Human cathepsin E produced in E. coli.
A cDNA for procathepsin E was generated from human gastric adenocarcinoma (AGS) cells, amplified by PCR and inserted into the T7 dependent vector pET 22b for expression in E. coli. Purification of the resultant product was accomplished simply, without the need to resort to column chromatography. The recombinant protein displayed comparable properties to those of its naturally occurring counterpart. The yield of homogeneous active enzyme obtained was approximately 3 mg per 40 g of cells. This is sufficient to permit crystallisation and structural analysis to begin and a mutagenesis programme to examine structure/activity relationships now to be undertaken.
Hill
J
J
Department of Biochemistry, University of Wales College, Cardiff, UK.
Montgomery
D S
DS
Kay
J
J
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D020059:Cathepsin E; D002403:Cathepsins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013274:Stomach Neoplasms; D014169:Transformation, Bacterial; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D004247:DNA; D002403:Cathepsins; D020059:Cathepsin E