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Biochemistry (Moscow)

, Volume 81, Issue 2, pp 152–162 | Cite as

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

  • Haining YuEmail author
  • Shasha Cai
  • Jiuxiang Gao
  • Chen Wang
  • Xue Qiao
  • Hui Wang
  • Lan Feng
  • Yipeng WangEmail author
Article

Abstract

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.

Key words

Anseriformes Anas platyrhynchos trypsin molecular cloning expression structure and function 

Abbreviations

cDNA

complementary DNA

EST

express sequence tags

FPLC

fast protein liquid chromatography

ORF

open reading frame

PCR

polymerase chain reaction

RACE

rapid-amplification of cDNA ends

5′-UTR

5′-untranslated region

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Copyright information

© Pleiades Publishing, Ltd. 2016

Authors and Affiliations

  1. 1.Institute of Marine Biological Technology, School of Life Science and BiotechnologyDalian University of TechnologyDalian, LiaoningChina
  2. 2.College of Pharmaceutical SciencesSoochow UniversitySuzhou, JiangsuChina

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