Journal of Bacteriology & Parasitology

Cloning, sequencing, expression and purification of Staphylococcus simulans lysostaphin enzyme gene

3^rd International Congress on Bacteriology and Infectious Diseases

August 04-06, 2015 Valencia, Spain

Hossein Vazini, Sajjad Yazdansetad, Nasibeh Afsari and Rasool Pazhohesh

Posters-Accepted Abstracts: J Bacteriol Parasitol

Abstract:

Lysostaphin is a zinc-metalloprotease glycylglycine endopeptidase enzyme which is secreted by Staphylococcus simulans
biovar staphylolyticus. Lysostaphin specifically disrupts pentaglycine crosslinks of peptidoglycan (between the third and
fourth glycine residues) of Staphylococcus aureus cell wall. The present study was aimed to clone, sequence, express and purify
of S. simulans lysostaphin enzyme gene for biological experiments and prospect of drug development.
Plasmid pACK1 of S. simulans was extracted using alkaline lysis method. The lysostaphin gene was amplified by PCR and
directly cloned into pTZ57R/T vector and transformed into Escherichia coli BL21 by cold CaCl2 shock method. In the next
time, the amplified fragment was digested from the recombinant pTZ57R/T vector using PstI and Xba�? enzymes. The vector
pWB980 was also digested by the same enzymes and purified. The ligation reaction was done between the restricted fragment
and vector by standard protocols. The recombinant plasmid was transformed into B. subtilis WB600 by electroporation
method at 950 V. The lysostaphin protein production was estimated by Bradford and SDS-PAGE methods. At last the protein
was confirmed by Western blot analysis. PCR amplification of the lysostaphin gene resulted in an amplicon of 750 bp. The
gene fragment was cloned into pTZ57R/T vector then sub-cloned into pWB980 vector of B. subtilis WB600. Several colonies
containing recombinant plasmid selected on medium supplemented with the kanamycin. PCR and enzymatic digestion
methods confirmed the process of gene cloning; moreover sequencing data exhibited no significance in the sequence of
recombinant gene in comparison with the sequence of wild type gene. The recombinant protein with 250 amino acids was
expressed in B. subtilis WB600 cells with significant yield. SDS-PAGE revealed a 27-kDa protein band on polyacrylamide
gel. The protein was purified using Ni-NTA agarose column and was recognized by Western blotting method with polyclonal
rabbit antiserum. In the present study, we report on cloning, expression and purification of the lysostaphin enzyme gene from
S. simulans to B. subtilis WB600 using expression vector pWB980 for the first time. The recombinant product can be safely used
as a novel antimicrobial agent in the treatment of staphylococcal infections.

Biography :

Hossein Vazini presently working at department of Nursing, Islamic Azad University, Hamedan, Iran.