Construction, cloning, and expression of Melittin antimicrobial peptide using Pichia pastoris expression system

Gene Reports ◽  
2020 ◽  
Vol 21 ◽  
pp. 100900 ◽  
Author(s):  
Khadijeh Moridi ◽  
Mohammad Hemmaty ◽  
Mohammad Reza Akbari Eidgahi ◽  
Mohsen Fathi Najafi ◽  
Hosna Zare ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Antimicrobial Peptide ◽  
Expression System ◽  
Cloning And Expression ◽  
Pichia Pastoris Expression System ◽  
Pastoris Expression System

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

scholarly journals Immunoassays Based on Penicillium marneffei Mp1p Derived from Pichia pastoris Expression System for Diagnosis of Penicilliosis

PLoS ONE ◽  
2011 ◽  
Vol 6 (12) ◽  
pp. e28796 ◽  
Author(s):  
Yan-Fang Wang ◽  
Jian-Piao Cai ◽  
Ya-Di Wang ◽  
Hui Dong ◽  
Wei Hao ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Expression System ◽  
Penicillium Marneffei ◽  
Pichia Pastoris Expression System ◽  
Pastoris Expression System

scholarly journals Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

2011 ◽  
Vol 10 (1) ◽  
pp. 24 ◽  
Author(s):  
Hidetsugu Asada ◽  
Tomoko Uemura ◽  
Takami Yurugi-Kobayashi ◽  
Mitsunori Shiroishi ◽  
Tatsuro Shimamura ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Pichia Pastoris ◽  
Expression System ◽  
Pichia Pastoris Expression System ◽  
Pastoris Expression System

scholarly journals Characterization of a highly stable α-galactosidase from thermophilic Rasamsonia emersonii heterologously expressed in a modified Pichia pastoris expression system

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Jian-Lu An ◽  
Wei-Xin Zhang ◽  
Wei-Ping Wu ◽  
Guan-Jun Chen ◽  
Wei-Feng Liu
Keyword(s):  
Pichia Pastoris ◽  
Promoter Activity ◽  
Expression System ◽  
Maximum Activity ◽  
Heterologous Proteins ◽  
Biotechnological Applications ◽  
Pichia Pastoris Expression System ◽  
Pastoris Expression System ◽  
Structurally Stable

Abstract Background Structurally stable α-galactosidases are of great interest for various biotechnological applications. More thermophilic α-galactosidases with high activity and structural stability have therefore to be mined and characterized. On the other hand, few studies have been performed to prominently enhance the AOX1 promoter activity in the commonly used Pichia pastoris system, in which production of some heterologous proteins are insufficient for further study. Results ReGal2 encoding a thermoactive α-galactosidase was identified from the thermophilic (hemi)cellulolytic fungus Rasamsonia emersonii. Significantly increased production of ReGal2 was achieved when ReGal2 was expressed in an engineered Pastoris pichia expression system with a modified AOX1 promoter and simultaneous fortified expression of Mxr1 that is involved in transcriptionally activating AOX1. Purified ReGal2 exists as an oligomer and has remarkable thermo-activity and thermo-tolerance, exhibiting maximum activity of 935 U/mg towards pNPGal at 80 °C and retaining full activity after incubation at 70 °C for 60 h. ReGal2 is insensitive to treatments by many metal ions and exhibits superior tolerance to protein denaturants. Moreover, ReGal2 efficiently hydrolyzed stachyose and raffinose in soybeans at 70 °C in 3 h and 24 h, respectively. Conclusion A modified P. pichia expression system with significantly enhanced AOX1 promoter activity has been established, in which ReGal2 production is markedly elevated to facilitate downstream purification and characterization. Purified ReGal2 exhibited prominent features in thermostability, catalytic activity, and resistance to protein denaturants. ReGal2 thus holds great potential in relevant biotechnological applications.

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