TWI541350B - A Method for Highly Efficient Expression of Recombinant Protein from Engineering Bacteria and Its Application - Google Patents

Description

Method for efficiently expressing recombinant protein of engineering bacteria and application thereof

The invention relates to the field of biotechnology, in particular to a method for efficiently expressing recombinant protein of engineering bacteria and an application thereof.

In the previous research, the inventors of the present invention obtained a series of novel types of recognition polypeptides (naturally or artificially designed) which can be operatively linked to other target cells by means of co-experimental and experimental verification. Recombinant polypeptide, such as the application number 200910092128.4, the novel antibiotic PMC-AM1 disclosed in the article "A new antibiotic containing antibody mimics and its preparation method and application" has broad-spectrum antibiotic properties, against meningococcus, multiple resistance Pseudomonas aeruginosa, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus have obvious antibacterial effects compared with existing antibiotics; application number is 200910157564.5, the name is "a new antibiotic and its nucleotide sequence, preparation method A series of new antibiotics against staphylococcus, such as PMC-SA1, PMC-SA2, PMC-SA3, PMC-SA4, PMC-SE and PMC-PA, are disclosed in the application. In vitro and in vivo tests, it shows superior targeting and killing efficiency compared to existing antibiotics, antifungal antibiotics, and chemotherapeutic drugs. These new antibiotics not only have obvious antibacterial effects, but also have biosafety comparable to other existing antibiotics. Resistance to resistance.

Since the above series of recombinant proteins are a class of water-soluble proteins, generally It consists of more than 600 amino acid residues, but has a hydrophobic segment of 40 amino acid residues near the carboxy terminus in the peptide chain of this protein. Compared with other single water-soluble proteins, the assembly and expression of the protein is more difficult, which inevitably affects its productivity, improves the expression process of the recombinant protein, and increases the expression rate of the protein, thereby solving the large-scale problem. Production has the technical problem of pushing the above series of recombinant proteins into practical clinical applications and practices.

The present invention is directed to a structure and a property characteristic of a recombinant polypeptide disclosed by the inventors in the prior patent application, and provides a method for efficiently expressing an engineered protein.

A method for efficiently expressing an engineered recombinant protein, wherein the polypeptide of the engineered recombinant protein has hydrophilicity at one end and is a polypeptide end of the colicin; the other end is hydrophobic, and the polypeptide end is recognized as a target substance, and is characterized in that it includes the following Step: (1) transfecting the recombinant mutant plasmid expressing the recombinant protein of the engineered bacteria into the E. coli pET system engineering bacteria to obtain a positive monoclonal, (2) enriching the positive monoclonal to obtain a seed bacterial liquid, and inducing the seed bacterial liquid A large amount of bacteria is grown, the obtained supernatant contains the expressed recombinant protein of the engineered bacteria, and (3) the recombinant protein of the engineered bacteria contained in the supernatant is separated and purified, The E. coli pET system engineered bacteria refers to E. coli B834 (DE3).

The medium used for the enrichment is: NaCl 6.0~6.7g/L, peptone 25.0g/L, yeast powder 7.5g/L, glucose 0.6~2.0g/L, Na ₂ HPO ₄ . 7H ₂ O 6.8~18.3g/L, KH ₂ PO ₄ 3.0~4.3g/L, NH ₄ Cl 1.0~1.4g/L, MgSO ₄ 0.2~0.4g/L, CaCl ₂ 0.01g/L, methylthioamide Acid 0~40mg/L, with water as solvent.

The medium used for the enrichment is: NaCl 6.0g/L, peptone 25.0g/L, yeast powder 7.5g/L, glucose 2.0g/L, Na ₂ HPO ₄ . H ₂ O 6.8g / L, KH ₂ PO ₄ 3.0g / L, NH ₄ Cl 1.0g / L, MgSO ₄ 0.2g / L, CaCl ₂ 0.01g / L, methionine 0 ~ 40mg / L, Water is the solvent. In the process of using E. coli B834 (DE3) as an engineered bacteria, methionine was 40 mg/L.

The induction is induced by thermal shock. The operation mode is as follows: after the seed bacteria liquid is put into the tank, the growth is started at 30 ° C for 2 to 3 hours, and when the OD value is 0.4-0.6, the thermal shock is performed at 42 ° C for 30 minutes, and then the temperature is lowered. To 37 ° C, the regrowth was 1.5-2 hours after the collection.

In the thermal shock induction step, PTG having a final concentration of 0.5 mM was added to the medium.

The recombinant protein in the purified cell supernatant was subjected to a CM ion exchange column, wherein the amount of the supernatant was 2.5 mg/ml of the supernatant protein amount/gel particle volume.

The recombinant protein in the supernatant of the purified cell was subjected to a CM ion exchange column, and the concentration of NaCl in the boric acid buffer used for elution was 0.2M.

The recombinant mutant plasmid refers to pBHC-SA1, pBHC-SA2, pBHC-SA3 pBHC-SA4, pBHC-SE, pBHC-PA or pBHC-PorA1.

Use of any of the above methods in the preparation of recombinant polypeptides PMC-SA1, PMC-SA2, PMC-SA3, PMC-SA4, PMC-SE, PMC-PA or PMC-AM.

A medium for engineering bacteria of Escherichia coli pET system, the formula is NaCl 6.0~6.7g/L, peptone 25.0g/L, yeast powder 7.5g/L, glucose 0.6~2.0g/L, Na ₂ HPO ₄ . 7H ₂ O 6.8~18.3g/L, KH ₂ PO4 3.0~4.3g/L, NH ₄ Cl 1.0~1.4g/L, MgSO ₄ 0.2~0.4g/L, CaCl ₂ 0.01g/L, methionine 0~40mg/L, using water as solvent.

The Escherichia coli pET system engineering bacteria is E. coli B834 (DE3), and the formulation of the medium is: NaCl 6.0 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 2.0 g/L, NaHPO ₄ . 7H ₂ O 6.8g / L, KH ₂ PO 43.0g / L, NH ₄ Cl 1.0g / L, MgSO ₄ 0.2g / L, CaCl ₂ 0.01g / L, methionine 40mg / L, with water as solvent .

Novagen's pET system is a popular system for the current cloning and expression of recombinant proteins in E. coli. In the present invention, the recombinant mutant plasmid is first transfected into a series of BL-21 (DE3) cells, and the original TG1 is obtained. Better protein expression yield in cells. From experimental data comparison, we found that the parent strain B834 (DE3) as BL-21 (DE3) has a more desirable expression yield than BL-21 (DE3). The experimental data confirmed that plasmid B834 (DE3) can achieve a protein expression yield that is ten times higher than the body plasmid TG1 body coefficient.

The culture medium provides the carbon source, nitrogen source and inorganic salt required for bacterial growth and reproduction, and the invention improves the growth rate of the engineering bacteria by improving the composition of the culture medium, thereby improving the expression yield of the target protein. By gradually improving and adjusting the content of each component in the medium, the optimal medium formula for the fermentation production of the engineered bacteria was found. In the present invention, our improved FB-M9 complex medium moderately increases the content of carbon source and nitrogen source, and adds the amino acids required for the growth of MgSO ₄ , CaCl ₂ and pET system bacteria, and moderately increases the bacterial growth rate and protein expression. effectiveness. The cost of the improved medium formulation is relatively low, which provides a large research space and development value for large-scale production in the future.

The carrying rate of CM ion gel particles in the purification system used in the present invention does not reach the ideal level described in the product manual, which limits the recovery rate of the target protein, and the present invention reduces the amount of sample and moderately increases the gel. The volume and other means have resulted in a significant improvement in recovery. This result suggests that there is still a need to find more efficient ion exchange gels for more efficient large scale industrial production. In addition, the present invention optimizes the concentration of the eluate in the ion exchange step to make the resulting recombinant protein impurities more less.

In summary, the present invention provides a plurality of alternative and more optimized processes for expressing recombinant proteins of Escherichia coli engineering bacteria by selecting an ideal engineered strain, optimizing medium composition, improving purification, and recovering efficiency, which is also found in the end. Efficient expression of the optimal combination of the desired fusion protein provides a possible development direction and technical route. Compared with the original expression system, the expression system developed by the Institute increased the expression and yield of the fusion protein by several tens of times, which provided a useful theoretical and practical basis for the subsequent large-scale industrial production.

The embodiments of the present invention are described by way of specific examples, and those skilled in the art can readily appreciate the other advantages and advantages of the present invention.

