阅读说明：本技术 一种适用于谷氨酸棒杆菌的重组表达载体、外源蛋白表达系统、应用和木聚糖酶的制备方法 (It is a kind of suitable for the recombinant expression carrier of Corynebacterium glutamicum, exogenous protein expression system, the preparation method of application and zytase ) 是由 刘秀霞 张伟 白仲虎 杨艳坤 于 2018-08-31 设计创作，主要内容包括：本发明提供了一种适用于谷氨酸棒杆菌的重组表达载体,该表达载体适用于在谷氨酸棒杆菌中表达外源蛋白并具有较高的表达水平。一种适用于谷氨酸棒杆菌的重组表达载体,其特征在于：所述重组表达载体中外源蛋白基因的启动子为?cspB启动子、信号肽为?cspB信号肽,所述?cspB启动子的核苷酸序列如SEQ ID NO：3所示,?cspB信号肽的核苷酸序列如SEQ ID NO：4所示。本发明中的外源蛋白在表达过程中,在信号肽的引导下,表达的外源蛋白能分泌到胞外,不需要经过菌体破碎,分泌出的外源蛋白相对于胞内表达易于纯化；且无胞外蛋白酶,分泌出的外源蛋白在培养基中较为稳定。此外,本发明还提供了外源蛋白的外源蛋白表达系统、应用和外源蛋白的制备方法。(The present invention provides a kind of recombinant expression carrier suitable for Corynebacterium glutamicum, which is suitable for expressing foreign protein and expression with higher in corynebacterium glutamicum.A kind of recombinant expression carrier suitable for Corynebacterium glutamicum, it is characterized by: the promoter of foreign protein genes is cspB promoter in the recombinant expression carrier, signal peptide is cspB signal peptide, the nucleotide sequence of the cspB promoter is as shown in SEQ ID NO:3, and the nucleotide sequence of cspB signal peptide is as shown in SEQ ID NO:4.Foreign protein in the present invention is during expression, and under the guidance of signal peptide, the foreign protein of expression can be secreted into extracellular, needs not move through bacterial cell disruption, the foreign protein secreted out of is easy to purify relative to intracellular expression；And without extracellular protease, the foreign protein secreted out of is relatively stable in the medium.In addition, the present invention also provides the exogenous protein expression systems of foreign protein, the preparation method of application and foreign protein.)

1. a kind of recombinant expression carrier suitable for Corynebacterium glutamicum, it is characterised in that: external source in the recombinant expression carrier The promoter of protein gene is cspB promoter, signal peptide is cspB signal peptide, the nucleotide of the cspB promoter Sequence is as shown in SEQ ID NO:3, and the nucleotide sequence of cspB signal peptide is as shown in SEQ ID NO:4.

2. a kind of recombinant expression carrier suitable for Corynebacterium glutamicum as described in claim 1, it is characterised in that: described heavy The foreign protein genes downstream of group expression vector is added with histidine tag.

3. a kind of recombinant expression carrier suitable for Corynebacterium glutamicum as claimed in claim 2, it is characterised in that: described outer Source protein gene is xylanase gene, and the carrier framework of the recombinant expression carrier is pxmj19 plasmid.

4. a kind of recombinant expression carrier suitable for Corynebacterium glutamicum as claimed in claim 3, it is characterised in that: described heavy The nucleotide sequence of group expression vector is as shown in SEQ ID NO:5.

5. exogenous protein expression system comprising Corynebacterium glutamicum, the Corynebacterium glutamicum are transferred to just like claim 1 ~ 4 In any recombinant expression carrier.

6. the application of carrier as described in any in claim 1 ~ 4 secreting, expressing zytase in corynebacterium glutamicum.

7. the preparation method of zytase, which is characterized in that this method comprises the following steps:

(1) xylanase gene is obtained, the nucleotide sequence of the xylanase gene is as shown in SEQ ID NO:1；

(2) histidine tag is added in the xylanase gene downstream, obtains improved xylanase gene；

(3) improved xylanase gene and cspB promoter and signal peptide are connected on carrier pxmj19, are weighed Group expression vector pxmj19- cspB- cspB-xynA, the nucleotide sequence of the carrier pxmj19 such as SEQ ID NO:2 institute Show, the nucleotide sequence of the cspB promoter is as shown in SEQ ID NO:3, and the nucleotide sequence of cspB signal peptide is such as Shown in SEQ ID NO:4；

(4) the recombinant expression carrier pxmj19- cspB- cspB-xynA is transferred to Corynebacterium glutamicum, is recombinated Bacterium；

(5) recombinant bacterium, secreting, expressing zytase are cultivated；

(6) purifying of zytase, comprising:

A, the culture of recombinant bacterium is centrifuged, obtains the supernatant containing zytase；

B, the supernatant by described containing zytase carries out affinity chromatography, and medium is nickel column, obtains the elution containing zytase Liquid；

C, the eluent by described containing zytase carries out desalination, obtains the xylanase solution of purifying.

