Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.
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 Sigma-Aldrich (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.
N. F. Gamaleya Federal Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, 123098, Russia. boksha_irina@mail.ru.
I S
IS
Boksha
N V
NV
Lavrova
A V
AV
Grishin
A V
AV
Demidenko
A M
AM
Lyashchuk
Z M
ZM
Galushkina
R S
RS
Ovchinnikov
A M
AM
Umyarov
L R
LR
Avetisian
M Iu
MIu
Chernukha
I A
IA
Shaginian
V G
VG
Lunin
A S
AS
Karyagina
D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018533:Biomass; D003001:Cloning, Molecular; D052978:Disk Diffusion Antimicrobial Tests; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008248:Lysostaphin / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D010457:Peptidoglycan / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D013210:Staphylococcus / Q000187:drug effects / Q000235:genetics*; D013211:Staphylococcus aureus / Q000187:drug effects; D041162:Staphylococcus haemolyticus / Q000187:drug effects; D013696:Temperature
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010457:Peptidoglycan; D011994:Recombinant Proteins; D008248:Lysostaphin