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Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
Gene cloning and expression of MAP30 in Pichia pastoris
Fang Wang1, Chun-Yu Chi1, Li-Yuan Wang1
1Heilongjiang Provincial Key Laboratory of Plant Biology, Life Science and Technology College, Harbin Normal University , Harbin , China.
Abstract:
MAP30, a single-stranded type-I ribosome inactivating protein found in Momordica charantia, shows anti-HIV and anti-tumour activity. It could significantly inhibit the HIV-1 and herpes simplex virus infection. In this study, we tried a safe and convenient expression system supplying MAP30 protein for medical practice. The gene encoding MAP30 was cloned into pMD18-T vector. The pMD18-MAP30 plasmid was transformed into competent Escherichia coli JM109 by a chemical method. The MAP30 gene was obtained from the pMD18-MAP30 plasmid digested with NotI and SnaBI and the MAP30 gene was ligated into pGAPHα. Then, pGAPHα-MAP30 was transformed into Pichia pastoris GS115 by electroporation. GS115 transformants were analysed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and Western blot. SDS-PAGE revealed an extra band of approximately 32 kDa in the supernatant protein of the GS115 transformants and in their intracellular protein fraction. The result of Western-blot analysis showed that the supernatant and the cell pellet from GS115 with pGAPHα-MAP30 could specially bind to monoclonal antibodies against His in the 32 kDa site. These results demonstrated that the expression of MAP30 in P. pastoris was successful; the process of the expression did not need methanol induction or introduction of an antibiotic-resistance gene. The study may provide a new way for MAP30 synthesis. Owing to its safety, this new approach is expected to be widely used in the medical field.
Insights
Researchers successfully expressed MAP30 protein in Pichia pastoris, offering a safe and convenient method for producing this anti-HIV and anti-tumour agent for medical use.
Area of Science:
- Biotechnology
- Molecular Biology
- Biochemistry
Background:
- Momordica charantia contains MAP30, a ribosome-inactivating protein with demonstrated anti-HIV and anti-tumour properties.
- Existing methods for MAP30 production may lack safety and convenience for widespread medical application.
Purpose of the Study:
- To develop a safe and convenient expression system for producing MAP30 protein suitable for medical practice.
- To investigate the successful expression of MAP30 in Pichia pastoris.
Main Methods:
- Cloned the MAP30 gene into pMD18-T vector and transformed into Escherichia coli JM109.
- Subcloned the MAP30 gene into the pGAPHα vector.
- Transformed Pichia pastoris GS115 with the pGAPHα-MAP30 construct and analyzed expression using SDS-PAGE and Western blot.
Main Results:
- SDS-PAGE confirmed an approximately 32 kDa protein band in both supernatant and intracellular fractions of Pichia pastoris GS115 transformants.
- Western blot analysis showed specific binding of the expressed protein to anti-His antibodies at the 32 kDa site.
- Successful MAP30 expression in Pichia pastoris was achieved without methanol induction or antibiotic-resistance genes.
Conclusions:
- The study successfully demonstrated the expression of MAP30 in Pichia pastoris, providing a novel and safe synthesis pathway.
- This expression system is convenient and does not require methanol induction or antibiotic-resistance genes, making it suitable for medical applications.
- The developed method offers a promising approach for the large-scale production of MAP30 for therapeutic use.
