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Cloning and Expression of the GOD Gene from the Aspergillus Niger in Pichia Pastoris GS115

Author: WangYanJiang
Tutor: MaJianZhong
School: Lanzhou University of Technology
Course: Biochemistry and Molecular Biology
Keywords: Aspergillus niger GOD ligation of blunt-end DNA fragments pPIC9K-His-GOD Pichia pastoris GS115 gene expression
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 36
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Abstract


Glucose oxidase is widely used in medicine, food, fermentation, biological researches and other fields. At present, glucose oxidase is mainly produced by fermentation of Aspergillus niger and Penicillin. Considering the shortage of the production of glucose oxidase, some researchers have been engaged in using genetic engineering means to achieve foreign glucose oxidase gene over-expression in Escherichia coli and Pichia pastoris. Among these means, the Pichia pastoris expression system can regulate the expression of foreign protein, process and modify the expressed products. Besides, it can express foreign protein to out of cells. Therefore, this method is favored by many researchers. In this research, Aspergillus niger GOD gene with the sequence of 6×His was cloned and pPIC9K-His-GOD was constructed. After linearized with restriction enzymes BlgII, it was transfirmed into Pichia pastoris and attained yeast strains with the incorporated GOD in genomes.The main research results are as following:1. Cloning of GOD gene amplified from Aspergillus niger DNA: GOD gene was attained through PCR, total DNA of Aspergillus niger as the template, forward and reverse primers designed and synthesized according to the both ends of the GOD sequence. The forward primer was designed a 6×His at its 5’-end. The blunt-end GOD DNA was ligated to pUC19 that was digested with SmaI and pUC19-His’s-GOD recombinant was constructed. The sequencing result showed that the whole length of the GOD gene of Aspergillus niger ZM-8 was 1749 bp. It was 1779bp if include the tag of 6×His, and coded for 593 amino acids.2. The construction of GOD expression vector and transformation of P. pastoris: The recombinant of pUC19-His-GOD was digested with NotI. After purification, the GOD-containing fragment was inserted into cloning site of pPIC9k digested with NotI and pPIC9K-His-GOD was constructed. According to yeast expression manual, the recombinant was lined with BlgII, and transformed Pichia pastoris GS115, so the GOD gene was integrated into chromosome of Pichia pastoris GS115. The transformed strains were transferred to medium with G418, and the grown strains were named Pichia pastoris GSGOD, through yeast-cell PCR, the GSGOD strains were confirmed to have the incorporated GOD gene. 3. Designing of ligation of blunt-end DNA fragments: During the experiment of cloning of GOD gene, the traditional method of ligation of blunt-end DNA fragments was improved. To increase the recombinant ratio of cloning blunt DNA fragments and simplify the operation, blunt-end GOD DNA was directly ligated to pUC19 digested by a blunt-end producing restriction enzyme (SmaI) with SmaI presence. In this method, GOD DNA phosphorylation and vector dephosphorylation are not necessary. The transformation of ligation results showed that the recombinant ratio can up to 86.8%. Compared to traditional ligation of blunt-end DNA fragments reaction, the results showed that the restriction enzyme SmaI in the ligation reaction could efficiently minimize the self-ligated vector and keep a high recombinant ratio.

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