Gene, 112 (1992) 107-112 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

107

GENE 06338

Cloning, sequencing and expression o f the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus (Recombinant DNA; protein export; prepropeptide; nucleotide sequence; expression vector)

Victor A. David a, Alan H. Deutch a, Alan Sloma b*, Diane Pawlyk b*, Abdul Ally b'* and Don R. Durham c "BioMolecular Research Department, Washington Research Center, W.R. Grace and Co.-Conn., Columbia, MD 21044 (U. S.A .); ~ Bio Technica International, Inc., 85 Bolton St., Cambridge, MA 02140 (U.S.A.) Tel. (617)864-0040; and ~ Biological Chemistry Department, Washington Research Center, W.R. Grace and Co.-Conn, Columbia, MD 21044 (U.S.A.) Tel. (410)531-4439 Received by W.M. Holmes: 24 June 1991 Revised/Accepted: 22 August/2 October 1991 Received at publishers: 20 December 1991

SUMMARY

The gone (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrioproteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coil produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long "pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.

INTRODUCTION

The Gram- marine microorganism Vibrio proteolyticus secretes large amounts of a thermostable, neutral protease (Npr) (Prescott and Williams, 1960) referred to in this Correspondence to: Dr. A.H. Deutch, BioMolecular Rose: ch Department, W.R. Grace and Co.-Conn, Columbia, MD 21044 (U.S.A.) Tel. (410) 531-4439; Fax (410) 531-4068. *Present addresses: (A.S., A.A.) Omnigene, Inc., 85 Bolton St., Cambridge, MA 02140 (U.S.A.) Tel. (617) 864-0040;(D.P.) Roche Institute of Molecular Biology, Department of Molecular Oncology and Virology, Nutley, NJ 07110 (U.S.A.) Tel. (201)235-3846. Abbreviations: aa, amino acid(s); Ap, ampieillin; apr, gene (DNA) encoding subtilisin; B., Bacillus; bp, base pair(s); cat, gone (DNA) encod-

present investigation as vibriolysin (NprV). The protease has been purified to homogeneity and has been shown to be a metalloenzyme that contains one atom of zinc (Griffin and Prescott, 1970). Many of the physicochemical properties defined for NprV also characterize the thermostable ing CAT; CAT, Cm acetyltransferase; Cm, chloramphenicol; FAAFA, N-[3-(2-furyl)acryloyl]-L-alanyl-phenylalanineamide; kb, kilobase(s) or 1000 bp; L (medium), Luria's (medium); Npr, neutral protease; NprV, vibriolysin neutral protease; nprV, gone (DNA) encoding NprV; nt, nucleotidc(s); ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; R, resistance/resistant; RBS, ribosome-binding site(s); sacQ, gone encoding regulatory protein for B. subtilisprotease genes; Tc, tetracycline; Tml, thermolysin (a neutral protease; see Fig. 4); V., Vibrio; [ ], denotes plasmid-carrier state.

108 neutral proteases elaborated by the Gram + microorganisms B. thermoproteolyticus (Endo, 1962) and B. stearothermophilus (Fujii et al., 1983). The enzyme from B. thermoproteolyticus, thermolysin, has been studied in detail (Villafranca and Raushel, 1979) and its aa sequence (Titani et al., 1972) and three-dimensional structure (Matthews et al., 1972) have been elucidated. The Npr-encoding gene orB. stearothermophilus has been cloned, sequenced (Kubo and lmanaka, 1988) and shown to encode a mature enzyme with an aa sequence containing only two differences from that of thermolysin. Since the primary structures of functionally or structurally important regions in enzymes sharing the same function but differing in origin are often highly conserved, we have isolated ond sequenced the nprV gene, encoding NprV, to enable a comparison of the deduced aa sequence with the sequence of the two neutral proteases from Bacillus. Further, we have obtained expression of NprV, when placed in the appropriate expression/secretion vector and transformed into B. subtilis.

was monitored spectrophotometrically at 335 nm. Nominal activity (

Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus.

The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolate...
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