CN1313612C

CN1313612C - Method for producing recombinant human granulocyte colony stimulating factor

Info

Publication number: CN1313612C
Application number: CNB2005100453888A
Authority: CN
Inventors: 陈文芳; 宋磊; 刘红军
Original assignee: QUANGANG MEDICINE CO Ltd SHANDONG PROV
Current assignee: QUANGANG MEDICINE CO Ltd SHANDONG PROV
Priority date: 2005-12-20
Filing date: 2005-12-20
Publication date: 2007-05-02
Anticipated expiration: 2025-12-20
Also published as: CN1814779A

Abstract

The present invention discloses a method for producing recombinant human granulocyte colony stimulating factors, which relates to the field of biological techniques. The production technology comprises the steps of fermentation, breaking of bacterium, extraction of inclusion bodies, molecular sieve chromatography, recoverability, anion exchange and cation exchange to obtain stock solution. BL21 serves as a host bacterium, and an engineering bacterium strain which has the characteristics of fast reproduction speed, stable passage and high expression quantity can be obtained. The bacterial endotoxin residual quantity level of the stock solution is obviously reduced by a plurality of purification procedures, and the specific activity is enhanced in a large range. The inclusion bodies are firstly and preliminarily purified, and then, are recovered by a dialysis method. The concentration of denaturant is slowly reduced, and the recoverability efficiency is kept at a high level. The method of the present invention can obtain the requirements of stability, high efficiency and mass production.

Description

A kind of production method of recombinant methionyl human G-CSF

Technical field

The present invention relates to biological technical field, relate in particular to the production method of recombinant methionyl human G-CSF.

Background technology

Granulocyte colony-stimulating factor (granulocyte colony-stimulatingfactor, G-CSF) having the promotion grain is hemopoietic stem cell proliferation differentiation and the effect that strengthens ripe granulocyte function, and the neutrocyte deficiency disease that serious neutrophil leucocyte deficiency disease that causes when promoting neutrophilic leukocyte increase, cancer chemotherapy during for bone marrow transplantation and aplastic anemia are followed all has obvious curative effects.

The biologic activity of genetically engineered reorganization G-CSF can be mass-produced, to meet clinical needs to natural similar.Escherichia coli expression foreign protein in the existing production method, most forms with insoluble inclusion body exist, and inclusion body is not have biologic activity, needs in process of production to handle through sex change, renaturation, recovers its biologic activity.Ubiquitous problem is that renaturation yield is low, and the production technique majority of bibliographical information is with the direct renaturation of inclusion body, and renaturation yield is about 20%.

Summary of the invention

In order to overcome the low and high defective of production cost of recombinant methionyl human G-CSF renaturation yield that existing production method exists, the invention provides a kind of production method of recombinant methionyl human G-CSF.

The production method of a kind of recombinant methionyl human G-CSF of the present invention is characterized in that operation steps is followed successively by: fermentation, broken bacterium and extraction inclusion body, sieve chromatography, renaturation, anionresin, cationic exchange, stoste.

Select the host bacterium of BL21 as expression G-CSF for use: the G-CSF gene adopts chemical synthesis, according to its gene order, the codon of coding N end several amino acid is changed into the codon of intestinal bacteria preference, be reconstituted among the expression vector PBV220 after full gene is synthetic, transform dissimilar intestinal bacteria, its expression amount and mitotic stability are investigated, filtered out the stable bacterial classification that efficiently expresses and go down to posterity, as the bacterial classification of engineering bacteria.The engineering bacteria that recombinant methionyl human G-CSF (rhG-CSF) production is used etc. selects for use e. coli bl21 to do the host bacterium, and recombinant plasmid PBV220-G-CSF goes down to posterity stable therein and can realize efficiently expressing.The most outstanding characteristics are to heat up in the fermenting process to induce the back thalline still can continue breeding, and its cell density is further improved.By relatively find G-CSF in BL21, go down to posterity stable, expression amount is high and growth and breeding is very fast.Table 1 is for expressing the comparison of G-CSF with DH5 α and BL21.

Two kinds of host bacterium of table 1. are expressed the comparison of G-CSF

The host bacterium	Cell density OD600 during end	The wet thallus amount (g/L) of every liter of nutrient solution gained	The wet thallus amount mg of gained behind the broken bacterium of every gram wet thallus
The host bacterium	Cell density OD600 during end			DH5α	6.3±1.0	8.2±1.6	43±6
BL21	13.2±1.5	12.8±1.8	55±8	DH5α	6.3±1.0	8.2±1.6	43±6

During the fermentation the engineering bacteria bacterial classification inoculation in shake the bottle the LB liquid nutrient medium in 30 ℃ of shaking culture, amplification obtains primary seed solution, obtain secondary seed solution by 1% transferred species enlarged culturing again, then by 10% transferred species in fermentor tank in the sterilized LB+M9 substratum, 30 ℃ of cultivations, and by air flow and stirring velocity control dissolved oxygen, use the adjusting PH with base value, suitably feed supplement further increases bacterium to OD ₆₀₀When 2.5-3.0, be warming up to 42 ℃ of thermal inductions, induce 4h to express target protein.By the control of fermentation parameter, make bacterial growth vigorous, and in logarithmic phase, heat up and induce, realize that high-density efficiently expresses.