The embodiments of the present invention are described below with reference to the drawings, and the following drawings are simplified schematic diagrams, and only the basic concept of the present invention is illustrated in a schematic manner, and only the structures related to the present invention are illustrated in the drawings. Rather than drawing according to the number, shape and size of the components in actual implementation, the types, quantities and proportions of the components in actual implementation are not limited to the illustrations, and can be changed according to the actual design requirements.

The invention is illustrated by the following examples. The experimental materials and instruments used in the present invention are as follows:

1. Strain:

E.coli TG1 engineering bacteria (AECOM, K.Jakes gift); E.coli BL-21 (DE3), B834 (DE3), NovaBlue (DE3) and 618 engineering bacteria were purchased from Novagen; Staphylococcus aureus ATCC BAA -42 was purchased from ATCC (American Type Culture Collection).

Plasmids: pBHC-SA1, pBHC-SA2, pBHC-SA3, pBHC-SA4, pBHC-SE, pBHC-PA, pBHC-PorA1 (described in the specification of two invention applications with application numbers 200910092128.4, and 200910157564.5)

The above biomaterials are known before the application date, and can be easily obtained by a person skilled in the art according to the prior art (commercially purchased or prepared), and the applicant's laboratory is also preserved, and can be distributed to the public within 20 years from the filing date. In the verification test).

2. Main reagents and drugs:

Yeast powder (OXIOD LP0021), peptone (OXIOD LP0042), and other chemical reagents are domestically analyzed pure; dialysis bag Snake Skin Dialysis Tubing (Pierce, molecular weight cutoff 1×104, Lot#KD32324); Streptomyces sulfate injection (North China Pharmaceutical); ampicillin sodium AMP for injection (Harbin Pharmaceutical); anion exchange column gel (Pharmacia Biotech CM) Sepharose Fast Flow Lot No. 225016).

LB liquid medium: 100 ml of sodium chloride 1 g, peptone 1 g, yeast 0.5 g, weighed the various reagents into a 250 ml flask, added 100 ml of tap water, dissolved it, and sterilized in an autoclave at 120 ° C for 8 min.

LB solid medium: 100ml sodium chloride 0.5~1.5g, peptone 0.5~2g, yeast 0.3~1g, agar 0.8~3g, LB solid medium is used for culturing monoclonal colonies after strain recovery. The various reagents were weighed and placed in a 250 ml flask, 100 ml of tap water was added, dissolved, and sterilized in an autoclave at 120 ° C for 8 min.

FB-M9 complex medium: NaCl 6.0~6.7g/L, peptone 25.0g/L, yeast powder 7.5g/L, glucose 0.6~2.0g/L, Na ₂ HPO ₄ . 7H ₂ O 6.8~18.3g/L, KH ₂ PO ₄ 3.0~4.3g/L, NH ₄ Cl 1.0~1.4g/L, MgSO ₄ 0.2~0.4g/L, CaCl ₂ 0.01g/L, methylthioamide Acid 0~40mg/L.

Modified FB-M9 complex medium: NaCl 6.0g/L, peptone 25.0g/L, yeast powder 7.5g/L, glucose 2.0g/L, Na ₂ HPO ₄ . 7H ₂ O 6.8 g / L, KH ₂ PO ₄ 3.0 g / L, NH ₄ Cl 1.0 g / L, MgSO ₄ 0.2 g / L, CaCl ₂ 0.01 g / L, methionine 0 ~ 40 mg / L. In the process of using E.coli B834 (DE3) as the engineering bacteria, the methionine is 40mg/L.

3. Main instruments:

Bio-Rad Protein Chromatography Purification System (BioLogic Duo Flow, BioLogic Maximizer, BioLogic QuadTec UV-Vis Detector, BioLogic Econo Pump); Sonicrep 150, 5 cm diameter purified ion exchange column (Pharmacia Biotech XK50), 11 cm diameter purified ion exchange column (Shanghai Huamei); centrifuge (Beckman Coulter Avanti J-20XP, Beckman Coulter Avanti J-25); Bio-Rad Smart Spect Plus spectrophotometer; fully automatic fermenter (Switzerland LP351-42L); bacterial high pressure homogenizer (Niro Soavi, Italy) NS1001L2KSN 6564).

