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Cloning, Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus

文献类型： 外文期刊

作者： Yin, Chaomin 1 ; Zheng, Liesheng 2 ; Chen, Liguo 2 ; Tan, Qi 3 ; Shang, Xiaodong 3 ; Ma, Aimin 1 ;

作者机构： 1.Huazhong Agr Univ, Coll Food Sci & Technol, Minist Agr, Key Lab Agromicrobial Resources & Utilizat, Wuhan 430070, Peoples R China

2.Huazhong Agr Univ, Coll Plant Sci & Technol, Wuhan 430070, Peoples R China

3.Shanghai Acad Agr Sci, Natl Res Ctr Edible Fungi Biotechnol & Engn, Key Lab Appl Mycol Resources & Utilizat,Minist Ag, Shanghai Key Lab Agr Genet & Breeding,Inst Edible, Shanghai 201403, Peoples R China

关键词： Pleurotus ostreatus;Aspartic protease (Po-Asp);Bioinformatics analysis;Pichia pastoris;Expression;Milk clotting

期刊名称：APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY （ 影响因子：2.926； 五年影响因子：2.685 ）

ISSN：

年卷期：

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收录情况： SCI

摘要： An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3′ portion of cDNA in our previouswork. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. Themature polypeptide with 327 amino acid residues had a calculated molecularmass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68–80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfatepolyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.

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