G-CSF in intestinal bacteria with the formal representation of insoluble inclusion body, behind the centrifugal collection thalline, break bacterium, extract inclusion body with N,O-Diacetylmuramidase and ultransonic method, and wash, the inclusion body urea denaturing treatment of 8mol/L, inclusion body rhG-CSF purity after the dissolving can reach more than 70%, and by the extraction and the washing of inclusion body, G-CSF has obtained preliminary purification.

In order to improve the renaturation effect, before renaturation, be further purified with sieve chromatography, removed part macromole impurity.

RhG-CSF is that form with insoluble inclusion body exists at the prokaryotic cell prokaryocyte expression in escherichia coli, needs sex change, renaturation to handle and recovers its biologic activity.Renaturation adopts the refolding method of dialysis, slowly reduces the concentration of denaturing agent, and adds G-SH/GSSG, promotes the formation of disulfide linkage, makes metaprotein recover its space conformation, has improved renaturation yield.

Be further purified by anion-exchange chromatography, remove remaining impurities and polymer, and renaturation solution directly goes up the anionresin column purification, do not need to concentrate.

Purifying process is on the basis of sieve chromatography and anion-exchange chromatography two-step purifying, further cationic exchange, make the bacterial endotoxin residual quantity level of stoste obviously reduce, raising is by a relatively large margin arranged than living, can obtain high purity, height ratio G-CSF stoste alive.Present single band on the stoste SDS-PAGE purity check behind the purifying, the HPLC purity check presents simple spike, bacterial endotoxin by＜3EU/300 μ g (national standard 10EU/300 μ g) standard inspection is still qualified, tropina residual quantity＜0.01% (national standard is 0.1%).Ratio work behind the anion-exchange column is 1.1 * 10 ⁸U/mg, the ratio work behind the cationic exchange coloum reaches 1.5 * 10 ⁸U/mg.Total yield from the inclusion body to stoste is more than 20%.

Description of drawings

Expression amount when inducing different time in Fig. 1 fermenting process

1 molecular weight Marker (molecular weight is 94,67,43,30,21 respectively, 14.4KD, and following Marker is identical)

2,3,4,5 is respectively the expression amount of 42 ℃ of G-CSF when inducing 1h, 2h, 3h, 4h

The sieve chromatography curve of Fig. 2 rhG-CSF

The SDS-PAGE analytical results of the sieve chromatography of Fig. 3 .rhG-CSF

The decent at 6 second peaks of ascending branch at the 2nd peak, first peak 5 behind preceding 3 purifying of 1 molecular weight Marker, 2 purifying in the 4 chromatography curves

Fig. 4 DEAE-Sepharose FF ion exchange chromatography curve and SDS-PAGE analytical results

1. go up the sample peak; 2.0.1mol/L NaCl elution peak; 3.1mol/L NaCl elution peak; 4. molecular weight marker

Fig. 5 .CM-Sepharose FF ion exchange chromatography curve

SDS-PAGE in Fig. 6 .rhG-CSF purge process analyzes

5 molecular weight Marker behind the 4 cation seperation column purifying behind the 3 anion column purifying after 1 inclusion body, 2 molecular sieve purification

The SDS-PAGE purity detecting result of Fig. 7 .rhG-CSF stoste

Embodiment

Embodiment 1

The fermentation: with the engineering bacteria bacterial classification inoculation in the LA flat board, 30 ℃ of cultivations, grow up to bacterium colony after, picking list colony inoculation is in the 10mlLA liquid medium, 30 ℃ of shaking culture obtain primary seed solution, and in the LA liquid nutrient medium, shaking culture obtains secondary seed solution by 1% transferred species.

The preparation fermention medium, can high voltage bearing part add the sterilization of fermentor tank mesohigh, with not high voltage bearing composition filtration sterilization, add into after waiting the liquid cooling in the fermentor tank, and the seed liquor that will newly cultivate is inoculated in the fermentor tank in 10% ratio and ferments.The control culture temperature be 30 ℃, with the ammoniacal liquor adjust pH make pH be stabilized in 6.8 and 7.4 between, by regulating air flow and rotating speed control dissolved oxygen＞70%, and suitable feed supplement.Increase bacterium to OD ₆₀₀When 2.5-3.0, elevated temperature to 42 ℃ carries out thermal induction, and the expression amount when inducing different time is seen Fig. 1.Induce 4h to finish fermentation, centrifugal collection thalline ,-70 ℃ are frozen standby.Every liter can get more than the wet thallus 10g, and expression amount reaches 50%.