Disclaimer: The biological materials used in the present invention are known before the application date, and the unit has been preserved, and the verification test is issued to the public within 20 years from the filing date.

Example 1. Optimized selection test of engineering strains

The classical plasmid carrying the colicin Ia and its immune protein gene (GenBank M13819) was obtained from the Dr. Finkelstein laboratory (Qiu XQ et al. An engineered multidomain bactericidal peptide as a model for targeted antibiotics against specific bacteria. Nat Biotechnol, 2003; 21(12): 1480-1485), by Seven recombinant mutant plasmids were obtained after transformation in this laboratory: pBHC-SA1, pBHC-SA2, pBHC-SA3 pBHC-SA4, pBHC-SE, pBHC-PA, pBHC-PorA1.

Step 1. Transform competent cells

Recombinant mutant plasmid pBHC-SA1 100ng was transfected into 4040ul Novagen several pET system engineering bacteria BL-21 (DE3), B834 (DE3), Nova Blue (DE3), 618, ice incubation for 5 minutes, thermal shock at 42 °C After 30 seconds, put it in ice for 2 minutes, add SOC medium 160 ul, 220 rpm, shake at 37 ° C for 1 hour, then plate (LB medium plus 1% agar, add 50 ug / ml ampicillin, 37 ° C overnight), pick a single Colonization enrichment, obtaining strains, and cryopreserving strains;

Step 2. Strains recovery

Strain recovery

The prepared strain was thawed at 4 ° C, and 1.5 ml was poured into 10 ml of LB medium (containing AMP 50 μg/ml), and cultured at 220 rpm and 37 ° C for 5 to 8 hours.

2. Inoculation of monoclonal bacteria

The bacterial solution after the resuscitation was diluted 104 or 105 times, and 10 ul of the diluted bacterial solution was added to the prepared LB solid medium (AMP 50 μg/ml) plate, and plated. In a humidification box, culture at 37 ° C in a constant temperature incubator After 10 to 12 hours, a single round colony grows on the surface of the medium.

Step 3. Pick and expand bacteria

(1) Select a regular round, smooth-edged single colony from a long plate with a sterilized toothpick or a ring to be placed in 1.5 ml of LB medium, and shake culture at 220 rpm and 37 ° C for 5-8 hours.

(2) 1.5 ml of LB bacterial solution was turned into 100 ml of LB medium, and cultured by shaking: 220 rpm, and cultured at 37 ° C for 5 to 8 hours.

(3) One-stage expansion culture: 100 ml of the above-mentioned bacterial liquid was added to 700 ml of the modified FB-M9 complex medium, and cultured by shaking: 220 rpm, 37 ° C for 5 to 8 hours.

(4) Two-stage expansion culture: 700 ml of the above-mentioned bacterial liquid was separately added to 6×700 ml of modified FB-M9 complex medium, and cultured by shaking: 220 rpm, 37° C. for 5 to 8 hours.

(5) Three-stage expansion culture: the above 6×700ml bacterial solution was added to 20L modified FB-M9 complex medium, and cultured in a fermenter: stirring speed 220 rpm, maximum oxygen permeation, culture at 37 ° C for 3 to 5 hours .

(6) Engineering bacteria fermentation and induced expression of protein: The above step 20L bacterial solution was added to 200L modified FB-M9 complex medium, and the protein was cultured and induced in the fermenter: stirring speed 220 rpm, maximum oxygen permeation, 30 ° C 2~4 hours; 42 °C, 0.5 hours; 37 °C, 1~2 hours, Note: IPTG with a final concentration of 0.5 mM was added at 42 °C.

Step 4, centrifugation

The culture solution was 6000 g and centrifuged at 4 ° C for 20 min. The precipitate after centrifugation was collected and placed in 50 mM boric acid buffer (pH 9.0) to suspend the bacterial body weight in boric acid buffer. Note: 2 mM PMSF was added to the boric acid buffer (Chinese name: phenylmethylsulfonyl fluoride) The serine protease inhibitor, after the suspension of the bacterial body weight, must be carried out at 4 ° C.)

Step 5, broken bacteria

After the cells were completely suspended in the pH 9.0 borate buffer solution, the cells were disrupted by high-pressure homogenizer 500-600 bar, and the cells were repeatedly broken 7 times, each time between 3 and 5 minutes.

