NDLI: Construction of a Rhizopus arrhizus glucoamylase gene suitable for expression in distinct host: introns spliced artificially by PCR

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Construction of a Rhizopus arrhizus glucoamylase gene suitable for expression in distinct host: introns spliced artificially by PCR

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Construction of a Rhizopus arrhizus glucoamylase gene suitable for expression in distinct host: introns spliced artificially by PCR

Content Provider	Springer Nature Link
Author	Yang, Liquan Dai, Xiaojun Hou, Jianhua Ma, Chunxiao Wang, Cuiyan Wu, Zhiqiang Li, Minggang
Copyright Year	2007
Abstract	Glucoamylase is an industrially extremely important enzyme in the fermentative production of ethanol, used in the enzymatic conversion of starch into high glucose and fructose syrups. The aim of this study is to construct a Rhizopus arrhizus glucoamylase gene (RaGA)—introns artificially spliced by PCR—suitable for expression in S. cerevisiae host and tried expressing in Picha pastoris. In previous work, we failed in amplifying glucoamylase gene from R. arrhizus by RT-PCR, so several primers were designed to splicing the introns by PCR in vitro. Sequence analysis shown that all introns in the RaGA were deleted correctly and no mutant was induced in the extrons compared with the RaGA gene originally cloned. The RaGA gene artificially constructed was transferred into P. pastoris integrative expression vectors pPIC9 (containing а-factor). Consequently, the plasmids pPIC9-RaGA was lineared by SacI and inserted into P. pastoris GS115 (His$^{−}$) genome downstream of the 5′AOX1 promoter by the method of electroporation. Induction by 0.75% methanol for 72 h led to synthesis of secreted glucoamylase. So it is demonstrated that the glucoamylase gene has been expressed in and secreted from P. pastoris.
Starting Page	9
Ending Page	15
Page Count	7
File Format	PDF
ISSN	03014851
Journal	Molecular Biology Reports
Volume Number	35
Issue Number	1
e-ISSN	15734978
Language	English
Publisher	Springer Netherlands
Publisher Date	2007-06-20
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Glucoamylase Picha pastories Rhizopus arrhizus Enzyme activity SDS-PAGE Western-blotting Animal Biochemistry Animal Anatomy / Morphology / Histology
Content Type	Text
Resource Type	Article
Subject	Genetics Medicine Molecular Biology

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