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Title: Production of talaromyces (Penicillium) marneffei glyceraldehyde-3-phosphate dehydrogenase recombinant protein expressed by pichia pastoris
Authors: Sophit Khanthawong
Nongnuch Vanittanakom
Keywords: Biochemistry, Genetics and Molecular Biology
Materials Science
Physics and Astronomy
Issue Date: 1-Jan-2016
Abstract: © 2016, Chiang Mai University. All Rights reserved. Talaromyces (Penicillium) marneffei is a thermal dimorphic fungus that can cause a fatal disseminated disease in human immunodeficiency virus-infected patients. The routes of infection and factors that affect the pathogenicity of this fungus remain unclear. The previous studies demonstrated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an adherence factor in T. marneffei during early phase of infection. In this study, recombinant GAPDH of T. marneffei was produced in a eukaryotic expression system, Pichia pastoris. The full-length gpdA gene of T. marneffei was amplified from cDNA clones using primers with restriction sequences. Then the amplified fragments were cloned into pPICzA plasmid and transformed into TOP10 E. coli competent cells. The fusion plasmids were purified, linearized and subsequently transformed into P. pastoris X-33 competent yeast cells by electroporation. The positive clones were then checked with PCR and sequencing. The selected clone was cultured and induced for recombinant protein expression. After purification, proteins of approximately 34 and 37 kDa were detected by coomassie blue stained gel. Only 37-kDa rGAPDH was identified by using western blot to detect his-tag. The rGAPDH was purified by using Ni-NTA reducing condition to remove the protein contamination. We have successfully cloned and produced GAPDH recombinant protein of T. marneffei in a yeast expression system. This protein will be further characterized for its virulence property.
ISSN: 01252526
Appears in Collections:CMUL: Journal Articles

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