BPTI and N-terminal extended analogues generated by factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli.
A recombinant gene for BPTI (bovine pancreatic trypsin inhibitor) is expressed in Escherichia coli using a MBP (maltose-binding protein) fusion vector. BPTI is fused through an FXa (blood coagulation factor Xa protease) target sequence (Ile-Glu-Gly-Arg) to the C-terminus of MBP. The MBP moiety of the hybrid protein enables purification in one step utilizing MBP's affinity to cross-linked amylose, and the FXa target sequence allows specific cleavage of the hybrid protein. Effective FXa cleavage is achieved by spacing the FXa target sequence and Arg-1 of the BPTI sequence with four residues (Met-Glu-Ala-Glu). The resulting N-terminal extended BPTI is readily converted to the wild-type sequence by trimming with cathepsin C exopeptidase, for the activity of which the spacing tetrapeptide is optimized. FXa cleavage is prohibited when the target sequence is placed next to Arg-1. In this construction, off-target cleavage at a somewhat homologous sequence (Val-Pro-Gly-Arg) results in five- or six-residue extended BPTI, indicating new details of the FXa specificity. The yield of highly purified recombinant BPTI is 3-6 mg/liter of culture, making the MBP-BPTI expression system convenient for the production of sufficient amounts of protein for NMR studies. 1H NMR is used to analyze the N-extended BPTI analogues.
Lauritzen
C
C
Institute of Life Sciences and Chemistry, University of Roskilde, Denmark.
Tüchsen
E
E
Hansen
P E
PE
Skovgaard
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020789:Cathepsin C; D002417:Cattle; D004247:DNA / Q000235:genetics; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D004926:Escherichia coli / Q000235:genetics*; D015951:Factor Xa; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; D004247:DNA; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; D015951:Factor Xa