3.8 Article

Combination of Genetic Manipulation Improved Saccharomycopsis fibuligera α-Amylase Secretion by Pichia pastoris

期刊

INDONESIAN JOURNAL OF CHEMISTRY
卷 19, 期 2, 页码 305-318

出版社

GADJAH MADA UNIV, DEPT CHEMISTRY
DOI: 10.22146/ijc.33140

关键词

Sfamy; signal peptide; gene dosage; folding; PDI; Pichia pastoris

资金

  1. Ministry of Research, Technology and Higher Education of the Republic of Indonesia [031/SP2H/PP/DP2M/III/2017]

向作者/读者索取更多资源

This study assessed the combinations of genetic manipulation; signal peptide modification, gene dosage increment and co-expression of folding component, to increase Saccharomycopsis fibuligera R64 alpha-amylase (Sfamy) secretion in Pichia pastoris. Sfamy native signal peptide was replaced with modified signal peptide which contained 15 amino acid of mouse salivary alpha-amylase signal peptide fused to the pro-region of the signal peptide of Saccharomyces cerevisiae alpha-mating factor (alpha-MF). Increase in gene dosage was identified by screening for P. pastoris harboring multicopy of the Sfamy gene. Whereas, co-expression of folding component was done by addition of Protein Disulfide Isomerase (PDI). Expression plasmids harboring Sfamy containing a modified signal sequence (pPICZA-MS-Sfamy) was used to transform P. pastoris GS115, and gene dosage increment was screened using zeocin. Effect of PDI co-expression on secretion levels of Sfamy was assessed by constructing the pPIC3.5K-Pdi1 plasmid and introducing into P. pastoris harboring multicopy of MS-Sfamy for expression of Sfamy. Signal peptide modification consequently increased Sfamy secretion by P. pastoris by 3.3-fold compared to the native signal peptide. Gene dosage increment had improved Sfamy secretion by 11-fold in P. pastoris [MS-Sfamy] resistant to 2000 mu g/mL zeocin, compared to P. pastoris harboring one copy of WT-Sfamy. Hence, PDI co-expression increased the secretion of Sfamy by 2-fold as compared without PDI co-expression. In summary, the combination of genetic manipulation successfully increased Sfamy secretion by 20-fold compared to P. pastoris harboring one copy of WT-Sfamy.

作者

我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。

评论

主要评分

3.8
评分不足

次要评分

新颖性
-
重要性
-
科学严谨性
-
评价这篇论文

推荐

暂无数据
暂无数据