ABSTRACT
A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion-exchange chromatography and gel-filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N-terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/l insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.
Subject(s)
Baculoviridae/genetics , Enzyme Precursors/genetics , Prekallikrein/genetics , Salivary Glands/enzymology , Amino Acid Sequence , Animals , Blotting, Western , Cell Line , Chromatography, DEAE-Cellulose , Chromatography, Gel , DNA/genetics , DNA/isolation & purification , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Humans , Kinetics , Molecular Sequence Data , Molecular Weight , Moths , Plasmids , Prekallikrein/isolation & purification , Prekallikrein/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping , TransfectionABSTRACT
A cDNA fragment encoding human salivary-gland kallikrein, including the kallikrein-owned signal peptide, was inserted into a baculovirus vector adjacent to the polyhedrin promoter and expressed in transfected insect cells. Biologically active kallikrein was isolated to homogeneity from serum-free culture supernatant using a four-step protocol. The N-terminal amino acid sequence of the insect-derived kallikrein was identical to that of the natural proteinase, thus indicating the proper removal of the mammalian signal peptide.
