Pichia pastoris expressing recombinant Thanatin antibacterial peptide

A Pichia pastoris and antibacterial peptide technology, applied in the fields of biotechnology and genetic engineering, can solve the problems of low expression of death factor and high production cost, and achieve the effects of reducing production cost and facilitating separation and purification

Inactive Publication Date: 2018-04-13
GUANGDONG HINAPHARM PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a Pichia pastoris expressing Thanatin antibacterial peptide,

Method used

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  • Pichia pastoris expressing recombinant Thanatin antibacterial peptide
  • Pichia pastoris expressing recombinant Thanatin antibacterial peptide
  • Pichia pastoris expressing recombinant Thanatin antibacterial peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of Deathin Expression Strain

[0059] 1.1 Design of the deadin THA gene based on the codon preference of Pichia pastoris

[0060] According to Pichia pastoris ( Pichia pastoris ) codon preference:

[0061] Amino Acid Codon Frequency ‰ Quantity

[0062] Phe UUU 24.1 1963

[0063] Phe UUC 20.6 1675

[0064] Leu UUA 15.6 1265

[0065] Leu UUG 31.5 2562

[0066] Ser UCU 24.4 1983

[0067] Ser UCC 16.5 1344

[0068] Ser UCA 15.2 1234

[0069] Ser UCG 7.4 598

[0070] Tyr UAU 16.0 1300

[0071] Tyr UAC 18.1 1473

[0072] STOP UAA 0.8 69

[0073] STOP UAG 0.5 40

[0074] Cys UGU 7.7 626

[0075] Cys UGC 4.4 356

[0076] STOP UGA 0.3 27

[0077] Trp UGG 10.3 834

[0078] Leu CUU 15.9 1289

[0079] Leu CUC 7.6 620

[0080] Leu CUA 10.7 873

[0081] Leu CUG 14.9 1215

[0082] Pro CCU 15.8 1282

[0083] Pro CCC 6.8 553

[0084] Pro CCA 18.9 1540

[0085] Pro CCG 3.9 320

[0086] His CAU 11.8 960

[0087] His CAC 9.1 737

[0088] H...

Embodiment 2

[0138] Example 2 Induced Expression of Recombinant Deathin

[0139] Pick the Pichia pastoris cells expressing deathin prepared in Example 1, inoculate them in 25 mL of BMGY medium, and culture them at 30° C. and 220 rpm for 24 hours to prepare a primary seed solution.

[0140] Inoculate 20 mL of primary seed liquid into 200 mL of BMGY medium to prepare secondary seed liquid, and culture at 30°C and 220 rpm for 24 hours.

[0141] All the secondary seed liquids were inoculated in a 5L fermenter (2L BSM medium), the temperature was controlled at 30°C±0.5°C, the dissolved oxygen was 20%±5%, and the pH=5.0±0.5.

[0142] After the basal glycerol is exhausted, add 10% glycerol. After the glycerol is exhausted, until the dissolved oxygen suddenly rises, starve for half an hour, add methanol to induce the expression of deadin, and induce the expression of death factor for a total of 120 hours. Centrifuge (6000×g, 5min) to take the fermentation supernatant to test.

Embodiment 3

[0143] Example 3 Detection of Recombinant Deathin Protein Concentration and Protein Electrophoresis

[0144] The total protein concentration of the supernatant of the fermentation broth in the example was measured by the Bradford method, and the results showed that the total protein concentration of the supernatant of the fermentation supernatant induced by the deadin expression strain for 120 hours was 0.67 g / L.

[0145] see image 3 , Tricine-SDS-PAGE protein electrophoresis analysis of the content of deathin in the fermentation supernatant showed that after protein electrophoresis in the fermentation supernatant, there was a protein band with a target size between 3 and 6KD, and the molecular weight of the protein band was similar to that of deathin. The molecular weight is similar, about 2.4KD; through software analysis, the content of death protein is more than 90%. The total protein concentration of the fermentation supernatant was 0.67g / L, and the corresponding death p...

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Abstract

The invention discloses a pichia pastoris expressing a recombinant Thanatin antibacterial peptide. The recombinant Thanatin antibacterial peptide comprises the amino acid sequence of GSKKPVPIIYCNRRTGKCQRM. The pichia pastoris is used for the secretory expression of Thanatin, large-scale expression of Thanatin is realized, and production cost is reduced. By the adoption of the pichia pastoris production strain, the highest expression level of Thanatin reaches 0.6 g/L, the highest level in public reports; besides, a Thanatin antibacterial peptide exists in a fermentation supernatant, there is noneed to break cells, and a Thanatin sample containing few impure proteins can be obtained simply through filtration to remove thalli, which facilitates subsequent separation and purification of Thanatin and reduces production cost. The prepared Thanatin sample has a significant bacteriostatic effect on escherichia coli and salmonella.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and genetic engineering, in particular to a Pichia pastoris expressing recombinant Thanatin antimicrobial peptide. Background technique [0002] Thanatin (THA), also known as death peptide, is a kind of insect in the stink bug ( Podisus maculiventris ) is one of the antimicrobial peptides with the broadest antibacterial spectrum found so far. It not only has killing activity against Gram-positive bacteria and Gram-negative bacteria, but also has inhibitory effect on some fungi. Animal cells do not exhibit hemolysis and are a potential alternative to antibiotics. Moratin consists of 21 amino acid residues, containing Cys residues at the 11th and 18th positions, which can form intramolecular disulfide bonds, and the disulfide bonds play an important role in the stability of dietin. NMR and molecular dynamics simulated the three-dimensional structure of deadin in aqueous solution. The dead...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/66C12P21/02C12R1/84
CPCC07K14/43563C12N15/66C12N15/815C12P21/02
Inventor 梁伟凡周玉岩李洪金丁小云林绿淼丘春尚
Owner GUANGDONG HINAPHARM PHARMA CO LTD
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