A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autogrupha calijornicu nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera,frugiperdu 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.Human salivary-gland kallikrein is a member of the tissue or glandular kallikreins, which comprise a subfamily of trypsin-like serine proteinases of closely related structure with molecular masses between 25 -40 kDa due to the heterogeneity of their carbohydrate content [l]. They share common biological features, especially the highly selective cleavage of the plasma protein kininogen to release the vasoactive and spasmogenic decapeptide kallidin (Lys-bradykinin) [2]. Tissue kallikreins are found in a variety of tissues like pancreas [3], salivary glands [4], kidney, intestine and prostate [5], but also in secretions of these tissues and different body fluids [6].Comparison of full-length cDNA sequences of human salivary-gland kallikrein [7], pancreatic [8] and kidney [9] kallikrein, as well as the sequence of the kallikrein gene [lo], has shown that tissue kallikrein is synthesized as a precursor with a 17-amino-acid signal peptide and an activation peptide of seven amino acids in length with the sequence motif AlaPro-Pro-Ile-Gln-Ser-Arg (Fig. l), which is identical in all examined kallikrein species. The mature enzyme of 238 amino acids revealed a complete sequence identity with the exception of two amino acid exchanges in position 121 from Gln to Glu and position 162 from Glu to Lys in salivary-gland kallikrein. [12], kidney [13] and in urine, where both latent and active forms are found in equal amounts [14]. In contrast, in salivary tissue and secretions only the activated form occurs. As kallikrein is easily inhibited by various proteins and peptides [15], it was unknown whether an enhancement in kallikrein activity was due to the activation of the proenzyme or to the removal of an inhibitor. The establishment of isolation procedures for prokallikrein from porcine pancreas [16], human urine [17] and rat urine [18] allowed the investigation of the activation ...

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