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Host cell and method for efficient expression and secretion thereof in recombinant protein

A host cell, secretion and expression technology, which is applied in the field of microbiology and fermentation engineering, can solve the problems of high efficiency, secretion and expression, induction, and long culture time of recombinant proteins, so as to reduce the environmental pressure of fermentation industry, reduce the cost of fermentation manufacturing, The effect of simplifying the fermentation manufacturing process

Active Publication Date: 2009-04-29
福建福大百特生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that, like all other host cells, yeast host cells cannot satisfy the high-efficiency and secretory expression of most recombinant proteins; another disadvantage is that the culture time is long, and the production process requires the continuous induction of harmful substances such as methanol
The above studies also suggest that there may be a secretion capacity limit in the protein secretion ability of specific Bacillus licheniformis strains

Method used

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  • Host cell and method for efficient expression and secretion thereof in recombinant protein
  • Host cell and method for efficient expression and secretion thereof in recombinant protein
  • Host cell and method for efficient expression and secretion thereof in recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Screening of Bacillus licheniformis host cells

[0072] The selection process of Bacillus licheniformis host cells is shown in the figure below (attached figure 1 ).

[0073] 526 strains of Bacillus licheniformis were isolated from 3050 natural samples collected in China, and screened against Bacillus licheniformis ATCC14580 and CICIM B0204. All collected Bacillus licheniformis strains were inoculated with LB medium supplemented with 4% glucose, cultured at 45°C for 72 hours, observed the color of the colonies and the formation of pigments around the colonies, and determined the formation of lichenin and polyglutamic acid by staining. 24 strains of Bacillus licheniformis were selected for the next test. 24 strains were inoculated on 0.01 mg / mL tetracycline or 0.005 mg / mL kanamycin resistance plates respectively, and after culturing at 37°C for 24 hours, 18 of them were determined to be antibiotic-sensitive. The possible self-plasmids were extracted and ide...

Embodiment 2

[0078] Example 2: Cloning of restriction modification system encoding gene cluster and construction of gene deletion plasmid

[0079] Analysis of the genome sequence of B. licheniformis ATCC 14580 shows that in the region of about 17.3 kb from 4157995 bp to 4175263 bp of the genome, there is a B. licheniformis specific type I DNA restriction modification system coding gene cluster (Veith B, et al. J Mol Biotechnol, 2004). Design the following steps to delete it (attached image 3 ).

[0080] Using primers PR1 (SEQ ID NO: 1) and PR2 (SEQ ID NO: 2), PCR technology was used to amplify the upstream sequence RM2-1 of the restriction modification system gene cluster from the chromosomal DNA of Bacillus licheniformis 3008, with a size of about 560bp, ( Figure 4A); Similarly, using primers PR3 (SEQ ID NO: 3) and PR4 (SEQ ID NO: 4), the downstream sequence RM2- 2, the size is about 610bp ( Figure 4 A). Fragment RM2-1 was digested with HindIII and EcoRV and fragment RM2-2 was di...

Embodiment 3

[0081] Example 3: Deletion of DNA Restriction Modification System Encoding Gene Clusters

[0082] The recombinant plasmid pUC-RM2'::Km R Amplification was carried out in recombinant bacteria Ec(met) (Wang Zhengxiang, Niu Dandan. Chinese invention patent, publication number: CN 101230329A). After the preparation of the recombinant plasmid was digested with HindIII, the Bacillus licheniformis 3008 cells were transformed by electroporation. The transformants were spread on LB plates containing 0.025 mg / mL kanamycin and cultured for 48 hours. After the growth colony was subcultured and purified on the LB medium of 0.025 mg / mL kanamycin, it was then subcultured in the LB medium without kanamycin, and the bacterial strain that kanamycin resistance disappeared was screened, and the primer PR7( SEQ ID NO: 7) and PR8 (SEQ ID NO: 8) identify kanamycin-sensitive strains, and the restriction modification system coding gene cluster in Bacillus licheniformis 3008 is deleted, then primer P...

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Abstract

The invention relates to a host cell and a method for the host cell applied in efficient secretion expression of a recombinant protein, which belongs to the filed of microbiological engineering and fermentation engineering. The invention provides a bacillus licheniformis host cell CBB3008 which is preserved in the China Center for Type Culture Collection with the preserving number of CCTCC NO: M 208236. The invention transforms an expression plasmid of an industrial enzyme which is obtained by a gene cloning technology into the host cell to synthesize an industrial enzyme preparation in the host cell efficiently, and then secretes the synthesized enzyme protein into a culture medium efficiently through a protein secretion system of the host cell, thus the invention can guide efficient secretion production of the industrial enzyme preparation. Therefore, the invention is helpful to reduce the fermentative production cost of the industrial enzyme preparation, simplify the fermentative production process and reduce the environmental pressure of the fermentation industry. A method for host cell screening and genetic improvement of the invention can also be used for other types of host cells, in particular for the breeding of the host cells of bacillus subtilis, bacillus megaterium, bacillus pumilus, bacillus starch solution and the like.

Description

technical field [0001] The invention is a bacillus host cell used for high-efficiency secretion and expression of recombinant protein. The host cell in the present invention is a bacillus obtained through microbial strain selection. The host cell of the present invention can satisfy the high-efficiency synthesis of recombinant protein and secrete the synthetic recombinant protein into the culture medium. The recombinant protein efficiently prepared by the host cell of the present invention is an enzyme with industrial application value, and further is bulk enzyme preparations such as α-amylase and phytase with starch hydrolysis activity. The invention belongs to the field of microbiology and fermentation engineering. Background technique [0002] Important proteins obtained from nature, such as enzymes, bioactive peptides, and pharmaceutical proteins, often fail to reach commercial development value due to their low content under natural conditions, and they need to be pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/56C12N15/55C12N15/75C12N9/26C12N9/42C12N9/16C12R1/10
Inventor 王正祥牛丹丹石贵阳
Owner 福建福大百特生物科技有限公司
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