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

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Abstract

An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3′ portion of cDNA in our previous work. 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. The mature polypeptide with 327 amino acid residues had a calculated molecular mass 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 sulfate-polyacrylamide 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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Acknowledgments

We are grateful to Qi Tang for assistance in Western blot experiment, Lifen Huang for assistance in skimmed milk clotting test, and Jihong Zhu for critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (31172011) and the Chinese National Science and Technology Support Program (2013BAD16B02).

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Authors and Affiliations

  1. Key Laboratory of Agro-Microbial Resources and Utilization, Ministry of Agriculture, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Chaomin Yin & Aimin Ma

  2. College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Liesheng Zheng & Liguo Chen

  3. National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, 201403, China

    Qi Tan & Xiaodong Shang

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  1. Chaomin Yin
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  2. Liesheng Zheng
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  3. Liguo Chen
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  4. Qi Tan
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  5. Xiaodong Shang
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Correspondence to Xiaodong Shang or Aimin Ma.

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Yin, C., Zheng, L., Chen, L. et al. Cloning, Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus . Appl Biochem Biotechnol 172, 2119–2131 (2014). https://doi.org/10.1007/s12010-013-0674-4

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