CN110387394B - Method for expressing antibody Fab fragments - Google Patents

Abstract

The invention discloses a method for expressing an antibody Fab fragment. The specific method comprises activating the preserved recombinant genetic engineering Escherichia coli seed, shake-flask culturing, transferring into 5L fermentation tank, ventilating, stirring, and culturing when thallus OD is in the fermentation tank₆₀₀When about 40 days are reached, a certain amount of MgSO is added₄And cooling to 25 ℃, continuing fermentation, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction when the OD of the thalli reaches about 90, adding a supplemented medium for supplementing, and collecting fermentation liquor after fermentation is carried out for 70-90 hours. According to the invention, the expression quantity of the anti-VEGF antibody Fab fragment can be greatly improved by optimizing dissolved oxygen DO, the final concentration C of an inducer IPTG, the feed supplement speed V, the temperature T of continuous fermentation after the addition of IPTG and the like in the fermentation process, the highest expression quantity can reach 360mg/L, and the production cost of the anti-VEGF antibody Fab fragment can be greatly reduced.

Description

Method for expressing antibody Fab fragments

Technical Field

The invention relates to the technical field of protein medicine bioengineering, in particular to a fermentation process of an anti-VEGF antibody Fab fragment.

Background

Vascular endothelial growth factor(VEGF) is a protein isolated from bovine pituitary follicular cells in 1989 by Gospodalow, and when the human VEGF gene is transcribed and translated in vivo, 5 isoforms are produced due to different splicing patterns of VEGF mRNA, encoding 121, 145, 165,189 and 206 amino acids, respectively, wherein VEGF is a novel polypeptide₁₆₅Are the main effector molecules. Can specifically act on vascular endothelial cells, cause the proliferation of the vascular endothelial cells and induce the formation of blood vessels in vivo, and VEGF is the blood vessel formation promoting factor with the strongest known effect at present. VEGF plays a key role in ontogeny and in diseases such as tumors, rheumatoid arthritis, and macular degeneration.

The overexpression of VEGF is closely related to the occurrence of diseases such as tumor, exudative macular degeneration (AMD), Diabetic Retinopathy (DR), Retinal Vein Occlusion (RVO) macular edema, myelodysplastic abnormality, etc., and anti-VEGF antibodies and Fab fragments thereof play a role by interfering the binding of VEGF and cell receptors, and can make cells activated by VEGF lose functions or die, thereby inhibiting the growth of tumor cells or inhibiting the intraocular angiogenesis of ischemic retinal disease models, and anti-VEGF antibody drugs have become hot spots in research and development. The FDA has approved a humanized antibody on the market, namely bevacizumab which takes VEGF as a target, and ranibizumab has achieved great economic benefit and social value.

Ranibizumab approved by FDA for the treatment of macular degeneration in 2006, belonging to the second generation of humanized anti-VEGF antibody Fab fragments, was derived from the full-length anti-VEGF antibody-bevacizumab Fab fragment, which was mutagenized at HCDR1 and HCDR3 to make ranibizumab bind more strongly to VEGF. Due to its high fluidity and tissue penetration, Fab is advantageous for local administration by intraocular injection, which is not achieved by IgG, making it rapidly the first choice for the treatment of the ophthalmic disease macular degeneration.

The Fab antibody fragment is an antigen-binding fragment of an IgG molecule, and is a heterodimer formed by the disulfide bond binding of the Fd chain and the L chain of the heavy chain. Since Fab antibody fragments are free of Fc fragments, complex glycosylation and post-translational modifications are not required, and therefore need not be produced by mammalian cell systems. Escherichia coli becomes the most common prokaryotic expression system for preparing the recombinant antibody Fab fragment on a large scale by the facts that the self-genetic background is clear, the operation is easy, the production period is short, the cost is low, the expression quantity is high, the expression product is easy to separate and purify, and the like.

There are currently three main methods for expression of antibody Fab in e.coli: coli cytoplasmic expression, e.coli extracellular secretion expression and e.coli periplasmic soluble expression. The expression level of the expressed Fab is easy to hydrolyze by protease due to the low extracellular secretion expression of the escherichia coli, so that the expression strategy of the Fab expressed by the supernatant is rarely used and the research is less; the Fab fragment expressed by the escherichia coli mainly adopts cytoplasmic expression and periplasmic expression, the Fab protein expressed by the escherichia coli cytoplasm is usually gathered in the bacterial cytoplasm and hydrolyzed by protease, or the Fab protein cannot be correctly folded due to lack of oxidation environment required by disulfide bond pairing in the cytoplasm to generate an inclusion body, the denaturation and renaturation of the inclusion body are very inefficient, the subsequent treatment process is too complex, the renaturation of the Fab fragment inclusion body is only limited in basic research, and the Fab fragment is not beneficial to expanded production.

The protein which is less expressed by protease contained in the periplasm space of the escherichia coli is not easy to hydrolyze by the protease, an oxidation environment required by correct pairing of disulfide bonds can be provided, the thiol-disulfide oxidoreductase contained in the periplasm can catalyze the formation of the disulfide bonds, the expressed antibody Fab fragment protein is guided to enter the periplasm space through a section of signal peptide, and the signal peptide is cut by enzyme to form the antibody Fab fragment with biological activity.

At present, researchers have made extensive attempts to express antibody Fab fragments in the periplasm of Escherichia coli worldwide, and the anti-VEGF antibody Fab fragments (Ranitumumab) are obtained by soluble expression of Escherichia coli periplasm by Wangzhiling, Wangzongming and the like, but the expression amount is only 1.94 mg/L; similarly, the Fab fragment of the anti-VEGF antibody is obtained by periplasmic soluble expression of escherichia coli, such as Juglans mandshurica and the like, but the expression amount is only 0.8 mg/L.

Therefore, the current problem that the expression level of the Fab fragment of the anti-VEGF antibody is too low mainly exists in the periplasm soluble expression of the escherichia coli, and a lot of difficulties still exist in the problem of how to improve the expression level of the Fab fragment of the anti-VEGF antibody in the periplasm space of the escherichia coli so as to realize the high-efficiency expression of the Fab fragment.