8. the preparation method of zytase according to claim 7, it is characterised in that: the glycosides of the recombinant expression carrier Acid sequence is as shown in SEQ ID NO:5.

9. the preparation method of zytase according to claim 7, it is characterised in that: in step (6), in affinity chromatography Balance, the buffer solution A of loading are as follows: 300 mMNaCl, the buffer of 20 mMTris, pH 8.0, the buffer for elution B are as follows: 300 mMNaCl, 20 mMTris, 250 mM imidazoles, the buffer of pH 8.0.

10. the preparation method of zytase according to claim 7, which is characterized in that in step (6), the desalination Medium is desalting column.

Technical field

The present invention relates to a kind of suitable for the recombinant expression carrier of Corynebacterium glutamicum, exogenous protein expression system, application With the preparation method of zytase, belong to field of biotechnology.

Background technique

Corynebacterium glutamicum is a kind of gram-positive bacteria of high GC content, is widely used in commercial scale amino acid, Such as glutamic acid and organic acid.In recent years, in addition to the production as amino acid, Corynebacterium glutamicum is also used as external source Protein expression host.Successfully there are Fab, scFv and vhh etc. with the albumen that Corynebacterium glutamicum gives expression to.Relative to being commonly used to For the Escherichia coli for expressing foreign protein, since Corynebacterium glutamicum is a kind of GRAS(Generally Recognized As Safe) bacterium, endotoxin-free and its cell membrane are single layer, and foreign protein is easy to be secreted into extracellular, need not move through broken, and born of the same parents Interior expression needs to be crushed, and processing cost is higher before purification；It is less to detect that protease exists extracellular, so being secreted into extracellular Albumen is relatively stable.

Therefore, it is necessary to the key element in existing carrier, such as the corresponding promoter of foreign protein genes, signal Peptide is designed, so that foreign protein is expressed in corynebacterium glutamicum and expression with higher.

Summary of the invention

In view of the above-mentioned problems, the present invention provides a kind of recombinant expression carrier suitable for Corynebacterium glutamicum, the expression Carrier is suitable for expressing foreign protein and expression with higher in corynebacterium glutamicum.

Its technical solution is a kind of such, recombinant expression carrier suitable for Corynebacterium glutamicum, it is characterised in that: institute The promoter for stating foreign protein genes in recombinant expression carrier is cspB promoter, signal peptide is cspB signal peptide, described The nucleotide sequence of cspB promoter is as shown in SEQ ID NO:3, the nucleotide sequence of cspB signal peptide such as SEQ ID Shown in NO:4.

Further, the foreign protein genes downstream of the recombinant expression carrier is added with histidine tag.

Further, the foreign protein genes are xylanase gene, and the carrier framework of the recombinant expression carrier is Pxmj19 plasmid.

Further, the nucleotide sequence of the recombinant expression carrier is as shown in SEQ ID NO:5.

The present invention also provides exogenous protein expression systems comprising Corynebacterium glutamicum, the Corynebacterium glutamicum turn Enter to have any of the above-described recombinant expression carrier.

The present invention also provides the applications of above-mentioned recombinant expression carrier secreting, expressing zytase in corynebacterium glutamicum.

The present invention also provides the preparation methods of zytase, which is characterized in that this method comprises the following steps:

(1) xylanase gene is obtained, the nucleotide sequence of the xylanase gene is as shown in SEQ ID NO:1；

(2) histidine tag is added in the xylanase gene downstream, obtains improved xylanase gene；

(3) improved xylanase gene and cspB promoter and signal peptide are connected on carrier pxmj19, are weighed Group expression vector pxmj19- cspB- cspB-xynA, the nucleotide sequence of the carrier pxmj19 such as SEQ ID NO:2 institute Show, the nucleotide sequence of the cspB promoter is as shown in SEQ ID NO:3, and the nucleotide sequence of cspB signal peptide is such as Shown in SEQ ID NO:4；

(4) the recombinant expression carrier pxmj19- cspB- cspB-xynA is transferred to Corynebacterium glutamicum, is recombinated Bacterium；

(5) recombinant bacterium, secreting, expressing zytase are cultivated；

(6) purifying of zytase, comprising:

A, the culture of recombinant bacterium is centrifuged, obtains the supernatant containing zytase；

B, the supernatant by described containing zytase carries out affinity chromatography, and medium is nickel column, obtains the elution containing zytase Liquid；

C, the eluent by described containing zytase carries out desalination, obtains the xylanase solution of purifying.

