Open AccessCloning and expression of a mureinolytic enzyme from the mycobacteriophage TM4
Author(s) -
Henry Marine,
Begley Máire,
Neve Horst,
Maher Fiona,
Ross Reynolds Paul,
McAuliffe Olivia,
Coffey Aidan,
O'Mahony Jim M.
Publication year - 2010
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2010.02080.x
Subject(s) - peptidoglycan , microbiology and biotechnology , cloning (programming) , affinity chromatography , escherichia coli , biology , biochemistry , gene , gel electrophoresis , molecular cloning , amidase , lysin , gene expression , chemistry , enzyme , bacteriophage , computer science , programming language
Abstract 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.