Step 6. Precipitating bacterial DNA

The crushed bacterial solution was centrifuged at 55,000 g for 40 min at 4 °C. The supernatant was taken, streptomycin sulfate was added (16 bottles of 1 million units of streptomycin sulfate per 200 ml of liquid), and stirred on a magnetic stirrer for 1 hour.

Step 7, dialysis

55000 g of the above-mentioned bacterial solution was centrifuged at 4 ° C for 20 min, and the supernatant was taken, placed in a dialysis bag, dialyzed in boric acid buffer for 8 to 12 hours, and dialyzed every 4 hours.

Step 8. Purification of protein drugs to obtain antibacterial engineering polypeptide drugs

55,000 g of the lysate solution was centrifuged at 4 ° C for 20 min, and the supernatant was taken in a beaker, and the protein was purified by ion exchange. Take the supernatant and apply it to the CM ion exchange column, measure the protein concentration and calculate the protein content per unit volume. The ratio of the sample loading to the CM ion gel particles is as follows. After sufficient washing, the new antibacterial engineering was obtained by eluting with 50 mM boric acid buffer containing 0.2 M NaCl.

The results are shown in Table 1. E. coli B834 (DE3) has the highest expression efficiency for PMC-SA.

The same operation and comparison were performed for the other six recombinant mutant plasmids, and the results showed the same trend as the results shown in Table 1, that is, compared with the remaining engineering bacteria, E. coli B834 (DE3) for the seven recombinant mutant plasmids. The expression efficiency is the highest.

In this embodiment, the growth induction method different from the past is thermal shock induction: that is, after the seed bacteria liquid is put into the tank, the initial temperature of the large-scale enrichment is 30 ° C, the growth of the liquid is about 2 hours, and the OD value is reached. 0.4~0.6, thermal shock at 42 °C for 30 minutes, after the end of thermal shock, the temperature is lowered to 37 °C, and the regrowth can be collected after 1.5~2 hours. At this time, the OD value of the proliferating bacteria solution can reach 1~3 or even more. high. IPTG was also added to induce the pET series of engineered bacteria in an amount of 0.5 mM.

Prior to the present application, a conventional method for producing the above recombinant polypeptide is as follows: 100 ng of the mutant plasmid and 40 ul of the prepared BL-21 engineered competent cells are incubated for 5 minutes, heat shocked at 42 ° C for 30 seconds, and then placed in ice. 2 minutes, add SOC broth 160ul, 220rpm, shake at 37 °C for 1 hour, then plate (LB PBS plus 1% agar, add 50ug / ml ampicillin, 37 ° C overnight), pick a large number of colonies to increase bacteria; Enrichment: 8~10 liters of FB medium, 250 rpm, 37 ° C, 3-4 hours; add IPTG, 250 rpm, 28 ° C regrowth for 4 hours; 4 ° C, 6000 g, 20 minutes to pellet the cells, take 4 ° C, 50 mM boric acid Buffer (pH 9.0, 2mM EDTA) 80-100ml suspension cells, after adding 50SF of PMSF, sonicate the cells (4°C, 400W, 1 minute, repeat 4~5 times, intermittent 2~3 minutes to ensure the temperature of the bacteria) The crushed cells were precipitated by high-speed centrifugation (4 ° C, 75,000 g, 90 minutes), and the supernatant was added with 5 million units of streptomycin sulfate to precipitate DNA (stirred at 4 ° C for 1 hour), 10,000 g, 4 ° C, and centrifuged for 10 minutes. The supernatant was loaded into a molecular weight 15,000 dialysis bag at 4 ° C, 10 liters of 50 mM boric acid buffer solution for dialysis overnight, and again centrifuged at 10,000 g, 4 ° C, 10 minutes. Precipitate, the supernatant was loaded onto a CM ion exchange column was thoroughly rinsed, 0.3M NaCl + 50mM borate elution buffer to obtain novel antibiotics produced.

Example 2. Improved medium

The classic colistin Ia growth medium is FB medium (Qiu XQ et al. An engineered multidomain bactericidal peptide as a model for targeted antibiotics against specific bacteria. Nat Biotechnol, 2003; 21(12): 1480-1485/Karen Jakes, Charles Abrams , Alan Finkelstein, et al. Alteration of the pH-dependent Ion Selectivity of the Colicin E1 Channel by Site-directed Mutagenesis. JBC, 1990; 265(12): 6984-6991). The formulation is as follows: 25 g/L peptone, 7.5 g/L yeast powder, 6 g/L NaCl, 1 g/L glucose.

