Express Sequence Tag Analysis – Identification of Anseriformes Trypsin Genes from Full-Length cDNA Library of the Duck (Anas platyrhynchos) and Characterization of Their Structure and Function

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Haining Yu^1*, Shasha Cai^1#, Jiuxiang Gao^1#, Chen Wang¹, Xue Qiao¹, Hui Wang¹, Lan Feng¹, and Yipeng Wang^2*

¹Dalian University of Technology, Institute of Marine Biological Technology, School of Life Science and Biotechnology, 116024 Dalian, Liaoning, China; E-mail: joannyu@live.cn

²Soochow University, College of Pharmaceutical Sciences, 215123 Suzhou, Jiangsu, China; fax: + 86 (411) 847-08850; E-mail: yipengwang@suda.edu.cn

^# These authors contributed equally to this work.

^* To whom correspondence should be addressed.

Received March 4, 2015; Revision received May 25, 2015

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Trypsins are key proteins important in animal protein digestion by breaking down the peptide bonds on the carboxyl side of lysine and arginine residues, hence it has been used widely in various biotechnological processes. In the current study, a full-length cDNA library with capacity of 5·10⁵ CFU/ml from the duck (Anas platyrhynchos) was constructed. Using express sequence tag (EST) sequencing, genes coding two trypsins were identified and two full-length trypsin cDNAs were then obtained by rapid-amplification of cDNA end (RACE)-PCR. Using Blast, they were classified into the trypsin I and II subfamilies, but both encoded a signal peptide, an activation peptide, and a 223-a.a. mature protein located in the C-terminus. The two deduced mature proteins were designated as trypsin-IAP and trypsin-IIAP, and their theoretical isoelectric points (pI) and molecular weights (MW) were 7.99/23466.4 Da and 4.65/24066.0 Da, respectively. Molecular characterizations of genes were further performed by detailed bioinformatics analysis. Phylogenetic analysis revealed that trypsin-IIAP has an evolution pattern distinct from trypsin-IAP, suggesting its evolutionary advantage. Then the duck trypsin-IIAP was expressed in an Escherichia coli system, and its kinetic parameters were measured. The three dimensional structures of trypsin-IAP and trypsin-IIAP were predicted by homology modeling, and the conserved residues required for functionality were identified. Two loops controlling the specificity of the trypsin and the substrate-binding pocket represented in the model are almost identical in primary sequences and backbone tertiary structures of the trypsin families.

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KEY WORDS: Anseriformes, Anas platyrhynchos, trypsin, molecular cloning, expression, structure and function

DOI: 10.1134/S0006297916020097

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