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Method for effectively expressing cationic antibacterial peptides in pichia pastoris

A Pichia pastoris, cationic technology, applied in the field of group engineering, can solve the problems of toxicity of host cells, protease attack, consumption of large manpower and material resources, etc., and achieve the effects of reducing toxic effects, increasing expression, and good application prospects.

Active Publication Date: 2010-12-15
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, since antimicrobial peptides are basic polypeptides with small molecules containing multiple basic amino acids, they are extremely vulnerable to protease attacks
The products expressed at the same time are often toxic to the host cells, which will directly inhibit the growth of the host bacteria and affect the expression efficiency
Therefore, the exogenous expression of antimicrobial peptides is more difficult than other peptide drugs
In addition, when screening high-efficiency-expressing engineering bacteria for antibacterial peptides, antimicrobial peptides are usually used to have special antibacterial activity. Screening through antibacterial experiments requires thousands of transformants to be identified each time. Preliminary screening is carried out, and the engineering bacteria with relatively high antibacterial activity are screened by antibacterial, and then re-screened, which consumes a lot of manpower and material resources
Therefore, how to protect antimicrobial peptides from protease attacks, reduce their toxicity to host cells, and how to screen yeast engineering bacteria that express antimicrobial peptides efficiently have become bottlenecks in the development of antimicrobial peptides.

Method used

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  • Method for effectively expressing cationic antibacterial peptides in pichia pastoris
  • Method for effectively expressing cationic antibacterial peptides in pichia pastoris
  • Method for effectively expressing cationic antibacterial peptides in pichia pastoris

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Embodiment 1

[0020] Cloning and sequence analysis of the acid xylanase gene of embodiment 1

[0021] Total RNA was extracted from Bispora sp.MEY-1 using a total RNA extraction kit for RT-PCR. Use 5 μg of total RNA as the template oligo (dT) as the primer to synthesize the first strand of cDNA, and use the upstream and downstream primers xyl11A-F and xyl11A-R (see Table 1) of acid xylanase XYL11A to obtain xylanase by PCR Full-length fragment of cDNA of gene xyl11A. Cloning the fragment into pEASY-T 3 On the vector, get pEASY-T 3 -xyl11A, after sequencing proved to be correct, it was used as a template to clone the gene sequence of xylanase mature protein.

[0022] Design primers according to the cloned xylanase XYL11A mature protein gene coding sequence: xyl11A-m-F and xyl11A-m-R, and introduce enzyme cutting sites EcoR I and Not I at the end of the primers to sequence the correct pEASY-T 3 The -xyl11A plasmid was used as a template for PCR amplification. The PCR reaction parameters a...

Embodiment 2

[0028] Example 2 Synthesis of the gene of cationic antimicrobial peptide limulusin precursor and KEX2 enzyme cleavage site

[0029] Artificial synthesis of antimicrobial peptide genes

[0030]

[0031] **Note**

[0032] KR: It is the recognition site of yeast's own protease KEX2, which is cut at the rear end of R

[0033] EA: It is the recognition site of yeast's own protease STE13, which is cut at the back end of A

[0034] KWCFRVCYRGICYRRCKR: is the sequence of the cationic antibacterial peptide limulusin Ta I with antibacterial activity

[0035] GKRNEVRQYRDRGYDVRAIPDETFFTRQDEDEDDDEE: Antimicrobial peptide precursor partial sequence

[0036] According to the codon preference of Pichia pastoris, the sequence modification was carried out without changing its amino acid sequence, and the appearance of AT-rich sequences was avoided as far as possible, and these sequences were related to the stability of mRNA. And design the multiple cloning sites BamH I-Xho I and EcoR I ...

Embodiment 3

[0057] Embodiment 3 Construction of Fusion Gene Ta1-xyl Expression Plasmid Vector

[0058] First, the gene of the precursor of Tachyplesin I containing the KEX2 restriction site was connected to the Pichia pastoris expression vector pPIC9 to obtain pPIC9-Ta1, that is, pUC19-Ta1 and pPIC9 were first treated with Xho I and EcoR I restriction endonucleases, The vector pPIC9 and Ta1 fragments were recovered after electrophoresis, and pPIC9-Ta1 positive clones were selected after ligation transformation. Using the same method again, acid xylanase xyl11A was connected to pPIC9-Ta1, that is, the acid xylanase xyl11A obtained by EcoR I and Not I restriction endonuclease treatment pPIC9-Ta1 and PCR amplification In the protein region, the vector pPIC9-Ta1 and xyl11A fragments were recovered after electrophoresis, and the fusion expression vector pPIC9-Ta1-xyl was obtained after ligation and transformation. The positive clones of pPIC9-Ta1-xyl were selected by enzyme digestion verificat...

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Abstract

The invention relates to the field of genetic engineering, in particular to a method for effectively expressing cationic antibacterial peptides in pichia pastoris. In the method, the cationic antibacterial peptides can be driven to be expressed by acid enzyme; a large amount of acidic amino acid of the acid enzyme can neutralize the alkaline amino acid of the cationic antibacterial peptides to keep the cationic antibacterial peptides out of the attack of host protease; the yield of antibacterial peptides can be judged directly by the activity of expressed recombinase; moreover, expressed products and the antibacterial peptides can be directly applied at the same time.

Description

technical field [0001] The invention relates to the field of group engineering, in particular, the invention relates to a method for effectively expressing cationic antibacterial peptides in Pichia pastoris. Background technique [0002] Cationic antibacterial peptides are cationic (containing too much lysine and arginine) amphoteric small molecule polypeptides generally composed of 12 to 50 amino acid residues. Widely present in animals, plants and microorganisms, it is an important part of the host immune defense system. It has a wide antibacterial spectrum, low immunogenicity, and is not easy to produce drug resistance and has the effect of inhibiting the growth of cancer cells. This makes antibacterial The development and utilization of peptides has become a research hotspot in recent years. However, the development and utilization of cationic antimicrobial peptides is not easy. The natural resources of antimicrobial peptides are limited, and the extraction is very comp...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/81C12N1/19C07K19/00C07K14/435C12R1/84
Inventor 姚斌黄火清杨培龙罗会颖石鹏君袁铁铮王亚茹柏映国孟昆史秀云
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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