In the present invention, we have adopted FB medium containing no glucose, and the formula is: peptone 25.0 g / L, yeast powder 7.5 g / L, NaCl 6.0 g / L, and the above-mentioned sugar-free FB culture medium and M9 culture The liquid was compounded in a predetermined volume to obtain the FB-M9 complex medium of the present invention.

The mother liquor of M9 medium is 5×M9, and its formula is: Na ₂ HPO ₄ . 7H ₂ O 64.0 g/L, KH ₂ PO ₄ 15.0 g/L, NH ₄ Cl 5.0 g/L, NaCl 2.5 g/L, MgSO ₄ 1.5 g/L, CaCl ₂ 0.05 g/L, 2% glucose.

Initially try to compound the two media: FB-M9: FB: M9 volume ratio = 7:10, the formula is: NaCl 6.7g / L, peptone 25.0g / L, yeast powder 7.5g / L, Na ₂ HPO ₄ . 7H ₂ O 18.3 g/L, KH ₂ PO ₄ 4.3 g/L, NH ₄ Cl 1.4 g/L, MgSO ₄ 0.4 g/L, CaCl ₂ 0.01 g/L, glucose 0.6 g/L.

In the present invention, the fermentation process is carried out using this formulation, and the fermentation step is the same as that in the first step of the first embodiment. The results are shown in Table 2. The weight of the wet bacteria per liter of the culture solution is significantly higher than that of the wet bacteria obtained only by the FB medium. The recovered protein yield is significantly improved, with an average yield of 30 mg/L.

The present invention further obtains the final modified FB-M9 medium by repeated comparison, and the target protein yield of 34 mg/L can be achieved under the fermentation conditions as in Example 1, as shown in Table 3.

The modified FB-M9 formula is: NaCl 6.0g / L, peptone 25.0g / L, yeast powder 7.5g / L, glucose 2.0g / L, Na ₂ HPO ₄ . 7H ₂ O 6.8g / L, KH ₂ PO ₄ 3.0g / L, NH ₄ Cl 1.0g / L, MgSO ₄ 0.2g / L, CaCl ₂ 0.01g / L, due to the need for methionine in the growth of B834 engineering bacteria Therefore, in the process of using B834 as an engineered bacteria, methionine (40 mg/L) was also added to the FB-M9 medium.

Table 3 Comparison of the yield of modified FB-M9 medium and other media

Example 3 Optimization of Protein Purification Conditions

The basic structures of the recombinant polypeptides (PMC-SA1, PMC-SA2, PMC-SA3, PMC-SA4, PMC-SE, PMC-PA, PMC-AM) prepared in the present invention are colicin Ia, and colicin Ia, etc. The electrical point is about 9.15, so the classical purification method uses the ion affinity chromatography exchange method (Qiu XQ et al. An engineered multidomain bactericidal peptide as a model for targeted antibiotics against specific bacteria. Nat Biotechnol, 2003; 21 (12): 1480-1485).

The working principle is as follows: in the boric acid buffer system of pH 9.0, most of the PMC-SA molecules exist in the form of positively charged ions, which flow through the negatively charged CM gel particles in the column. The above-mentioned recombinant protein molecules of positive charge are hung on the negatively charged CM gel particles due to the attraction between charges. Other miscellaneous proteins are washed out of the gel column.

In this embodiment, the other steps are the same as in the first embodiment, and the protein is washed. After clean, respectively borate buffer with a 0.1 ~ 0.3M NaCl gradient elution of a gel column.

Since the Na+ ion is more positively charged than the recombinant protein molecule ion, the recombinant protein ion is displaced from the CM gel particle by the Na+ ion. In the ion exchange purification, there are two variables that can be manipulated to obtain better recovery of the target protein: 1. Different concentrations of NaCl can be selected in the range of 0.05~1M, respectively, and eluted on the CM gel particles. Protein molecules with different positive charge intensities. The amount of CM gel particles used can be further optimized: in a certain ionic strength environment, the amount of protein carried by each CM gel particle is relatively constant, and in order to increase the amount of protein carried by the gel column, It is necessary to increase the volume of the gel column.

