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.