title: Bacterial expression and refolding of human trypsinogen.
abstract: 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.
authors.affiliation: Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
authors.forename: Hubertus
authors.initials: H
authors.lastname: Hohenblum
authors.forename: Karola
authors.initials: K
authors.lastname: Vorauer-Uhl
authors.forename: Hermann
authors.initials: H
authors.lastname: Katinger
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
mesh_terms: 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
chemical_list: D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; C104967:gene 10 protein, phage T7; D014362:Trypsinogen; D014357:Trypsin