CM Sepharose Fast Flow is an anion exchange column gel produced by GE. According to its manual, it can be combined with up to 9 mM cation per 100 ml gel. The binding capacity actually available during the dynamic binding process varies with the nature of the sample, and the molecular weight is inversely related to the binding capacity. The closest to the molecular weight of the recombinant polypeptide prepared in the present invention in its standard sample is Bovine COHb-(Mr69kD). Its theoretical dynamic binding capacity is 30 mg/ml. That is, when a recombinant protein molecule sample is recovered by using a 100 ml-capacity CM Sepharose Fast Flow gel, the theoretical maximum recovery is about 300 mg (0.004 mM), which is operated according to the manual thereof, and between the CM gel particles and the recombinant protein molecule in the present invention. The actual dynamic binding capacity is 3 mg/ml, which is only 10% of the theoretical value.

In the experiment, we found that during the second half of the heteroprotein washing process, the conductance curve showed a transient small rising peak (as shown in Figure 1a). Based on this phenomenon, we speculate that when the amount of recombinant protein in the sample is large, only a part of the recombinant protein molecule in the sample can be recovered due to the limited binding capacity of the CM gel particles to the target protein. The recombinant protein that failed to mount on the CM gel particles was flushed out of the gel column along with the hybrid protein. Since the recombinant protein is positively charged, a brief rising peak of the conductance curve occurs.

In the optimization scheme of the present invention: in order to reduce the loss of recombinant protein of interest, we reduce the amount of each batch of the sample to 1/3 of that specified in the manual, and increase the volume of the gel used. The volume of the gel is increased from 150 ml to 600 ml, that is, The amount of protein in the serum: gel particle volume = 2.5 mg / ml, the amount of recombinant protein lost during the elution decreased, the experimental data showed that the recovery rate of the recombinant protein was increased by 3.5 times, the results are shown in Figure 1b.

In addition, we set the NaCl gradient in the borate buffer used for elution to 0.1M-0.2M-0.3M, with the highest elution efficiency and protein purity of 0.2M, as shown in Figure 2b.

Example 4. Protein purity and activity assay

Step 1. SDS-PAGE electrophoresis

The fusion protein sample extracted by the optimization method of Example 4 was subjected to SDS-PAGE electrophoresis, and silver nitrate staining was performed. As shown in Figure 2, in the electropherogram a, there is a clear protein at a relative molecular mass of about 70 kD. The imprint strip is the PMC-SA1 prepared in the present invention. Panel b shows that the protein received by the improved gradient elution method of Example 4 eliminated the band and increased purity. The optimized method of the present invention produced the same six recombinant proteins showing the same improvement in purity.

Step 2. Detection of antibacterial activity

The recombinant proteins PMC-SA1 and PMC-AM prepared after the preparation methods were modified by Examples 1, 2, and 3, and the following antibacterial activity tests were carried out.

Inoculate 10 ul of methicillin-resistant Staphylococcus aureus (MRSA, ATCC BAA-42) in 10 ml of BM medium (105 CFU/ml), add antibacterial drugs, and divide into 6 parallel groups according to antibacterial drugs: ampicillin sodium 2 ug/ Ml, oxacillin 4 ug/ml, wild-type colicin Ia, PMC-SA and Ph-NM (4 ug/ml), blank control group. Incubate at 37 ° C, shaking at 210 rpm, and measure the optical density value (595 nm) of the bacterial liquid every hour to draw a growth curve, as shown in Fig. 3.

The bacteriostatic test curve shows that the recombinant polypeptide prepared by the improved method of the present invention has good antibacterial activity.

While the foregoing description of the preferred embodiments of the invention, the embodiments of the invention The spirit and scope of this creative principle. Familiar with the technology of the present invention It will be appreciated by those skilled in the art that the present invention may be modified in many forms, structures, arrangements, ratios, materials, components and components. Therefore, the embodiments disclosed herein are to be considered as illustrative and not restrictive. The scope of the present invention is defined by the scope of the appended claims, and the legal equivalents thereof are not limited to the foregoing description.

Figure 1. Protein elution process for different volume gel columns Eluent conductance value a. Protein elution process of a 150 ml CM gel column. b. Protein elution process of a 600 ml CM gel column.

