MOLECULAR-CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION OF THE GENE ENCODING A TRYPSIN-LIKE PROTEASE FROM STREPTOMYCES-ERYTHRAEUS

Streptomyces erythraeus produces an extracellular mammalian-type serin e protease bearing trypsin-like substrate specificity. The gene encodi ng the protease was cloned and sequenced as an initial step for invest igating its structure-function relationship by site-specific mutagenes is. The cloned gene is composed of an 816-bp open reading frame encodi ng 272 amino acid residues, suggesting that it is synthesized as a pre cursor protein containing a 42-residue prepropeptide. In the N-termina l prepropeptide portion, the tract of 30 residues from the initiator m ethionine has a typical signal sequence for Streptomyces and the remai ning 12 residues are thought to comprise a propeptide. The cloned gene was replaced downstream of a strong promoter in a high expression pla smid, pSEV2, and expressed in Streptomyces lividans TK24. The gene pro duct was secreted extracellularly and identified as an inactive precur sor which consists of the mature enzyme and the 12-residue N-terminall y extended peptide chain. The precursor protein was converted to a ful ly active mature form by limited proteolysis with alpha-chymotrypsin a t the Phe-(-1)-Ile-1 bond. Protein sequence analysis revealed that, ex cept for the C-terminal three residues, recombinant SET is identical w ith the native enzyme.