Staphylococcus simulans recombinant lysostaphin: Production, purification, and determination of antistaphylococcal activity

• Published in: Biochemistry (Moscow) 2016-05, Vol.81 (5), p.502-510
• Main Authors: Boksha, I. S., Lavrova, N. V., Grishin, A. V., Demidenko, A. V., Lyashchuk, A. M., Galushkina, Z. M., Ovchinnikov, R. S., Umyarov, A. M., Avetisian, L. R., Chernukha, M. Iu, Shaginian, I. A., Lunin, V. G., Karyagina, A. S.
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - biosynthesis; Anti-Bacterial Agents - isolation & purification; Anti-Bacterial Agents - pharmacology; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biomass; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Cloning, Molecular; Disk Diffusion Antimicrobial Tests; E coli; Electrophoresis, Polyacrylamide Gel; Enzymatic activity; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Life Sciences; Lysostaphin - biosynthesis; Lysostaphin - isolation & purification; Lysostaphin - pharmacology; Microbiology; Peptides; Peptidoglycan - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; Staphylococcus - drug effects; Staphylococcus - genetics; Staphylococcus aureus; Staphylococcus aureus - drug effects; Staphylococcus haemolyticus; Staphylococcus haemolyticus - drug effects; Staphylococcus infections; Staphylococcus simulans; Temperature
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297916050072

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.