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Heterologous expression of non-specific lipid transfer proteins (nsLTPs) from Physcomitrella patens in yeast Pichia pastoris
Linköping University, Department of Physics, Chemistry and Biology. (Molecular Genetics)
2012 (English)Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
Abstract [en]

This study focused on utilization of Pichia pastoris system for the expression of type G non-specific lipid transfer protein (nsLTPs) from moss Physcomitrella patens, a small, soluble, basic protein which is capable of in vitro transfer of phospholipid molecules between membranes. A 250-265 bp DNA fragment which contains the coding region of the nsLTP gene was amplified by Polymerase Chain Reaction (PCR).  The nsLTPs were inserted into the expression vector pPCIZαA which contains the alcohol oxidase gene (AOX1) promoter. The AOX1 promoter under methanol induction allows for the expression of heterologous protein gene. The recombinant plasmid was transformed into the host strain P.pastoris KM71H by electroporation. PCR analysis showed a clear 800-900 bp fragment with AOX1 primers and a 250-265 bp fragment with gene specific primers suggesting that nsLTP gene has been successfully integrated into the P.pastoris genome. The expressed nsLTP had a molecular mass of 11-13 KDa when analyzed with SDS-PAGE and Western blot. Western blot analysis also confirmed that nsLTPs were N-glycosylated with a molecular mass of ~33KDa. A simple affinity purification procedure was adopted to purify nsLTPs from the culture supernatant, yielding 10 mg of nsLTPG2 (purity 46%), 8.8 mg of nsLTPG4 (purity 30.5%) and 9 mg of nsLTPG8 (purity 52%) from 9 mL of the supernatant. These results clearly indicate that P.pastoris is an efficient system for expression of nsLTPs.

Place, publisher, year, edition, pages
2012. , 37 p.
Keyword [en]
AOX1, Glycosylation, KM71H, nsLTPs, Pichia pastoris, pPCIZαA expression vector
National Category
Biological Sciences
URN: urn:nbn:se:liu:diva-77480ISRN: LITH-IFM-EX--12/2636--SEOAI: diva2:528562
Subject / course
2012-05-25, Planck, IFM, Linköpings universitet, Linköping, 10:40 (English)
Life Earth Science
Available from: 2012-06-05 Created: 2012-05-20 Last updated: 2012-06-05Bibliographically approved

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