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Method for increasing expression of CP4-EPSPS in Hansenula polymorpha

A technology of CP4-EPSPS and Hansenula, which is applied to peptide preparation methods, microbial-based methods, chemical instruments and methods, and can solve problems such as increasing the expression level of CP4-EPSPS

Active Publication Date: 2012-07-11
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] Before the present invention, there was no report on the constitutive expression of CP4-EPSPS in Hansenula using codon-optimized CP4-EPSPS gene, and no one used a composite enhancer sequence to greatly increase the expression of CP4-EPSPS in Hansenula. The expression level in Sclerotinia cells

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  • Method for increasing expression of CP4-EPSPS in Hansenula polymorpha
  • Method for increasing expression of CP4-EPSPS in Hansenula polymorpha
  • Method for increasing expression of CP4-EPSPS in Hansenula polymorpha

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Embodiment 1

[0040] Embodiment 1: Artificial synthesis of TYMV-HCMV composite enhancer

[0041] According to the published DNA sequences of Turnip yellowmosaic virus (TYMV) (see GenBank, accession number: X07441) and Human cytomegalovirus (HCMV) (see GenBank, accession number: K03104), according to the codon preferred by yeast (Sharp P M, et al., 1986), artificially designed and synthesized a new composite enhancer sequence (see SEQ ID NO: 1), in this sequence, 105 nucleotide bases are the 3' non-coding region of TYMV (The 6214th to 6318th nucleotide base of the TYMV genome) (see attached figure 1 The single underlined part in the middle), there are 490 nucleotide bases derived from the core part of the early gene enhancer of HCMV (the 201st to 690th nucleotide bases) (see appendix figure 1 At the same time, in order to facilitate the construction of yeast expression vectors, in the process of artificially synthesizing the composite enhancer sequence, a restriction enzyme site EcoR was sy...

Embodiment 2

[0042] Embodiment 2: Artificial synthesis of CP4-EPSPS gene after codon optimization

[0043] The CP4-EPSPS gene is derived from the Agrobacterium CP4 strain, and its codons are preferred by prokaryotes, while Hansenula belongs to eukaryotes, so there are certain differences in their gene codon preferences, and this difference is likely to cause It affects the stability and expression efficiency of CP4-EPSPS gene and its transcription product in Hansenula cells. In order to improve the biological yield of CP4-EPSPS, according to the DNA sequence (originally plant-preferred codon) and amino acid sequence (see GenBank, accession number: JF445290) of the published CP4-EPSPS gene, without changing its amino acid sequence Next (see SEQ ID NO: 2), according to the preferred codons of yeast (Sharp P M, et al., 1986), the DNA coding sequence of the new CP4-EPSPS mature protein was artificially designed and synthesized, codon optimization and transformation Compared with the original ...

Embodiment 3

[0044] Embodiment 3: Cloning of TYMV-HCMV composite enhancer and CP4-EPSPS gene

[0045] The above artificially synthesized TYMV-HCMV composite enhancer sequence and CP4-EPSPS fusion protein gene DNA fragment were directly inserted into the T site of pEASY-T1 (purchased from Beijing TransGenic Company) plasmid, according to the method provided by the company , to obtain bacterial clones containing intermediate plasmid vectors TYMV-HCMV-T and CP4-EPSPS-T (see attached image 3 ), and then, through DNA sequencing, it was determined that the TYMV-HCMV-T composite enhancer and CP4-EPSPS-T gene sequences contained in it were correct and complete (DNA sequencing was completed by Beijing Biaokai Technology Co., Ltd.).

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Abstract

The invention relates to the field of genetic engineering, particularly to a method for realizing high expression of CP4 (cysteine proteinase 4)-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) fusion protein gene in a constitutive manner in Hansenula polymorpha cells by using a composite enhancer. The method comprises the following steps: (1) improving the yield of codon-optimized CP4-EPSPS fusion protein gene in Hansenula polymorpha by using the composite enhancer; (2) regulating and controlling CP4-EPSPS fusion protein gene to express in a constitutive manner in polytype Hansenula by adopting Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter; (3) fermenting optimized Hansenula polymorpha; and (4) rapidly purifying the recombinant heterologous protein by employing nanofiltration and nickel column affinity chromatography. The CP4-EPSPS recombinant fusion protein prepared by the method can serve as an alternative protein standard substance in the field of study on the origin of protein and the like.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a method for using a composite enhancer to realize the high constitutive expression of the CP4-EPSPS fusion protein gene in Hansenula polymorphic cells, including: 1) using a composite enhancer to Improve the yield of CP4-EPSPS fusion protein gene after codon optimization in Hansenula; Constitutive expression; 3) optimized Hansenula fermentation and growth conditions; 4) a method for rapidly purifying the recombinant exogenous protein by using nanofiltration and nickel column affinity chromatography. The CP4-EPSPS recombinant fusion protein prepared by this method can be used as a candidate protein standard substance for protein traceability research and other fields. Background technique [0002] The lack of measurement methods and measurement standards for genetically modified plant proteins makes the detection results untraceable, so it is difficult to guarantee the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/67C12N15/81C12N15/62C12N15/113C12P21/00C07K1/34C12R1/78
Inventor 刘德虎王楠李刚强刘树鹏
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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