Production of bovine-pancreatic-trypsin-inhibitor homologues in Escherichia coli and their characterization.
Biologically active bovine pancreatic trypsin inhibitor (BPTI) was produced in Escherichia coli using an OmpA leader-peptide fusion-protein system, and BPTI homologues were generated by cassette mutagenesis. Amino acids in the reactive loop of alpha 1-proteinase inhibitor (alpha 1-PI) were incorporated into the reactive loop of BPTI in a stepwise approach such that the contribution of individual amino acids could be assessed. The introduction of mutations into BPTI diminished the yield of heterologous protein relative to wild-type BPTI. However, for three BPTI homologues sufficient material was isolated to allow characterization of the proteins by electrospray MS and N-terminal peptide sequencing.
Chesshyre
J A
JA
Dyson Perrins Laboratory, University of Oxford, U.K.
Kraunsoe
J A
JA
Lowe
G
G
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005285:Fermentation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D011498:Protein Precursors; D000515:alpha 1-Antitrypsin; C057184:outer membrane protein A precursor (E coli); D014359:Trypsin Inhibitor, Kazal Pancreatic; D004247:DNA