Expression and purification of a cold-adapted group III trypsin in Escherichia coli.
The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and purification of a recombinant HP-thioredoxin-trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its purification by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the first biochemically characterized group III trypsin. It has dual substrate specificity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 degrees C with a complete inactivation at 30 degrees C. The aim of the study was to facilitate further studies of recombinant trypsin Y by finding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an efficient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod.
Pálsdóttir
Helga Margrét
HM
Science Institute, University of Iceland, Laeknagardi, Vatnsmýrarvegi 16, Reykjavík IS-101, Iceland.
Gudmundsdóttir
Agústa
A
D000222:Adaptation, Physiological; D000818:Animals; D015153:Blotting, Western; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D048389:Gadus morhua; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D014357:Trypsin / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014357:Trypsin