Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification.
Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.
Jónsdóttir
Gudrún
G
Department of Food Science, University of Iceland, Science Institute, Vatnsmýrarvegi 16, IS-101 Reykjavík, Iceland.
Bjarnason
Jón Bragi
JB
Gudmundsdóttir
Agústa
A
D000222:Adaptation, Physiological; D000818:Animals; D015153:Blotting, Western; D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003080:Cold Temperature; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005399:Fishes / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D002614:Chelating Agents; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006639:Histidine; D013879:Thioredoxins; D014357:Trypsin