ABSTRACT
This article has been retracted consistent with Elsevier Policy on Article Withdrawal. Please see . The Publisher apologizes for any inconvenience this may cause.
ABSTRACT
The paper entitled "Constitutive expression and characterization of Hepatitis B surface antigen purified by metal affinity precipitation", which was published online on 19 May 2006, was withdrawn at the author's request.
ABSTRACT
Methicillin-resistant Staphylococcus aureus is a major problem in the world, causing hospital acquired infections and the infections/pathogenesis in community. Lysostaphin is a novel therapeutic molecule to kill the multidrug-resistant S. aureus. Mature lysostaphin is a single polypeptide (approximately 27 kDa) chain metalloprotease glycylglycine endopeptidase, capable of specifically hydrolyzing penta-glycine crosslinks present in the peptidoglycan of the S. aureus cell wall. The mature lysostaphin gene of Staphylococcus simulans has been cloned and overexpressed in the cytoplasm of E. coli with amino terminal hexa-histidine as a fusion partner under the transcriptional control of bacteriophage T7 phi 10 promoter/lac operator and ribosome binding site. The transformed E. coli BL21 (lambdaDE3) cells produced catalytically active soluble (His)6-lysostaphin fusion protein in the cytoplasm representing approximately 20% of the total cellular proteins. The fusion protein was purified to homogeneity using a single chromatographic step of IMAC on Ni-NTA agarose. The present cloning, expression, and purification procedure of recombinant lysostaphin from a non-pathogenic organism E. coli enables preparation of large quantity of r-lysostaphin for structure function studies and evaluation of its clinical potential in therapy and prophylaxis of staphylococcal infections.
Subject(s)
Cytoplasm/metabolism , Escherichia coli/genetics , Histidine/chemistry , Lysostaphin/chemistry , Lysostaphin/metabolism , Staphylococcus/enzymology , Amino Acid Sequence , Catalysis , Cloning, Molecular , Enzyme Activation , Escherichia coli/metabolism , Gene Expression Regulation, Enzymologic , Histidine/genetics , Hydrogen-Ion Concentration , Lysostaphin/isolation & purification , Molecular Sequence Data , Molecular Weight , Phylogeny , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Solubility , Staphylococcus/classification , Staphylococcus/genetics , Structure-Activity Relationship , Temperature , Time FactorsABSTRACT
A hypothetical open reading frame from Bacillus subtilis genome, yjbI [NCBI genome database Accession No. ] having homology to many globin and globin-like proteins from different microbial genomes, was selectively amplified from the chromosomal DNA of B. subtilis strain DB104 based on genome sequence database of B. subtilis strain 168. The gene was cloned and over-expressed in Escherichia coli under the transcriptional control of tandem lambda P(L) and P(R) promoters, and the protein was purified to homogeneity. The single-chain monomeric hemoglobin-like protein is stable to the extent of 5.45 kcal/mol at 25 degrees C, binds carbon mono-oxide, and shows optical spectra characteristic of hemoproteins. The protein also exhibits peroxidase-like activity. This is the first report of a truncated bacterial globin endowed with peroxidase-like activity. The activity is enhanced in the presence of urea and guanidine hydrochloride, more so in the presence of the latter. Presumably, only a small portion of the protein is involved in peroxidase activity, which is exposed with increasing concentration of the denaturants.