Disclosure of Invention

The invention aims to provide a fermentation process for expressing an anti-VEGF antibody Fab fragment in the periplasm of escherichia coli, which can efficiently obtain a soluble anti-VEGF antibody Fab fragment from an escherichia coli fermentation system and provide reference for efficiently expressing other antibody Fab fragments in the periplasm of the escherichia coli.

In a first aspect of the present invention, there is provided a method for expressing a protein in the periplasm of E.coli, comprising the steps of:

(1) providing an escherichia coli strain for expressing a target protein;

(2) a fermentation step, namely fermenting the escherichia coli strains in the step (1) by using a fermentation medium;

(3) an induced expression step, feeding a supplemented medium and IPTG (isopropyl-beta-thiogalactoside) and continuing fermentation to perform induced expression of the target protein; the temperature of the continuous fermentation is T, the dissolved oxygen is DO, and the final concentration of IPTG is C;

wherein the feeding medium has a feeding speed V of 6mL/h to 20mL/h, preferably 6mL/h to 12mL/h, more preferably 7mL/h to 10 mL/h.

In another preferred embodiment, the E.coli is a recombinant E.coli strain expressing the Lanitumumab Fab protein, such as E.coli W3110/pTack-OmpA-Fab.

In another preferred embodiment, in said step (3), the final concentration C of IPTG after the addition of IPTG is 0.1mmol/L to 1mmol/L, preferably 0.1mmol/L to 0.5mmol/L, more preferably 0.2mmol/L to 0.4 mmol/L.

In another preferred embodiment, in the step (3), the temperature T for continuing the fermentation after adding IPTG is 15-40 ℃, preferably 20-37 ℃, and more preferably 25-30 ℃.

In another preferred embodiment, in the step (3), the dissolved oxygen DO after adding IPTG is 10% to 60%, preferably 15% to 60%, and more preferably 30% to 60%.

In another preferred embodiment, in the step (3), DO, T or C is two or more selected from the group consisting of: dissolved oxygen DO is 10% -60%, the temperature T of continuous fermentation is 15 ℃ -40 ℃, and the final concentration C of IPTG is 0.1-1 mmol/L.

In another preferred embodiment, in the step (3), DO, T or C is selected from two or more of the following groups: dissolved oxygen DO is 15% -60%, the temperature T for continuous fermentation is 20 ℃ -35 ℃, and the final concentration C of IPTG is 0.1-0.5 mmol/L.

In another preferred example, in the step (2), fermentation is carried out to OD₆₀₀The step (2) is carried out after a value of 60 to 100 (preferably 80 to 90; more preferably 82 to 90).

In another preferred embodiment, the protein of interest is selected from the group consisting of: fab fragments, Protein A Protein, IGF-1 (insulin-like growth factor 1).

In another preferred embodiment, the protein of interest is an antibody Fab fragment.

In another preferred embodiment, the antibody Fab fragment is a VEGF antibody Fab fragment, preferably a ranibizumab Fab fragment.

In another preferred embodiment, the pH of the fermentation medium is 6.5-7.5, preferably 6.8-7.2, more preferably 6.9.

In another preferred embodiment, the fermentation medium comprises: 10-20g/L Yeast extract (Yeast extract), 5-10g/L Tryptone (Tryptone), 2-3g/L (NH)₄)₂SO₄1-3g/L KCl, 80-120g/L glycerin, 2-4g/L citric acid monohydrate (citric acid monohydrate).

In another preferred embodiment, the feed medium comprises: 60-90 wt% of glycerol and 1-5 wt% of yeast extract.

In another preferred embodiment, the feed medium contains 80 wt% glycerol, 2 wt% yeast extract.

In another preferred example, in the step (2), the initial fermentation parameters are set as follows: temperature: 30 ℃, pH: 6.95, rotation speed: 400r/min, ventilation: 2-3vvm, pot pressure: 0.1-0.12MPa, dissolved oxygen: the series rotation speed was 30%.

In another preferred example, in the step (2), during fermentation for 25-35h, the fermentation temperature is reduced from 30 ℃ to 25 ℃, and simultaneously 2.0-4.0g of anhydrous magnesium sulfate is added.

In another preferred example, the feeding of the feed medium and the addition of IPTG in step (3) are carried out when the fermentation in step (2) is carried out for 35-45 h.

In another preferred embodiment, in the step (3), when the fermentation is continued for 60-90h after adding IPTG, preferably 80-86h, the fermentation liquid is collected.

In another preferred embodiment, the expression level of the method is 2mg/L or more, preferably 10mg/L or more, more preferably 50 mg/L or more, still more preferably 100mg/L or more, most preferably 100mg/L or more.

In another preferred embodiment, the method has a maximum expression level of 360 mg/L.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a schematic diagram of the genetic elements constituting the pTack-OmpA-Fab plasmid.

FIG. 2 SDS-PAGE results of different phases of induction at 30 ℃ induction temperature: m: a molecular marker; 1: inducing for 15 h; 2: inducing for 20 h; 3: inducing for 31 h; 4: inducing for 36 h; 5: inducing for 41 h; 6: induction is carried out for 43 hours; 7: induction was carried out for 56 h.

FIG. 3 Effect of different final concentrations of IPTG on fermentation biomass and expression level of the target protein.

FIG. 4 influence of different feeding rates on fermentation biomass and expression level of target protein.

FIG. 5 Effect of different fermentation temperatures on fermentation biomass and expression level of target protein after addition of IPTG.

FIG. 6 shows the effect of different dissolved oxygen concentrations on fermentation biomass and expression level of target protein.

Detailed Description

The present inventors have made extensive and intensive studies and have unexpectedly designed for the first time a method for expressing a protein (e.g., a Fab fragment of a VEGF antibody) in the periplasm of E.coli at a high efficiency. According to the method, the speed V (6mL/h-20mL/h) of the fed-batch culture medium is optimized, dissolved oxygen DO (10% -60%) in the fermentation process after IPTG is added, the final concentration C (0.1mmol/L-1mmol/L) of an inducer IPTG is added, the fermentation temperature T (15 ℃ -40 ℃) after IPTG is added can greatly improve the expression quantity of the anti-VEGF antibody Fab fragment, the highest expression quantity can reach 360mg/L, the production cost of the anti-VEGF antibody Fab fragment is greatly reduced, and the requirement of industrial production can be met. In addition, the method for efficiently expressing the protein in the periplasm of the escherichia coli has certain broad spectrum and can also be applied to other proteins. The present invention has been completed based on this finding.

