caa a22 4500 397556632 CHVBK 20180308164804.0 cr unu---uuuuu 161202e199609 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021451 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021451 Cloning and Expression of an Isovaleryl Pepstatin-Insensitive Carboxyl Proteinase Gene from Xanthomonas sp. T-22 [Elektronische Daten] [Kohei Oda, Masaaki Ito, Ken-ichi Uchida, Yuji Shibano, Ken-ichi Fukuhara, Saori Takahashi] Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, is the second example of the unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to the classical aspartic proteinase inhibitor. The gene coding for XCP was cloned, sequenced, and expressed in Escherichia coli. The XCP gene contains an open reading frame of 2,481 base pairs encoding a protein of 827 amino acid residues with a Mr of 83,677. The XCP was synthesized as a large precursor consisting of three regions: NH2-terminal prepro (N-Prepro) (237 amino acid residues); mature XCP (398 a.a. residues); and COOH-terminal pro (C-Pro) (192 a.a. residues). The N-Prepro and mature XCP regions had no sequence similarity to any other proteins reported so far, except the carboxyl proteinase from Pseudornonas sp. 101 [Oda K., Takahashi, T., Tokuda., Y., Shibano, Y., and Takahashi, S. (1994) J. Biol. C 269, 26518-26524]. The C-Pro region showed high similarity to COOH-terminal regions of other microbial proteinase precursors. E. coli carrying a plasmid containing the cloned wild-type XCP gene produced an 84-kDa protein. This protein was processed into a mature, active form under acidic conditions. This process was completely blocked by tyrostatin, an XCP-specific inhibitor from Kitasatosporia sp. 55, indicating an autocatalytic processing. The purified recombinant XCP had the same characteristics as authentic XCP except for the NH2-terminal amino acid sequence. When the mutant XCP gene truncated in the C-Pro region was expressed in E. coli, an expected 64-kDa protein was detected in the cells, and also processed into the 42-kDa active form under the acidic conditions. Thus, the C-Pro region was not essential for the formation of active mature XCP. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence acid proteinase nationallicence photosynthesis nationallicence carboxyl protelnase nationallicence cloning nationallicence pepstatin nationallicence Xanthornonas nationallicence Oda Kohei Departmnent of Applied Biology, Faculty of Textile Science, Kyoto Institue of Technology Technology, Sakyo-ku, Kyoto 606 aut Ito Masaaki Departmnent of Applied Biology, Faculty of Textile Science, Kyoto Institue of Technology Technology, Sakyo-ku, Kyoto 606 aut Uchida Ken-ichi Department of Bioscience, School of Science and Engineering, Teikyo University Utsunomiyo, Tochigi 320 aut Shibano Yuji Department of Molecular Biology, Biomolecular Engineering Research Institute Furuedai Suita, Osaka 565 aut Fukuhara Ken-ichi Centra1 Research Laboratories Ajinomoto Co., Inc., Kawasaki 210 aut Takahashi Saori Departmnent of Applied Biology, Faculty of Textile Science, Kyoto Institue of Technology Technology, Sakyo-ku, Kyoto 606 aut The Journal of Biochemistry Oxford University Press 120/3(1996-09), 564-572 0021-924X 120:3<564 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021451 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021451 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Oda Kohei Departmnent of Applied Biology, Faculty of Textile Science, Kyoto Institue of Technology Technology, Sakyo-ku, Kyoto 606 aut NATIONALLICENCE 700 1- Ito Masaaki Departmnent of Applied Biology, Faculty of Textile Science, Kyoto Institue of Technology Technology, Sakyo-ku, Kyoto 606 aut NATIONALLICENCE 700 1- Uchida Ken-ichi Department of Bioscience, School of Science and Engineering, Teikyo University Utsunomiyo, Tochigi 320 aut NATIONALLICENCE 700 1- Shibano Yuji Department of Molecular Biology, Biomolecular Engineering Research Institute Furuedai Suita, Osaka 565 aut NATIONALLICENCE 700 1- Fukuhara Ken-ichi Centra1 Research Laboratories Ajinomoto Co., Inc., Kawasaki 210 aut NATIONALLICENCE 700 1- Takahashi Saori Departmnent of Applied Biology, Faculty of Textile Science, Kyoto Institue of Technology Technology, Sakyo-ku, Kyoto 606 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/3(1996-09), 564-572 0021-924X 120:3<564 1996 120 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