Expression of chymotrypsin(ogen) in the thioredoxin reductase deficient mutant strain of Escherichia coli AD494(DE3) and purification via a fusion product with a hexahistidine-tail.
A reliable protocol was designed for fast expression and purification of recombinant chymotrypsin(ogen). The zymogen was overexpressed in soluble form as a (His)6-fusion construct in the cytoplasm of the thioredoxin reductase deficient Escherichia coli strain AD494(DE3). This allowed purification of chymotrypsinogen in a highly selective affinity chromatography capture step using a Ni-NTA column. After activation with enterokinase, the enzymatically active chymotrypsin was purified in a polishing step using a modified soybean trypsin inhibitor agarose column. This expression system and the use of affinity chromatography for capture and polishing, offers an easier and faster route to recombinant chymotrypsin(ogen) than the previously described use of Saccharomyces cerevisiae.
Verheyden
G
G
Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Belgium.
Volckaert
G
G
Engelborghs
Y
Y
D001483:Base Sequence; D002918:Chymotrypsin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002919:Chymotrypsinogen / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D006639:Histidine; D004247:DNA; D002919:Chymotrypsinogen; D013880:Thioredoxin-Disulfide Reductase; D002918:Chymotrypsin