Expression and characterization of cathepsin B from tsetse (Glossina morsitans morsitans)

Jpn J Vet Res. 2013 Nov;61(4):137-47.

Abstract

Digestive enzymes in tsetse fly midgut are thought to modulate the development of African trypanosome which is a causative agent of trypanosomosis in human and animal. Cathepsin B is induced after the first blood meal ingestion and being higher in trypanosome infected flies. A DNA fragment encoding pro-cathepsin B (930 bp) (Accession No. AF329480_1) was cloned and expressed in E. coli and P. pastoris protein expression systems. An active recombinant cathepsin B (rGmcathB) produced by P. pastoris was migrating from 35 to 45 kDa under reducing condition, rGmcathB exhibited the highest proteolytic activity at pH 4.0 with wide range temperature 25-30 degrees C, also degraded bovine hemoglobin and serum albumin. rGmcathB exhibited hydrolysis preference for Z-Arg-Arg-MCA (K(cat)/K(M) 7.58 mM(-1)sec(-1)) and bovine hemoglobin (K(cat)/K(M) 3.77 x 10(3) mM(-1)sec(-1)). The proteolytic activity of rGmcathB was inhibited by specific cysteine protease inhibitor (E-64) confirmed belonging to papain-like cysteine protease family. These results indicated that rGmcathB shows the activity of cathepsin B and have high affinity with blood protein referring a role in blood meal digestion. In this study, the recombinant protein expressed by E. coli expression system was not enzymatically active as shown in the recombinant protein expressed by P. pastoris expression system. This finding implies that P. pastoris expression system is more suitable for expressing enzymatically active recombinant proteases than E. coli expression system.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cathepsin B / chemistry
  • Cathepsin B / genetics
  • Cathepsin B / metabolism*
  • Cloning, Molecular
  • Gene Expression Regulation / physiology*
  • Gene Expression Regulation, Enzymologic / physiology*
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Sequence Alignment
  • Temperature
  • Tsetse Flies / genetics
  • Tsetse Flies / metabolism*

Substances

  • Cathepsin B