eluent solution used for said 20 extracting and purifying in CM ion exchange column is boric acid buffer solution with 0.2 mol/L NaCl.

7. The method according to any one of claims 1 to 6, wherein the recombinant plasmid expressing the recombinant protein is selected from the group consisting of pBHC—SAl, pBHC—SAZ, pBHC—SA3, A4, pBHC—SE, A and pBHC—P or A1.

8. The method according to any one of Claims 1 to 7 wherein the recombinant protein is PMC—SAI, PMC-SAZ, PMC-SA3, PMC—SA4, PMC—SE, PMC—PA or

9. A medium for recombinant Ecoli bacteria with pET system, wherein the medium has water as solvent and ses the ing components: NaCl 6.0~6.7 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 0.6~2.0 g/L, 10 NazHPO4-7H20 6.8~18.3 g/L, KHQPO4 3.0~4.3 g/L, NH4C1 l.0~l.4 g/L, MgSO4 0.2~0.4 g/L, CaClz 0.01 g/L, and methionine 0~40 mg/L.

10. The medium according to claim 9, wherein said recombinant Ecoli bacteria with pET system is Ecoli B834 (DEB), and the medium has water as solvent and comprises the following components: NaCl 6.0 g/L, peptone 25.0 g/L, yeast 15 powder 7.5 g/L, glucose 2.0 g/L, NazHPO4.7H20 6.8 g/L, KH2P04 3.0 g/L, NH4C1 1.0 g/L, MgSO4 0.2 g/L, CaClz 0.01 g/L, and methionine 0~40 mg/L.