The curve indicated by the arrow in the figure represents the conductance value of the eluent, and the portion indicated by the arrow is the small conductance peak caused by PMC-SA lost during the loading process. When the gel volume is increased, the small area of the conductance caused by the loss of PMC-SA is reduced by 70%.

Another curve: the eluted protein OD value, Figure 2. The SDS-PAGE gel electrophoresis pattern of PMC-SA is from left to right: a. 1. Marker, 2. TG1 produces PMC-SA1, 3. BL-21 PMC-SA1 produced by PMC-SA1, 4. B834; b. 1. PMC-SA1, 3. 0.1M NaCl borate buffer eluted PMC-SA1, 3. 0.2M NaCl borate buffer eluted PMC-SA1, 4 PMC-SA1 eluted with 0.3 M NaCl borate buffer.

Figure 3. Antibacterial curve of PMC-SA against MRSA (BAA42) The ordinate is the absorbance value, the abscissa is the growth time of the bacteria Control: control group; Amp: ampicillin sodium; OXA: oxacillin; Ia-wt: wild type colicin; PMC-SA1: anti-S. aureus polypeptide; PMC -AM: Anti-meningococcal polypeptide.

Claims (6)

A method for efficiently expressing an engineered recombinant protein, wherein the polypeptide of the engineered recombinant protein has hydrophilicity at one end and is a polypeptide end of the colicin; the other end is hydrophobic, and the polypeptide end is recognized as a target substance, and is characterized in that it includes the following Step: (1) transfecting the recombinant mutant plasmid expressing the recombinant protein of the engineered bacteria into the E. coli pET system engineering bacteria to obtain a positive monoclonal, (2) enriching the positive monoclonal to obtain a seed bacterial liquid, and inducing the seed bacterial liquid A large amount of bacteria is grown, and the obtained supernatant contains the expressed recombinant protein of the engineered bacteria. The medium used for the enrichment is: NaCl 6.0~6.7g/L, peptone 25.0g/L, yeast powder 7.5g/ L, glucose 0.6~2.0g/L, Na ₂ HPO ₄ . 7H ₂ O 6.8~18.3g/L, KH ₂ PO ₄ 3.0~4.3g/L, NH ₄ Cl 1.0~1.4g/L, MgSO ₄ 0.2~0.4g/L, CaCl ₂ 0.01g/L, methylthioamide Acid 0~40mg/L, using water as solvent; (3) separating and purifying the expressed recombinant protein contained in the supernatant, the Escherichia coli pET system engineering bacteria refers to E.coli B834 (DE3); The recombinant mutant plasmid refers to pBHC-SA1, pBHC-SA2, pBHC-SA3 pBHC-SA4, pBHC-SE, pBHC-PA or pBHC-PorA1. The method according to claim 1, wherein the medium used for enriching the bacteria is: NaCl 6.0 g/L, peptone 25.0 g/L, yeast powder 7.5 g/L, glucose 2.0 g/L, Na ₂ HPO ₄ . 7H ₂ O 6.8g / L, KH ₂ PO ₄ 3.0g / L, NH ₄ Cl 1.0g / L, MgSO ₄ 0.2g / L, CaCl ₂ 0.01g / L, methionine 0 ~ 40mg / L, Water is the solvent. The method according to any one of claims 1 or 2, wherein the induction is induced by thermal shock, and the operation mode is as follows: after the seed bacteria liquid is put into the tank, the growth is started at 30 ° C for 2 to 3 hours, and the OD value is 0.4-0.6. , thermal shock at 42 ° C for 30 minutes, then the temperature is lowered to 37 °C, the regrowth is 1.5-2 hours after the collection. The method according to claim 3, wherein in the thermal shock inducing step, IPTG having a final concentration of 0.5 mM is added to the medium. The method according to claim 1, wherein the recombinant engineered recombinant protein contained in the supernatant is separated and purified, and a CM ion exchange column is used, wherein the sample loading amount is based on the amount of the supernatant protein and the gel particle volume. Determined by 2.5mg/ml. According to the method of claim 5, the recombinant engineered recombinant protein contained in the supernatant is separated and purified, and a CM ion exchange column is used, and the concentration of NaCl in the boric acid buffer used for elution is 0.2M.