The invention has the main advantages that:

(1) by optimizing important fermentation parameters (such as IPTG concentration, induction temperature, feeding speed and dissolved oxygen) in the fermentation process, the efficient expression of the anti-VEGF antibody Fab fragment (ranibizumab) in the periplasm of escherichia coli can be realized;

(2) the invention utilizes the optimized conditions, the highest expression quantity of the anti-VEGF antibody Fab fragment (ranibizumab) can reach 360mg/L, thereby effectively reducing the production cost and laying a solid foundation for the industrialization of the Fab fragment;

(3) the method also provides a thought for the high-efficiency expression of other antibody Fab in the escherichia coli;

(4) the expression quantity of the Fab fragment of the anti-VEGF antibody expressed by the method is obviously superior to that of 1.94mg/L in the prior art.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer (e.g., commercial instructions). Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Term(s) for

Antibody Fab fragments

Antibody Fab fragments are heterodimerically active proteins formed by light (VL-CL) and heavy (VH-CH; Fd fragment) chains via pairs of interchain and intrachain disulfide bonds. Compared to intact immunoglobulin molecules, Fab fragments have the following advantages: the antigen binding site of the full-length antibody is reserved with small molecular weight, and the antigen binding site has good tissue penetrability and can permeate to target tissues which cannot be reached by the full-length antibody; and the Fc fragment is not contained, glycosylation modification is not needed, and the method is particularly suitable for fermentation expression of escherichia coli, short in expression period and low in fermentation cost.

General procedure

The invention adopts the following technical scheme, which comprises the following steps:

(1) preparation of fermentation Strain

Activating strains: 20. mu.L of the stored recombinant strain E.coli W3110/pTack-OmpA-Fab (constructed according to the strain construction method in example 1) expressing the Ranitumumab Fab protein was inoculated into 30mL of LB liquid medium (containing 25mg/L kanamycin) and shake-cultured at 37 ℃ and 220r/min for 6 hours.

Culturing fermentation strains: taking 10 mu L of the bacterial liquid, inoculating the bacterial liquid into 100mL LB liquid culture medium (containing 25mg/L kanamycin), and shaking-culturing for 11h at 37 ℃ and 220r/min by a shaking table, wherein the obtained bacterial liquid is the upper tank seed liquid.

(2) Preparation of fermentation medium and feed medium:

preparation of a fermentation medium: taking Yeast extract (Yeast extract): 10.00g-20.00g, Tryptone (Tryptone): 5.00g-10.00g, (NH)₄)₂SO₄: 2.20g-3.20g, KCl: 1.20g-2.30g, glycerol (glycerol): 80.00g-112.00g, citric acid monohydrate (citric acid monohydrate): 2.50g to 3.70g of NaH₂PO₄·2H₂O：2.00g-3.00g，Na₂HPO₄·12H₂O：4.30g-6.30g，MgSO₄： 0.40g-0.51g，CaCl₂：0.10g-0.19g，ZnSO₄·7H₂O：20.60mg，MnSO₄·4H₂O：27.20mg， CuSO₄·5H₂O：8.10mg，CoCl₂.2H₂O:2.46mg，FeCl₃·6H₂O：100.60mg，H₃B0₃： 0.30mg，Na₂MoO₄·2H₂O: 0.20mg, water is added and mixed evenly, then 50% ammonia water is used for adjusting the pH value to about 6.90, and finally the volume is fixed to 1L by using a measuring cylinder. Adding defoaming agent in 0.5 per mill (V/V), and high pressure steam sterilizing at 121 deg.C for 30 min.

Preparation of a feed medium: taking 800g of glycerol (glycerol) and 20g of Yeast extract (Yeast extract), adding water until the total mass is 1000g, and fully stirring to obtain the supplemented medium. Steam autoclaving at 121 ℃ for 30 minutes.

(3) Fermentation: 2.5L of fermentation Medium was placed in a 5L fermenter and 200mL of OD was added₆₀₀About 4.0 escherichia coli fermentation strains are put into a fermentation tank, and initial fermentation parameters are set as follows: temperature: 30 ℃, pH: 6.95, rotation speed: 400r/min, ventilation: 2-3vvm, pot pressure: 0.1-0.12MPa, dissolved oxygen: the serial rotating speed is 30 percent; when the fermentation lasts for about 30-35h, the OD of the thallus₆₀₀About 40 ℃ at which time the fermentation temperature was decreased from 30 ℃ to 25 ℃ while adding about 2.0g of anhydrous magnesium sulfate. Continuing fermentation until the fermentation time is about 38-40h, and the thallus OD₆₀₀When the concentration is about 82-90 ℃, the dissolved oxygen rises sharply, which indicates that the carbon source (such as glycerol) in the culture medium is completely consumed, at the moment, the fed-batch culture medium is fed at the speed of 6-20 mL/h, IPTG (0.1-1 mmol/L) is added for the induced expression of the target protein, the fermentation temperature is adjusted to 20-37 ℃, and the setting value of the dissolved oxygen at the series rotating speed is changed from 30% to 15-60%. And after fermenting for 60-85h, collecting fermentation liquor, cleaning the fermentation tank, closing the fermentation equipment, and finishing fermentation.

Wherein in the step (3), the inducer IPTG is added in an amount of 0.1mmol/L-1mmol/L, preferably 0.2-0.4 mmol/L.

Wherein in the step (3), the feeding speed of the feed medium is 6mL/h-20mL/h, preferably 7mL/h-10 mL/h.

Wherein in the step (3), the fermentation temperature is adjusted within the range of 20-37 ℃ after the inducer IPTG is added, and preferably within the range of 25-30 ℃.

Wherein in the step (3), the set range of dissolved oxygen after adding IPTG is 15% -60%, preferably 30% -60%.

