New effective sources of the Staphylococcus simulans lysostaphin.
The gene encoding Staphylococcus simulans lysostaphin has been cloned into two Escherichia coli expression systems: pET23b+ (Novagen, UK) and pBAD/Thio-TOPO (Invitrogen, USA), which allow the overexpression of a target protein as a fusion protein. The enzyme produced in the pET system contains a cluster of six histidines at the C-terminus, and the protein produced in the pBAD system contains 133 additional amino acid residues at the N-terminus, including thioredoxin, a cluster of six histidines and a recognition site for endoprotease Factor Xa. The recombinant enzymes were purified by metal-affinity chromatography on a Co2+-Sepharose column. Approximately 20 mg of purified recombinant enzyme were obtained in the pET expression system and 39 mg in the pBAD system, from a 1-L culture. The obtained fusion protein from the pET system revealed specific activity that was approximately 10 times higher than that of the fusion protein from the pBAD system (970 U/mg versus 83 U/mg). The purified enzymes displayed maximum activity at close to 45 degrees C and pH 8.0 or 7.5 for the enzyme obtained from pET and pBAD system, respectively. The lysostaphin activity was strongly inhibited by Zn2+ or Cu2+ (2 mM) with a 70-80% decrease. The Ni2+ (2 mM) also inhibited the enzyme with a 60 and 20% activity decrease for enzyme from the pET and pBAD system, respectively. The Co2+ had no impact on enzymatic activity at the 2 mM concentration; however, 30 and 20% activity decreases were observed at the 10mM concentration for the enzyme obtained from the pET and pBAD expression systems, respectively. EDTA, known as a strong inhibitor of the native lysostaphin, had no impact on the antistaphylococcal activity of either recombinant enzyme.
Szweda
Piotr
P
Department of Food Chemistry, Technology and Biotechnology, Gdańsk University of Technology, ul. G. Narutowicza 11/12, 80-952 Gdańsk, Poland.
Kotłowski
Roman
R
Kur
Józef
J
D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D008248:Lysostaphin / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013210:Staphylococcus / Q000145:classification / Q000201:enzymology* / Q000235:genetics*
D011994:Recombinant Proteins; D008248:Lysostaphin