Method for increasing expression amount of pichia pastoris for secreting and expressing plectasin

A technology of mycelia and Pichia pastoris, which is applied in the field of biotechnology and genetic engineering, can solve the problems that mycelia engineering bacteria cannot fully exert protein secretion expression, retention, and protein folding is too late

Inactive Publication Date: 2018-05-04
GUANGDONG HINAPHARM PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the protein expression level exceeds the processing capacity of the cell itself, the excess protein will remain in the endoplasmic reticulum because it is too late to fold correctly (correctly form 3 pairs of disulfide bonds, etc.), thereby triggering the unfolded protein response (UPR), resulting in protein The expression level decreased, so that plectasin engineered bacteria failed to fully exert their ability to secrete and express proteins
At present, there are no case reports on protein folding and other related auxiliary expression of Plectasin

Method used

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  • Method for increasing expression amount of pichia pastoris for secreting and expressing plectasin
  • Method for increasing expression amount of pichia pastoris for secreting and expressing plectasin
  • Method for increasing expression amount of pichia pastoris for secreting and expressing plectasin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Construction of protein disulfide bond isomerase expression vector

[0063] According to Pichia pastoris protein disulfide bond isomerase PDI gene sequence, design amplification primers

[0064] PDI-F: 5'-TATTCGAAATGCAATTCAACTGGAATAT-3' and

[0065] PDI-R: 5'-GTGAATTCTTAAAGCTCGTCGTGAGCGT-3';

[0066] Add the BstBI restriction site TTCGAA at the 5' end of the gene, and add the EcoRI restriction site GAATTC at the 3' end of the gene;

[0067] Using the Pichia pastoris genome as a template, the protein disulfide bond isomerase gene PDI was amplified, and the gene size was 1554bp. The PDI fragment was digested with BstBI and EcoRI, and connected to the pPIC6αA vector fragment that was also digested with BstBI and EcoRI to obtain the protein disulfide bond isomerase expression vector pPIC-PDI. The ligation product was heat-shocked at 42°C and transformed into Escherichia coli Top10 competent cells, spread on an LB resistance plate, and cultured overnight at 37°C...

Embodiment 2

[0071] Example 2 Transformation of Plectasin Engineering Bacteria

[0072] Treat the pPIC-PDI expression vector with PmeI restriction endonuclease, cut the gel to recover the linearized expression vector, electroporate (1.5-2.5kv) to PLE-2 Pichia pastoris expression strain, and spread the bacterial solution on YPD plate (containing 0.1-0.5mg / ml Blasticidin), incubate at 30°C for 2-3 days until a single colony grows.

[0073] Pick a single colony, cultivate and evaluate the expression of plectasin, detect the size of the inhibition zone of the fermentation supernatant, and screen to obtain the PDI strain.

[0074] Bacterial inhibition zone detection: Inoculate the indicator bacteria (Staphylococcus aureus CMCC26003) into the MH medium to prepare the indicator bacteria solution. Dilute with normal saline to OD600=2.3, take 100μL diluted indicator bacteria solution to 100mL MH solid medium (temperature 50-55℃), mix well, take 10.5mL solid medium to a standard petri dish, cool an...

Embodiment 3

[0075] Example 3 Induced Expression of Recombinant Plectasin

[0076] A single colony of the PDI strain prepared in Example 2 was picked, inoculated in 25 mL of BMGY medium, and cultured at 30° C. and 220 rpm for 24 hours to prepare a primary seed solution.

[0077] 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.

[0078] All secondary seed liquids were inoculated in a 5L fermenter (2L BSM medium), controlled temperature 30°C±0.5°C, dissolved oxygen 20%±5%, pH=5.0±0.5; after the basic glycerin was exhausted, add 10% glycerol , after glycerol was exhausted until dissolved oxygen rose to 90%, starved for half an hour, fed with methanol to induce the expression of plectasin, induced for a total of 72 hours, centrifuged (6000×g, 5min) to take the fermentation supernatant for detection.

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Abstract

The invention discloses a method for increasing expression amount of pichia pastoris for secreting and expressing plectasin. The method is characterized in that a pichia pastoris genome is used as a template, a PDI (protein disulfide isomerase) gene is amplified, a PDI segment is treated by BstBI and EcoRI double enzyme digestion, and is connected to a pPIC6aphlaA carrier segment which is also treated by the BstBI and EcoRI double enzyme digestion, and an expression carrier pPIC-PDI is formed; the PDI is expressed in a plectasin expression strain, three pairs of disulfide bonds of the plectasin are promoted to form a correct space structure, the plectasin is smoothly secreted and expressed out of cells, and the purpose of increasing the expression amount of plectasin is realized. The method has the advantage that the expression amount of plectasin is increased by 30%.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and genetic engineering, in particular to a method for increasing the expression level of Pichia secreted and expressed by Pichia pastoris. Background technique [0002] Plectasin is the first fungal defensin isolated from the saprotrophic ascomycete Pseudolectania nigrella. Plectasin has obvious killing effect on Gram-positive bacteria, including: Streptococcus, Staphylococcus, Enterococcus, Corynebacterium and Bacillus. NZ2114 is a mutant of Plectasin, which contains 3 amino acid mutations. Compared with Plectasin, the inhibitory effect of NZ2114 mutant on Staphylococcus aureus is increased by 30 times. The NZ2114 gene was transformed into Pichia pastoris to obtain the PLE-2 engineering strain, which could secrete and express Plectasin, and the expression amount reached 4.9g / l. The prepared plectasin samples had obvious inhibitory effect on Staphylococcus aureus, Clostridium welchii, St...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N15/81C12N15/61C12R1/84
CPCC07K14/37C12N9/90C12Y503/04001
Inventor 梁伟凡周玉岩丁小云李洪金丘春尚林绿淼
Owner GUANGDONG HINAPHARM PHARMA CO LTD
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