caa a22 4500 397494467 CHVBK 20180308164517.0 cr unu---uuuuu 161202e199508 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a124912 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a124912 Molecular Cloning, Nucleotide Sequence, and Expression of the Gene Encoding a Trypsin-Like Protease from Streptomyces erythraeus [Elektronische Daten] [Yuko Nagamine-Natsuka, Shigemi Norioka, Fumio Sakiyama] Streptomyces erythraeus produces an extracellular mammalian-type serine protease bearing trypsin-like substrate specificity. The gene encoding the protease was cloned and sequenced as an initial step for investigating its structure-function relationship by site-specific mutagenesis. The cloned gene is composed of an 816-bp open reading frame encoding 272 amino acid residues, suggesting that it is synthesized as a precursor protein containing a 42-residue prepropeptide. In the N-terminal prepropeptide portion, the tract of 30 residues from the initiator methionine has a typical signal sequence for Streptomyces and the remaining 12 residues are thought to comprise a propeptide. The cloned gene was replaced downstream of a strong promoter in a high expression plasmid, pSEV2, and expressed in Streptomyces lividans TK24. The gene product was secreted extracellularly and identified as an inactive precursor which consists of the mature enzyme and the 12-residue N-terminally extended peptide chain. The precursor protein was converted to a fully active mature form by limited proteolysis with a-chymotrypsin at the Phe-(-1)-Ile-1 bond. Protein sequence analysis revealed that, except for the C-terminal three residues, recombinant SET is identical with the native enzyme © by the Journal of Biochemistry Regular Papers nationallicence Key words nationallicence gene cloning nationallicence proenzyme activation in vitro nationallicence recombinant protein expression nationallicence Streptomyces erythraeus nationallicence trypsin-like protease nationallicence Nagamine-Natsuka Yuko Division of Protein Chemistry and Research Center for Protein Engineering, Institute for Protein Research, Osaka University, Suita, Osaka 565 aut Norioka Shigemi Division of Protein Chemistry and Research Center for Protein Engineering, Institute for Protein Research, Osaka University, Suita, Osaka 565 aut Sakiyama Fumio Division of Protein Chemistry and Research Center for Protein Engineering, Institute for Protein Research, Osaka University, Suita, Osaka 565 aut The Journal of Biochemistry Oxford University Press 118/2(1995-08), 338-346 0021-924X 118:2<338 1995 118 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a124912 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a124912 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nagamine-Natsuka Yuko Division of Protein Chemistry and Research Center for Protein Engineering, Institute for Protein Research, Osaka University, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Norioka Shigemi Division of Protein Chemistry and Research Center for Protein Engineering, Institute for Protein Research, Osaka University, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Sakiyama Fumio Division of Protein Chemistry and Research Center for Protein Engineering, Institute for Protein Research, Osaka University, Suita, Osaka 565 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 118/2(1995-08), 338-346 0021-924X 118:2<338 1995 118 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford