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Application of pichia pastoris translation correlation factor Bcy1 to foreign protein expression

A technology of Pichia pastoris and related factors, which is applied in the field of bioengineering to achieve the effects of promoting cell growth, increasing cell biomass accumulation, and strong versatility

Active Publication Date: 2019-11-22
拜斯特药业(广州)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no report that Bcy1 promotes cell growth in the stationary phase of Pichia pastoris fermentation and increases the expression of foreign proteins

Method used

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  • Application of pichia pastoris translation correlation factor Bcy1 to foreign protein expression
  • Application of pichia pastoris translation correlation factor Bcy1 to foreign protein expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Construction of recombinant Pichia pastoris expressing enhanced green fluorescent protein eGFP and phytase Phy

[0061] After entrusting Shanghai Jierui Bioengineering Co., Ltd. to synthesize the eGFP gene, the eGFP gene fragment and the vectors pPIC9K and pPICZA were digested with EcoRI and NotI respectively. After recovery and purification, the vectors pPIC9K-eGFP and pPICZA- eGFP; and then use BamHI and MluⅠ to double digest the plasmids pPICZA-eGFP and pPICHKA respectively, recover and purify the fragments pPIC-eGFP and HKA (His-Kan-AOX1 fragment, about 5300bp), and use T4 DNA ligase to ligate to obtain the vector pPICZA-HKA -eGFP; at the same time, after entrusting Shanghai Jierui Bioengineering Co., Ltd. to synthesize the Phy gene, the Phy gene fragment and the vector pPIC9K were double-digested with EcoRI and NotI respectively, and after recovery and purification, the vector pPIC9K-Phy was obtained by T4DNA ligase connection; The vectors pPIC9K-eGFP, p...

Embodiment 2

[0070] Example 2: Cloning and expression of Pichia pastoris translation-related factor Bcy1 gene

[0071] (1) Cloning of Pichia pastoris translation-related factor Bcy1 gene

[0072] Design Bcy1 gene amplification primers ZA-Bcy1-S and ZA-Bcy1-A according to the gene sequence SEQ ID No.2 of Pichia pastoris translation-related factor Bcy1; wherein:

[0073] The amino acid sequence of Pichia pastoris translation-related factor Bcy1 is shown below (SEQ ID No.1):

[0074] MTAYTDVVNDLSRELSLKKPKDVIQFCADYFNNKLAEDPARQQPSTATAPSSRPAQTASGAPLFKDQFGGDPHEPLSHHPHRGSIFKDSFTHPEDPQSNKIRPLDNASPGSGFAAHLAKVKFNPERRISVSAESVNPEIFATSSWQPPVHDLSDEQLQRLNNIVRKSFLFSQLEDEALRTVIYALEEKKVPRGTEIIKQGDEGDYFYVLESGEVTFVVNGETKGQGKSGSTFGELALMYNSPRAATVISSQDCVLWALDRMTFRRILLEGTAKRRAMYDGFLKDVPVLRCLNSYERNKLADALESETYNKGDTIIREGEPGENFYFIEQGEAEVFKEGEGHMATLGKGDYFGELALLNDLPRQATVVAKSDVIKVVTLDKNGFQRLLGPAIEVLKLQDPTKI。

[0075] The nucleotide sequence of Pichia pastoris translation-related factor Bcy1 is as follows (SEQ ID No.2): ...

Embodiment 3

[0090] Example 3: The effect of translation-related factor Bcy1 on the expression of secreted and intracellularly expressed eGFP

[0091] Transform the plasmid pPICZA-Bcy1 into recombinant Pichia pastoris GS115 / pPIC9K-eGFP (secretion type) and GS115 / pPICZA-HKA-eGFP (intracellular expression type) competent cells respectively, use YPDZ plate for screening, and pick transformants for use Primers 5'AOX1 and ZA-Bcy1-A were verified by PCR to obtain positive transformants. Then pick the positive transformant and culture it in a 50ml shake flask with 5ml BMGY medium and culture it for 16-20 hours. After the OD600 reaches 2-6, transfer it to a 250ml shake flask with 25ml BMMY medium. The initial OD600 of the strain is 1.0 , under the conditions of 30° C. and 250 rpm, 1% (v / v) methanol was used to induce fermentation for 120 hours, 1% (v / v) methanol was added every 24 hours, and samples were taken every 24 hours to determine the cell growth curve and fluorescence curve. Pichia pastor...

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Abstract

The invention discloses application of a pichia pastoris translation correlation factor Bcy1 to foreign protein expression. According to application, it is discovered that the biomass in the cell fermentation process can be significantly improved by the pichia pastoris translation correlation factor Bcy1 (annotation: protein kinase A regulated subunit), the expression quantity of enhanced green fluorescence protein eGFP is improved, the expression quantity of phytase Phy is improved, the cell growth of pichia pastoris during stable fermentation can be promoted, cell biomass accumulation is increased, it is indicated that the pichia pastoris translation related factor Bcy1 has the high universality, and the pichia pastoris translation related factor Bcy1 has good application prospects to aspects of cell biomass accumulation during fermentation and foreign protein yield improvement.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to the application of a Pichia pastoris translation-related factor Bcy1 in the expression of foreign proteins. Background technique [0002] The Pichia pastoris expression system is a general and efficient exogenous protein expression system, which is widely used in the expression and production of various exogenous proteins and in the study of protein expression and secretion regulation mechanisms. In addition to the research on the metabolic module of Pichia pastoris, the exogenous protein synthesis regulation module based on the "central dogma" of protein synthesis is mainly focused on the gene transcription module, protein processing and folding module and its transport and secretion module. However, the transcription level and protein expression level of genes are not completely consistent, and translation and post-translational modifications are required from mRNA to protein. As...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/81C12N1/19C12N9/16C07K14/435C12R1/84
CPCC07K14/43595C12N9/1205C12N9/16C12N15/815
Inventor 林影廖锡豪林雯炀梁书利
Owner 拜斯特药业(广州)有限公司
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