Further, the nucleotide sequence of the recombinant expression carrier is as shown in SEQ ID NO:5.

Further, in step (6), the buffer solution A of balance, loading in affinity chromatography are as follows: 300 mMNaCl, 20 The buffer of mMTris, pH 8.0, the buffer solution B for elution are as follows: 300 mMNaCl, 20 mMTris, 250 mM imidazoles, pH 8.0 buffer.

Closely a bit, in step (6), the medium of the desalination is desalting column.

Beneficial effects of the present invention are as follows: (1) using Corynebacterium glutamicum as the expressive host of the safe foreign protein of representative, Relative to can generate endotoxin and host e. coli with duplicature for, Corynebacterium glutamicum does not produce endotoxin, compares Safety, cell monolayer film are conducive to heterologous protein secretion to extracellular；(2) foreign protein in the present invention is during expression, In Under the guidance of signal peptide, the foreign protein of expression can be secreted into extracellular, need not move through bacterial cell disruption, the foreign protein secreted out of It is easy to purify relative to intracellular expression；And without extracellular protease, the foreign protein secreted out of is relatively stable in the medium；(3) In the end of foreign protein added with histidine tag, only need a step affinitive layer purification that can obtain purer foreign protein, Intracellular expression purifying relative to Escherichia coli is relatively simple.

Biological deposits explanation

Classification system: Corynebacterium glutamicum；

Chinese translation: Corynebacterium glutamicum

Depositary institution's full name: China General Microbiological culture presevation administrative center；

Depositary institution's abbreviation: CGMCC；

Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；

Preservation date: on May 3rd, 2016

Deposit number: CGMCC1.15647.

Detailed description of the invention

Fig. 1 is pxmj19- cspB- cspB-xynA carrier structure figure.

Fig. 2 is pxmj19-cspB-cspA-xynA carrier structure figure.

Fig. 3 is that the SDS-PAGE of expressed xylanase schemes, and swimming lane 1 is albumen Marker in figure, and swimming lane 2 is to contain xylan Enzyme supernatant, swimming lane 3 are that purifying flows through liquid supernatant, and remaining swimming lane be the zytase purified.Arrow meaning is zytase egg Informal voucher band.

Specific embodiment

According to following embodiments, the present invention can be better understood.However, as it will be easily appreciated by one skilled in the art that real It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited Invention.

Following embodiments are using zytase as purpose albumen, i.e. foreign protein, since promoter and signal peptide are secretion table The key element reached, so the destination protein of expression is not limited to zytase, carrier is also not necessarily limited to pxmj19.

Xylan is the main component of plant hemicellulose, be widely present in nature, content is only second to cellulose Renewable polysaccharide resource.Zytase is one of hydrolase crucial in xylan hydrolysis enzyme system, is by the inscribe mode of action β-Isosorbide-5-Nitrae-xylose glycosidic bond enzyme in degradation of xylan molecule, hydrolysate are mainly reduced sugar xylose and xylobiose, wood The xylo-oligosaccharides such as three pools, there are also a small amount of arabinoses.

In paper-making industry, (there is the paper pulp after Enzymatic Deinking zytase high whiteness, high-freedom degree and low-residual ink, reduction to subtract Slow chemical method bring environmental pollution, is a kind of method of environmental protection), feed industry (hemicellulose that cannot be digested of degrading, Be conducive to the degradation using polysaccharide, increase the utilization rate of animal feed) and food industry (wheat flour improvement, elimination fermentation Excessively harm, improves the processing equipment performance of dough, improves bread core quality and delaying aging etc.) etc. all have in industrial productions There is biggish application value.

Plasmid extracts AxyPrep Plasmid Miniprep Kit and AxyPrep DNA plastic recovery kit and is purchased from Axygen, EcoRV and EcoRI restriction endonuclease are purchased from Thermo, homologous recombination kit ClonExpress Ultra One Step Cloning Kit is purchased from Nanjing Vazyme Biotechnology Co., Ltd.；Nickel column is 1 ml His-Tag, is purchased from Roche；Desalting column For G25 desalting column, it is purchased from and adds Lake.Zytase detection kit endo-XYLANASE ASSAY PROCEDURE is purchased from Megazyme company；Pxmj19 is purchased from general such as spit of fland biotechnology (Beijing) Co., Ltd.