Wherein in the step (3), the feed medium is fed and (and) IPTG is added, and the feed medium can be fed and then IPTG (such as IPTG solution) is added; it is also possible to add IPTG (e.g.IPTG solution) first and then feed the medium; it is also possible to add together, for example, to dissolve IPTG in the feed medium (for example in a fed-batch manner); or added all at once, i.e. IPTG is added while feeding the feed medium. In addition, "adding IPTG" can also be adding IPTG in a feeding mode; IPTG may also be added in portions.

EXAMPLE 1 Strain construction

The construction method of recombinant strain E.coli W3110/pTack-OmpA-Fab for secretory expression of Ranitumumab Fab protein in periplasmic space refers to patent WO2014/178078A 2.

The construction process of the ranibizumab Fab recombinant expression gene engineering bacteria is as follows: the Fab light and heavy chain gene containing signal peptide was artificially synthesized (the synthetic gene elements are shown in FIG. 1, the gene sequence is shown in SEQ ID No.1), PCR amplified and ligated to pTack plasmid, i.e., pTack-OmpA-Fab, via BamHI/EcoRI restriction sites. The plasmid with correct sequencing is electrically transformed into E.coli W3110 cells, namely E.coli W3110/pTack-OmpA-Fab. The gene sequence and the corresponding amino acid sequence of the pTack-OmpA-Fab plasmid are shown in SEQ ID No. 1-8.

EXAMPLE 2 measurement of expression levels of target protein at different times in fermentation

(i) Sampling and extracting target protein at different times of fermentation:

taking 4mL of fermentation liquid induced in different periods in the fermentation process, putting the fermentation liquid into a 5mL centrifuge tube, centrifuging for 10 minutes by using a desktop high-speed centrifuge at 12000r/min, and discarding the supernatant to obtain thalli. Then adding 2mL of periplasmic protein extract (pH7.4, 100mmol/L Tris +10mmol/L EDTA) to fully break up the thalli, then using the periplasmic protein extract to fix the volume to 4mL, placing the mixture into a water bath after uniform mixing, extracting overnight at 37 ℃, then centrifuging the mixture for 10 minutes by a desk-top high-speed centrifuge at 12000g of centrifugal force, and taking the supernatant, namely the periplasmic protein extract containing the target protein.

The results of SDS-PAGE electrophoresis at 30 ℃ for different periods are shown in FIG. 2.

(ii) (i) detecting the content of the target protein in the periplasmic protein extract:

preparing a mobile phase: phase A: (pH 7.2, 50mmol/L PBS +150mmol/L NaCl): weighing 12.89g Na₂HPO₄·12H₂O，2.27gNaH₂PO₄·2H₂O, 8.77g NaCl is dissolved in water, the pH is adjusted to 7.2 by 6mol/L HCl and 5mol/L NaOH, and the volume is adjusted to 1L by using a measuring cylinder.

Phase B: (pH 2.7, 0.1mol/L Gly-HCl): weighing 7.50g of Gly, dissolving in water, adjusting pH to 2.7 with 6mol/L HCl, and metering to 1L by using a measuring cylinder.

A detection instrument: waters1525-2489, column: GE; hiscreen en^TM capto^TML; column temperature: room temperature;

detection wavelength: 230 nm;

sample injection: (i) filtering the periplasm protein extract obtained in the step (2) through a 0.22 mu m filter membrane, and adjusting the pH to be about 7.2 by using 1mol/L hydrochloric acid and 1mol/L Tris; reference preparation (ranibizumab injection, norhua pharmaceutical, batch number: S0098): 10mg/mL of the reference formulation was diluted 100-fold with deionized water and 1mol/L Tris adjusted to pH 7.2; sample introduction volume: 3 mL;

the elution gradient is shown in table 1 below:

TABLE 1

Time	Flow rate	％A	％B
					2.00	100	0
3.0	2.00	100	0
				3.10	5.00	100	0
10.00	5.00	100	0
				10.05	5.00	0	100
16.00	5.00	0	100
				16.05	5.00	100	0
20.00	5.00	100	0
				20.05	2.00	100	0

And (4) analyzing results: and calculating the content of the target protein in the periplasmic protein extract according to the peak area ratio of the target protein peak elution peak of the periplasmic extract to the target protein peak elution peak in the reference preparation by using Empower3 data analysis software.

Example 3 Effect of inducer concentration on microbial Biomass and expression level of target protein

In this example, the effect of the concentration of the inducer on the biomass of the cells and the expression level of the target protein was compared by adding IPTG as the inducer at different concentrations.

(1) Preparation of fermentation Strain

Activating strains: the stored recombinant strain E.coli W3110/pTack-OmpA-Fab 20. mu.L expressing the Ranitumumab Fab protein was inoculated into 30mL of LB liquid medium (containing 25mg/L kanamycin) and shake-cultured at 37 ℃ for 6 hours at 220r/min with a shaker.

Culturing fermentation strains: taking 10 mu L of the bacterial liquid, inoculating the bacterial liquid into 100mL LB liquid culture medium (containing 25mg/L kanamycin), and shaking-culturing for 11h at 37 ℃ and 220r/min by a shaking table, wherein the obtained bacterial liquid is the upper tank seed liquid.

(2) Preparation of fermentation medium and feed medium:

preparation of a fermentation medium: yeast extract: 10.00g, tryptone: 5.00g (NH)₄)₂SO₄: 3.20g, KCl:2.30g, glycerol: 112.00g, citric acid monohydrate: 3.70g, NaH₂PO₄·2H₂O：3.00g， Na₂HPO₄·12H₂O：6.30g，MgSO₄：0.51g，CaCl₂：0.19g，ZnSO₄·7H₂O：20.60mg， MnSO₄·4H₂O：27.20mg，CuSO₄·5H₂O：8.10mg，CoCl_2·2H₂O:2.46mg，FeCl₃·6H₂O： 100.60mg，H₃B0₃：0.30mg，Na₂MoO₄·2H₂O：0.20mg, adding water, mixing uniformly, then adjusting the pH to about 6.90 by using 50% ammonia water, and finally metering to 1L by using a dosing cylinder. Adding defoaming agent according to 5 per mill (V/V), and sterilizing for 30 minutes at 121 ℃ by high-pressure steam.