Embodiment 2

Broken bacterium, washing inclusion body: wet thallus with the ratio suspension thalline of TE (50mmol/LTris-Cl, 5mmol/LEDTA) in 1: 10 (g/ml), is added N,O-Diacetylmuramidase to final concentration 1mg/ml, place 4h for 4 ℃, ultrasonication in the ice bath, 6min/ time, a 3min that has a rest, totally 7 times.4 ℃, the centrifugal 15min of 8500rpm abandon supernatant, and precipitation is resuspended in the TE damping fluid, repeat said process twice, and precipitation is washed two times with the 2mol/L urea.

Precipitation is used the 8mmol/L urea, 2mmol/L EDTA, and 1mmol/L DTT, the liquid dissolving of 50mmol/LTris-HCl pH 8.0,4 ℃, the centrifugal 15min of 8500rpm, supernatant is the inclusion body dissolution fluid.

Embodiment 3

Sieve chromatography: get above-mentioned inclusion body dissolution fluid, add DTT, 30 ℃ of water-bath 30min upper props to final concentration 5mmol/L, moving phase is the 8mmol/L urea, 2mmol/L EDTA, 1mmol/LDTT, 50mmol/L Tris-HCl pH 8.0, the about 0.7ml/min of flow velocity, fraction collection elution peak.The chromatography curve is seen Fig. 2, and target protein is arranged in second peak, through molecular sieve purification, can remove the foreign protein of molecular weight greater than 30KD, sees Fig. 3.

Embodiment 4

Renaturation: the 8mmol/L urea of the sample behind the preliminary purification, 1mmol/L EDTA, 1mmol/LDTT, 50mmol/L Tris-HCl pH 8.0 is diluted to 0D280=0.25, is loaded in the dialysis tubing, to 4mmol/L urea, the 20mmol/L Tris-HCl pH8.0 of 4 times of volumes, the 4mmol/LG-SH/1mmol/LGSSG liquid of dialysing, change 1mmol/L urea, 20mmol/L Tris-HCL, pH8.0, dialysis 24h, change the 20mmol/LTris-HCl of same volume, more than the pH8.0 dialysis 8h.With sample solution via hole diameter 0.45um membrane filtration, DEAE chromatography column in the preparation.

Embodiment 5

DEAE-SepharoseFF column purification: level pad: 20mmol/L Tris-HCl pH8.0, target protein under the salt concn of 0.1mol/L NaCl by wash-out, dense metaprotein and the foreign protein of taking off of 1mol/L NaCl, chromatography curve and SDS-PAGE see Fig. 4.

Embodiment 6

CM-SephroseFF cationic exchange column purification:

Level pad: 20mmol/L NaAc-HCL PH4.0

Wash post with the 20mM NaAc-HAc PH4.0 that contains 0.3M NaCL earlier, see Fig. 5 with the 20mM NaAc-HAc pH4.0 buffer solution elution target protein that contains 0.45M NaCL again.

Embodiment 7

G-CSF purity check: adopt SDS-PAGE and two kinds of methods of HPLC to carry out, the results are shown in Figure 6, Fig. 7, present single band and simple spike respectively.

Claims

1. the production method of a recombinant methionyl human G-CSF is characterized in that operation steps is followed successively by: fermentation, broken bacterium and extraction inclusion body, sieve chromatography, renaturation, anionresin, cationic exchange, stoste; Described bacterial strain selects for use BL21 as the host bacterium of expressing G-CSF; In the described fermenting process, the engineering bacteria bacterial classification inoculation is 30 ℃ of shaking culture in the LB liquid nutrient medium that shakes bottle, amplification obtains primary seed solution, obtain secondary seed solution by 1% transferred species enlarged culturing again, then by 10% transferred species in fermentor tank in the sterilized LB+M9 substratum, 30 ℃ of cultivations, and by air flow and stirring velocity control dissolved oxygen, use the adjusting PH with base value, suitably feed supplement further increases bacterium to OD ₆₀₀When 2.5-3.0, be warming up to 42 ℃ of thermal inductions, induce 4h to express target protein, by the control of fermentation parameter, make bacterial growth vigorous, and in logarithmic phase, heat up and induce; Adopt the broken bacterium of N,O-Diacetylmuramidase and ultransonic method in the step of described broken bacterium and extraction inclusion body, extract inclusion body, and wash, the inclusion body urea denaturing treatment of 8mol/L; Adopt the method for dialysis in the described renaturation step, slowly reduce the concentration of denaturing agent, and add G-SH/GSSG, promote the formation of disulfide linkage.

2. the production method of recombinant methionyl human G-CSF according to claim 1 is characterized in that anionresin adopts the DEAE-SepharoseFF column purification.

3. the production method of recombinant methionyl human G-CSF according to claim 1 is characterized in that cationic exchange adopts CM-SephroseFF cationic exchange column purification.