Journal of Biomolecular NMR, 14: 285–286, 1999. KLUWER/ESCOM © 1999 Kluwer Academic Publishers. Printed in the Netherlands. 285 Letter to the Editor: Backbone 1 H, 13 C, and 15 N resonance assignments of Streptomyces subtilisin inhibitor Hiroaki Sasakawa a , Atsuo Tamura a , Kazuyuki Akasaka a,∗ , Seiichi Taguchi b , Yoko Miyake c & Masatsune Kainosho c a Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan; b Department of Biological Science and Technology, Science University of Tokyo, Noda, Chiba 278, Japan; c Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Hachioji, Tokyo 192, Japan Received 19 April 1999; Accepted 22 April 1999 Key words: resonance assignment, Streptomyces subtilisin inhibitor Biological context Streptomyces subtilisin inhibitor (SSI) is a dimeric 23 kDa protease inhibitor isolated from Streptomyces albogriseolus (Murao et al., 1973; Hiromi et al., 1985). The crystal structure of SSI retains a unique dimeric structure with two identical subunits associ- ated through the β-sheet, each of the subunits con- sisting of 113 amino acids with two disulfide bridges (Mitsui et al., 1979). Complete assignments of 1 H N , 15 N and 13 C NMR signals have been hampered by the lack of an appropriate expression system for uni- form isotope labeling and the self-association of the molecules in a relatively low concentration range. In the present work, we have succeeded in construct- ing an efficient expression system that can produce a sufficient amount of soluble SSI labeled with 15 N and/or 13 C nuclei. The resultant protein has been used for 3D 13 C/ 15 N heteronuclear NMR measurements, which allowed nearly complete signal assignments of the backbone atoms. Methods and results The solubility of SSI produced from the secretory ex- pression vector for the SSI gene, pOS1t2 (Taguchi et al., 1993), turned out to be too low for complete signal assignments by heteronuclear 3D NMR mea- surements. The reason for this low solubility was considered to be the hydrophobicity of the Phe residue ∗ To whom correspondence should be addressed. in the N-terminal region, which was inevitably in- serted when the plasmid of the SSI was constructed. To improve the solubility of the recombinant SSI, gene engineering was performed in the region encoding the SSI N-terminus for truncation of the Phe residue. pOS1t2 was digested with EcoRI and HindIII to ob- tain the DNA fragment containing the SSI-mature- portion-encoding region. The isolated EcoRI/HindIII DNA fragment was inserted into the same sites of the secretion plasmid vector, pIN-III-ompA-3 (Ghrayeb et al., 1984). The resultant plasmid, pOS2t2, was di- gested with EcoRI and the 5 ′ -protruding 4 nucleotides (AATT) were deleted with mung bean nuclease, fol- lowed by self-ligation. E. coli JM109 was transformed to a wild-type pOS2t2 with the ligated sample af- ter EcoRI digestion to eliminate undesired derivatives such as the regenerated form. Six colonies exhibiting inhibitor activity were found to have the desired nu- cleotide sequence with a disrupted EcoRI site. One of these isogenic plasmids was designated to pOST. The newly obtained recombinant SSI showed a much higher solubility and practically the same 1 H-NMR spectrum with the same inhibitory activity against subtilisin as the wild-type SSI. SSI, uniformly labeled with 15 N or with 13 C/ 15 N isotopes was produced by cultivating E. coli JM109 carrying pOST plasmid in an M9 medium with ( 15 NH 4 ) 2 SO 4 and 13 C-D-glucose as the only nitrogen and carbon sources, respectively. For NMR measure- ments, lyophilized SSI was dissolved to a concen- tration of 1 mM in 95% 1 H 2 O/5% 2 H 2 O containing 25 mM phosphate buffer, pH 6.3. For sequential assignment, HSQC, CT-HNCO, CT-HNCA and CT- jn233127.tex; 12/07/1999; 9:40; p.1 PDF-OUTPUT DISK Gr.: 201022326, JNMR K9924A (jnmrkap:bio2fam) v.1.1