RESUMO
In this study, we describe the characterization, cloning, expression and purification of the lysin A gene of the mycobacteriophage TM4. The gene TM4_gp29 (gp29) is a 1644-bp gene that codes for a 58.6-kDa protein and contains peptidoglycan recognition protein, Zn-binding and amidase catalytic domains. The gene was cloned into Escherichia coli using the 'His-Tag' pQE60 vector. After affinity chromatography-mediated purification, the protein was concentrated and visualized using sodium dodecyl sulphate polyacrylamide gel electrophoresis. Evidence of peptidoglycan-degrading activity was observed initially by a chloroform assay and later by conventional zymogram analysis.
Assuntos
Clonagem Molecular , Expressão Gênica , Micobacteriófagos/enzimologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Micobacteriófagos/genética , Micobacteriófagos/fisiologia , Peptidoglicano/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/isolamento & purificação , Proteínas do Envelope Viral/metabolismoRESUMO
The endolysin LysK derived from staphylococcal phage K has previously been shown to have two enzymatic domains, one of which is an N-acetylmuramoyl-L-alanine amidase and the other a cysteine/histidine-dependant amidohydrolase/peptidase designated CHAP(k). The latter, when cloned as a single-domain truncated enzyme, is conveniently overexpressed in a highly-soluble form. This enzyme was shown to be highly active in vitro against live cell suspensions of S. aureus. In the current study, the IVIS imaging system was used to demonstrate the effective elimination of a lux labeled S. aureus from the nares of BALB/c mice.
