Cytoplasmic expression of mature glycylglycine endopeptidase lysostaphin with an amino terminal hexa-histidine in a soluble and catalytically active form in Escherichia coli.
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.
Sharma
Rahul
R
Bharat Biotech Foundation, Genome Valley, Turkapally, Shameerpet, Hyderabad, AP 500 078, India.
Sharma
Poonam R
PR
Choudhary
Manohar L
ML
Pande
Amit
A
Khatri
Ghan Shyam
GS
D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008248:Lysostaphin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013210:Staphylococcus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D006639:Histidine; D008248:Lysostaphin