Preparation of a feed medium: taking 800g of glycerol and 20g of yeast extract, then adding water until the total mass is 1000g, and fully and uniformly stirring to obtain the 80% glycerol feed supplement culture medium. Steam autoclaving at 121 ℃ for 30 minutes.

(3) Fermentation: 2.5L of fermentation Medium was placed in a 5L fermenter and 200mL of OD was added₆₀₀About 4.0 escherichia coli fermentation strains are put into a fermentation tank, and initial fermentation parameters are set as follows: temperature: 30 ℃, pH: 6.95, rotation speed: 400r/min, ventilation: 2-3vvm, pot pressure: 0.1-0.12MPa, dissolved oxygen: the serial rotating speed is 30 percent; when the fermentation lasts for about 32 hours, the OD of the cells₆₀₀About 40 ℃ at which time the fermentation temperature was decreased from 30 ℃ to 25 ℃ while adding about 2.0g of anhydrous magnesium sulfate. Continuing fermentation until the fermentation time reaches about 40h, and the thallus OD₆₀₀When the concentration is about 83.70, the dissolved oxygen rises sharply, which indicates that the carbon source in the culture medium is completely consumed, at this time, the feeding culture medium is fed at a speed of 12mL/h, and IPTG (isopropyl-beta-D-thiogalactoside) with final concentrations of 0.1mmol/L, 0.2mmol/L, 0.3mmol/L and 0.5mmol/L is added to perform induction expression of the target protein, the fermentation temperature is adjusted to 25 ℃, and the set value of the dissolved oxygen at the series rotating speed is 30%. And after fermenting for 82 hours, collecting fermentation liquor, cleaning the fermentation tank, closing fermentation equipment and finishing fermentation.

According to the test method in example 1, the test results are shown in FIG. 3 and the following Table 2:

TABLE 2

The results show that: the wet weight of the thalli can be reduced by high-concentration IPTG (isopropyl-beta-thiogalactoside), but the expression quantity of the target protein is greatly improved along with the increase of the concentration of the thalli in a certain range; however, if the IPTG concentration is too high, the expression level of the target protein is greatly reduced.

Example 4 Effect of different feed rates on microbial Biomass and expression level of target protein

In this example, the effect of different feeding rates on the biomass of cells and the expression level of the target protein was compared by feeding the feeding medium at different rates after induction.

The first two steps of this example are the same as example 3.

(3) Fermentation: 2.5L of fermentation Medium was placed in a 5L fermenter and 200mL of OD was added₆₀₀About 4.0 escherichia coli fermentation strains are put into a fermentation tank, and initial fermentation parameters are set as follows: temperature: 30 ℃, pH: 6.95, rotation speed: 400r/min, ventilation: 2-3vvm, pot pressure: 0.1-0.12MPa, dissolved oxygen: the serial rotating speed is 30 percent; when the fermentation lasts for about 32.62h, the OD of the cells is₆₀₀About 40 ℃ at which time the fermentation temperature was decreased from 30 ℃ to 25 ℃ while adding about 2.0g of anhydrous magnesium sulfate. Continuing fermentation until the fermentation time is about 39h, and the thallus OD₆₀₀When the concentration is about 89.20, the dissolved oxygen rises sharply, which indicates that the carbon source in the culture medium is completely consumed, at this time, the fed-batch culture medium is fed at the speed of 6mL/h, 9mL/h and 12mL/h, and IPTG (0.2 mmol/L) is added to perform induction expression of the target protein, the fermentation temperature is adjusted to 25 ℃, and the setting value of the dissolved oxygen at the series rotating speed is 30%. And after fermenting for 81 hours, collecting fermentation liquor, cleaning the fermentation tank, closing the fermentation equipment, and finishing the fermentation.

According to the test method in example 1, the test results are shown in FIG. 4 and the following Table 3:

TABLE 3

Feed supplement speed (mL/h)	Wet weight of thallus (g/L)	Fab concentration (μ g/mL)
			6	232.50	66.50
9	245.00	153.00
			12	248.00	72.00

The results show that: the feeding speed can influence the expression amount of the target protein to a great extent, and the excessively high or excessively low feeding speed is not favorable for the expression and synthesis of the target protein.

Example 5 Effect of different fermentation temperatures on microbial Biomass and expression level of target protein after addition of IPTG

In this example, the influence of different induction temperatures on the biomass of the cells and the expression level of the target protein was compared by adjusting different temperatures after induction.

The first two steps of this example are the same as example 3.

(3) Fermentation: 2.5L of fermentation Medium was placed in a 5L fermenter and 200mL of OD was added₆₀₀About 4.0 escherichia coli fermentation strains are put into a fermentation tank, and initial fermentation parameters are set as follows: temperature: 30 ℃, pH: 6.95, rotation speed: 400r/min, ventilation: 2-3vvm, pot pressure: 0.1-0.12MPa, dissolved oxygen: the serial rotating speed is 30 percent; when the fermentation is carried out for about 31 hours, the OD of the cells₆₀₀About 40 ℃ at which time the fermentation temperature was decreased from 30 ℃ to 25 ℃ while adding about 2.0g of anhydrous magnesium sulfate. Continuing fermentation until the fermentation time reaches about 40h, and the thallus OD₆₀₀When the concentration is about 88.50, the dissolved oxygen rises sharply, which indicates that the carbon source in the culture medium is completely consumed, at this time, the supplemented culture medium is fed at a speed of 7mL/h, IPTG with a final concentration of 0.2mmol/L is added for the induced expression of the target protein, and the set values of the dissolved oxygen at the series rotating speed of 20 ℃, 25 ℃, 30 ℃ and 35 ℃ are respectively adjusted to be 30%. After fermenting for 85h, collecting fermentation liquor, cleaning the fermentation tank, closing the fermentation equipment, and finishing the fermentation.

According to the detection method in example 1, the detection results are shown in fig. 5 and table 4 below.

