Bacterial expression and refolding of human trypsinogen.
The expression of recombinant trypsinogens from different mammalian origins in Escherichia coli typically leads to the formation of insoluble aggregates. This work describes the high level expression of human trypsinogen 1 in E. coli using the T7 expression system. Direct expression of trypsinogen was not possible, but the N-terminal fusion of the first 11 amino acids of the T7 protein 10 resulted in an expression level of 200 mg g(-1) bacterial dry mass. A refolding procedure was optimized, and a method using continuous feed of denatured product was developed. Thus the working concentration of trypsinogen could be raised four-fold, while the yield of active protein could be maintained at 20-35%. The refolded trypsinogen was converted to trypsin by autocatalytic activation, and the utility for the detachment of mammalian cells in culture was proven.
Hohenblum
Hubertus
H
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Vorauer-Uhl
Karola
K
Katinger
Hermann
H
Mattanovich
Diethard
D
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D036022:Capsid Proteins / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D002479:Inclusion Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014357:Trypsin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014362:Trypsinogen / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; C104967:gene 10 protein, phage T7; D014362:Trypsinogen; D014357:Trypsin