TABLE 4

Temperature (. degree.C.)	Wet weight of thallus (g/L)	Fab concentration (μ g/mL)
			20	267.00	120.00
25	282.50	205.00
			30	231.00	363.50
35	290	53.00

The results show that: the expression of the target protein can be greatly influenced by different induction temperatures, and the target protein is reduced in solubility to form insoluble inclusion bodies at overhigh temperature, so that the expression quantity of the target protein is reduced; too low a temperature may affect the activity of the enzyme and also decrease the expression of the protein of interest.

Example 6 Effect of different dissolved oxygen on microbial Biomass and expression level of target protein

In this example, the effect of different dissolved oxygen on the biomass of cells and the expression level of a target protein was compared by adjusting different dissolved Oxygen (OD) values after induction.

The first two steps of this example are the same as example 3.

(3) Fermentation: 2.5L of fermentation Medium was placed in a 5L fermenter and 200mL of OD was added₆₀₀About 4.0 escherichia coli fermentation strains are put into a fermentation tank, and initial fermentation parameters are set as follows: temperature: 30 ℃, pH: 6.95, rotation speed: 400r/min, ventilation: 2-3vvm, pot pressure: 0.1-0.12MPa, dissolved oxygen: the serial rotating speed is 30 percent; when the fermentation lasts for about 32 hours, the OD of the cells₆₀₀About 40 ℃ at which time the fermentation temperature was decreased from 30 ℃ to 25 ℃ while adding about 2.0g of anhydrous magnesium sulfate. Continuing fermentation until the fermentation time reaches about 38.50h, and the thallus OD₆₀₀When the concentration is about 85.70, the dissolved oxygen rises sharply, which indicates that the carbon source in the culture medium is completely consumed, at this time, the feeding of the culture medium is started at a speed of 9mL/h, IPTG (0.2 mmol/L) is added to perform induced expression of the target protein, the fermentation temperature is adjusted to 20 ℃, and the settings of the dissolved oxygen in the series rotating speed are 15%, 30% and 60% respectively. And after fermenting for 83 hours, collecting fermentation liquor, cleaning the fermentation tank, closing fermentation equipment and finishing fermentation.

According to the detection method in example 1, the monitoring results are shown in fig. 6 and the following table 5:

TABLE 5

Dissolved oxygen (%)	Wet weight of thallus (g/L)	Fab concentration (μ g/mL)
			15	269.50	106.00
30	245.00	153.00
			60	241.00	260.00

The results show that: the high-level dissolved oxygen can promote the transcription level of target protein genes, thereby improving the expression quantity of the target proteins.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence information of the present invention:

SEQ ID No.1 Synthesis of Fab Gene sequence containing Signal peptide

CATATGATGAAGAAAACCGCTATTGCGATTGCAGTGGCCCTGGCAGGTTTCGCTACCGTTGCACAGGCTGATATCCAGCTGACCCAGTCTCC GTCTTCTCTGTCTGCTTCTGTTGGTGATCGTGTGACCATCACCTGCTCTGCGTCTCAGGATATCTCCAACTACCTGAACTGGTACCAGCAGA AACCGGGTAAAGCTCCGAAAGTGCTGATCTACTTCACCTCTTCTCTGCATTCTGGTGTTCCGTCTCGCTTCTCTGGTTCTGGTTCTGGTACC GATTTCACCCTGACCATCTCTTCTCTGCAGCCGGAAGATTTCGCGACCTACTACTGCCAGCAGTACTCTACCGTTCCGTGGACCTTCGGTCA GGGTACCAAAGTGGAAATCAAACGCACCGTTGCTGCTCCGTCTGTGTTCATCTTCCCGCCGTCCGATGAACAGCTGAAATCTGGTACCGCTT CTGTGGTTTGCCTGCTGAACAACTTCTACCCGCGTGAAGCGAAAGTTCAGTGGAAAGTGGATAACGCTCTGCAGTCTGGTAACTCTCAGGAA TCCGTTACCGAACAGGATTCCAAAGATTCCACCTACTCTCTGTCTTCTACCCTGACCCTGTCCAAAGCGGATTACGAAAAACACAAAGTGTA CGCTTGCGAAGTTACCCATCAGGGTCTGTCTTCTCCGGTTACCAAATCCTTCAACCGTGGTGAATGCTAAGGATCCTGACGAGGCGTAAAAA ATGAAGAAAACCGCTATTGCGATTGCAGTGGCCCTGGCAGGTTTCGCTACCGTTGCACAGGCTGAAGTTCAGCTGGTGGAATCTGGTGGTGG TCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCGCTGCTTCTGGTTACGATTTCACCCATTACGGTATGAACTGGGTTCGTCAGGCTC CGGGTAAAGGTCTGGAATGGGTTGGTTGGATCAACACCTACACCGGTGAACCGACCTACGCTGCTGACTTCAAACGTCGCTTCACCTTCTCT CTGGATACCTCCAAATCTACCGCTTACCTGCAGATGAACTCTCTGCGTGCTGAAGATACCGCTGTGTACTACTGCGCGAAATACCCGTACTA CTACGGTACCTCTCATTGGTACTTCGATGTTTGGGGTCAGGGTACCCTGGTTACCGTTTCCTCTGCTTCTACCAAAGGTCCGTCTGTTTTCC CGCTGGCTCCGTCTTCCAAATCTACCTCTGGTGGTACCGCTGCTCTGGGTTGCCTGGTGAAAGATTACTTCCCGGAACCGGTTACCGTTTCT TGGAACTCTGGTGCTCTGACCTCTGGTGTTCATACCTTCCCGGCTGTTCTGCAGTCTTCTGGTCTGTACTCTCTGTCTTCTGTGGTTACCGT TCCGTCTTCCTCTCTGGGTACCCAGACCTACATCTGCAACGTGAACCATAAACCGTCTAACACCAAAGTGGATAAGAAAGTGGAACCGAAAT CCTGCGATAAAACCCATCTGTAAGAATTC

SEQ ID No.2 connecting peptide gene sequence

GGATCCTGACGAGGCGTAAAAA

SEQ ID No.3 Signal peptide Gene sequence

ATGAAGAAAACCGCTATTGCGATTGCAGTGGCCCTGGCAGGTTTCGCTACCGTTGCACAGGCT

SEQ ID No.4 Signal peptide amino acid sequence

MKKTAIAIAVALAGFATVAQA

SEQ ID No.5 light chain Gene sequence

GATATCCAGCTGACCCAGTCTCCGTCTTCTCTGTCTGCTTCTGTTGGTGATCGTGTGACCATCACCTGCTCTGCGTCTCAGGATATCTCCAA CTACCTGAACTGGTACCAGCAGAAACCGGGTAAAGCTCCGAAAGTGCTGATCTACTTCACCTCTTCTCTGCATTCTGGTGTTCCGTCTCGCT TCTCTGGTTCTGGTTCTGGTACCGATTTCACCCTGACCATCTCTTCTCTGCAGCCGGAAGATTTCGCGACCTACTACTGCCAGCAGTACTCT ACCGTTCCGTGGACCTTCGGTCAGGGTACCAAAGTGGAAATCAAACGCACCGTTGCTGCTCCGTCTGTGTTCATCTTCCCGCCGTCCGATGA ACAGCTGAAATCTGGTACCGCTTCTGTGGTTTGCCTGCTGAACAACTTCTACCCGCGTGAAGCGAAAGTTCAGTGGAAAGTGGATAACGCTC TGCAGTCTGGTAACTCTCAGGAATCCGTTACCGAACAGGATTCCAAAGATTCCACCTACTCTCTGTCTTCTACCCTGACCCTGTCCAAAGCG GATTACGAAAAACACAAAGTGTACGCTTGCGAAGTTACCCATCAGGGTCTGTCTTCTCCGGTTACCAAATCCTTCAACCGTGGTGAATGC SEQ ID No.6 light chain amino acid sequence

DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYS TVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID No.7 heavy chain Gene sequence

GAAGTTCAGCTGGTGGAATCTGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCGCTGCTTCTGGTTACGATTTCACCCA TTACGGTATGAACTGGGTTCGTCAGGCTCCGGGTAAAGGTCTGGAATGGGTTGGTTGGATCAACACCTACACCGGTGAACCGACCTACGCTG CTGACTTCAAACGTCGCTTCACCTTCTCTCTGGATACCTCCAAATCTACCGCTTACCTGCAGATGAACTCTCTGCGTGCTGAAGATACCGCT GTGTACTACTGCGCGAAATACCCGTACTACTACGGTACCTCTCATTGGTACTTCGATGTTTGGGGTCAGGGTACCCTGGTTACCGTTTCCTC TGCTTCTACCAAAGGTCCGTCTGTTTTCCCGCTGGCTCCGTCTTCCAAATCTACCTCTGGTGGTACCGCTGCTCTGGGTTGCCTGGTGAAAG ATTACTTCCCGGAACCGGTTACCGTTTCTTGGAACTCTGGTGCTCTGACCTCTGGTGTTCATACCTTCCCGGCTGTTCTGCAGTCTTCTGGT CTGTACTCTCTGTCTTCTGTGGTTACCGTTCCGTCTTCCTCTCTGGGTACCCAGACCTACATCTGCAACGTGAACCATAAACCGTCTAACAC CAAAGTGGATAAGAAAGTGGAACCGAAATCCTGCGATAAAACCCATCTG

SEQ ID No.8 heavy chain amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTA VYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL

Sequence listing

<110> Shanghai Domrei Biotechnology, Inc

Shanghai Pharmaceutical Industry Research Institute

<120> method for efficiently expressing Fab fragment of antibody

<130> P2018-0476

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 1501

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

catatgatga agaaaaccgc tattgcgatt gcagtggccc tggcaggttt cgctaccgtt 60

gcacaggctg atatccagct gacccagtct ccgtcttctc tgtctgcttc tgttggtgat 120

cgtgtgacca tcacctgctc tgcgtctcag gatatctcca actacctgaa ctggtaccag 180

cagaaaccgg gtaaagctcc gaaagtgctg atctacttca cctcttctct gcattctggt 240

gttccgtctc gcttctctgg ttctggttct ggtaccgatt tcaccctgac catctcttct 300

ctgcagccgg aagatttcgc gacctactac tgccagcagt actctaccgt tccgtggacc 360

ttcggtcagg gtaccaaagt ggaaatcaaa cgcaccgttg ctgctccgtc tgtgttcatc 420

ttcccgccgt ccgatgaaca gctgaaatct ggtaccgctt ctgtggtttg cctgctgaac 480

aacttctacc cgcgtgaagc gaaagttcag tggaaagtgg ataacgctct gcagtctggt 540

aactctcagg aatccgttac cgaacaggat tccaaagatt ccacctactc tctgtcttct 600

accctgaccc tgtccaaagc ggattacgaa aaacacaaag tgtacgcttg cgaagttacc 660

catcagggtc tgtcttctcc ggttaccaaa tccttcaacc gtggtgaatg ctaaggatcc 720

tgacgaggcg taaaaaatga agaaaaccgc tattgcgatt gcagtggccc tggcaggttt 780

cgctaccgtt gcacaggctg aagttcagct ggtggaatct ggtggtggtc tggttcagcc 840

gggtggttct ctgcgtctgt cttgcgctgc ttctggttac gatttcaccc attacggtat 900

gaactgggtt cgtcaggctc cgggtaaagg tctggaatgg gttggttgga tcaacaccta 960

caccggtgaa ccgacctacg ctgctgactt caaacgtcgc ttcaccttct ctctggatac 1020

ctccaaatct accgcttacc tgcagatgaa ctctctgcgt gctgaagata ccgctgtgta 1080

ctactgcgcg aaatacccgt actactacgg tacctctcat tggtacttcg atgtttgggg 1140

tcagggtacc ctggttaccg tttcctctgc ttctaccaaa ggtccgtctg ttttcccgct 1200

ggctccgtct tccaaatcta cctctggtgg taccgctgct ctgggttgcc tggtgaaaga 1260

ttacttcccg gaaccggtta ccgtttcttg gaactctggt gctctgacct ctggtgttca 1320

taccttcccg gctgttctgc agtcttctgg tctgtactct ctgtcttctg tggttaccgt 1380

tccgtcttcc tctctgggta cccagaccta catctgcaac gtgaaccata aaccgtctaa 1440

caccaaagtg gataagaaag tggaaccgaa atcctgcgat aaaacccatc tgtaagaatt 1500

c 1501

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

ggatcctgac gaggcgtaaa aa 22

<210> 3

<211> 63

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

atgaagaaaa ccgctattgc gattgcagtg gccctggcag gtttcgctac cgttgcacag 60

gct 63

<210> 4

<211> 21

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 4

Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala

1 5 10 15

Thr Val Ala Gln Ala

20

<210> 5

<211> 642

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

gatatccagc tgacccagtc tccgtcttct ctgtctgctt ctgttggtga tcgtgtgacc 60

atcacctgct ctgcgtctca ggatatctcc aactacctga actggtacca gcagaaaccg 120

ggtaaagctc cgaaagtgct gatctacttc acctcttctc tgcattctgg tgttccgtct 180

cgcttctctg gttctggttc tggtaccgat ttcaccctga ccatctcttc tctgcagccg 240

gaagatttcg cgacctacta ctgccagcag tactctaccg ttccgtggac cttcggtcag 300

ggtaccaaag tggaaatcaa acgcaccgtt gctgctccgt ctgtgttcat cttcccgccg 360

tccgatgaac agctgaaatc tggtaccgct tctgtggttt gcctgctgaa caacttctac 420

ccgcgtgaag cgaaagttca gtggaaagtg gataacgctc tgcagtctgg taactctcag 480

gaatccgtta ccgaacagga ttccaaagat tccacctact ctctgtcttc taccctgacc 540

ctgtccaaag cggattacga aaaacacaaa gtgtacgctt gcgaagttac ccatcagggt 600

ctgtcttctc cggttaccaa atccttcaac cgtggtgaat gc 642

<210> 6

<211> 214

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 6

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 7

<211> 693

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

gaagttcagc tggtggaatc tggtggtggt ctggttcagc cgggtggttc tctgcgtctg 60

tcttgcgctg cttctggtta cgatttcacc cattacggta tgaactgggt tcgtcaggct 120

ccgggtaaag gtctggaatg ggttggttgg atcaacacct acaccggtga accgacctac 180

gctgctgact tcaaacgtcg cttcaccttc tctctggata cctccaaatc taccgcttac 240

ctgcagatga actctctgcg tgctgaagat accgctgtgt actactgcgc gaaatacccg 300

tactactacg gtacctctca ttggtacttc gatgtttggg gtcagggtac cctggttacc 360

gtttcctctg cttctaccaa aggtccgtct gttttcccgc tggctccgtc ttccaaatct 420

acctctggtg gtaccgctgc tctgggttgc ctggtgaaag attacttccc ggaaccggtt 480

accgtttctt ggaactctgg tgctctgacc tctggtgttc ataccttccc ggctgttctg 540

cagtcttctg gtctgtactc tctgtcttct gtggttaccg ttccgtcttc ctctctgggt 600

acccagacct acatctgcaa cgtgaaccat aaaccgtcta acaccaaagt ggataagaaa 660

gtggaaccga aatcctgcga taaaacccat ctg 693

<210> 8

<211> 231

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Leu

225 230

Claims (11)

1. A method for expressing a protein in the periplasm of E.coli, comprising the steps of:

(1) providing an escherichia coli strain for expressing a target protein;

(2) a fermentation step, namely fermenting the escherichia coli strains in the step (1) by using a fermentation medium;

(3) an induced expression step, feeding a supplemented medium and IPTG (isopropyl-beta-thiogalactoside) and continuing fermentation to perform induced expression of the target protein; the temperature of the continuous fermentation is T, the dissolved oxygen is DO, and the final concentration of IPTG is C;

wherein the velocity V of the fed-batch medium is 6mL/h to 20mL/h, and the fed-batch medium contains: 60-90 wt% of glycerol, 1-5 wt% of yeast extract; the target protein is an antibody Fab fragment, and the antibody Fab fragment is a ranibizumab Fab fragment;

in the step (3), the final concentration C of IPTG after the addition of IPTG is 0.1mmol/L-1 mmol/L;

the temperature T for continuing fermentation after IPTG is added is 15-40 ℃;

adding IPTG and then dissolving oxygen DO to 10% -60%;

the fermentation medium contains: 10-20g/L yeast extract, 5-10g/L tryptone, 2-3g/L (NH)₄）₂SO₄1-3g/L KCl, 80-120g/L glycerin, 2-4g/L citric acid monohydrate.

2. The method of claim 1, wherein the E.coli is a recombinant E.coli strain expressing the Lanitumumab Fab protein.

3. The method of claim 1, wherein in step (3), the final concentration C of IPTG after the addition of IPTG is from 0.1 to 0.5 mmol/L.

4. The method of claim 1, wherein in step (3), the temperature T for continuing the fermentation after the addition of IPTG is from 20 ℃ to 37 ℃.

5. The method of claim 1, wherein in step (3), the dissolved oxygen DO is between 15% and 60% after the addition of IPTG.

6. The method of claim 1, wherein the fermentation medium has a pH of 6.5 to 7.5.

7. The method of claim 1, wherein the feed medium comprises 80 wt% glycerol and 2 wt% yeast extract.

8. The method of claim 1, wherein in step (2), the initial fermentation parameters are set as follows: temperature: 30 ℃, pH: 6.95, rotation speed: 400r/min, ventilation: 2-3vvm, pot pressure: 0.1-0.12MPa, dissolved oxygen: the series rotation speed was 30%.

9. The method of claim 1, wherein in the step (2), the fermentation temperature is reduced from 30 ℃ to 25 ℃ during fermentation for 25-35 hours, and 2.0-4.0g of anhydrous magnesium sulfate is added.

10. The method of claim 1, wherein the feeding of the feed medium and the addition of IPTG in step (3) are performed when the fermentation in step (2) is performed for 35-45 h.

11. The method of claim 1, wherein in step (3), the fermentation broth is collected after addition of IPTG and continued for 60-90 h.

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