A novel surface autolysin of Listeria monocytogenes serotype 4b, IspC, contains a 23-residue N-terminal signal peptide being processed in E. coli.
The 86-kDa protein IspC of 774 amino acids in Listeria monocytogenes serotype 4b has been recently identified as the target of humoral immune response to listerial infection and as a novel surface autolysin. A signal peptide is predicted at the N-terminal end of IspC, but no biochemical data has been shown to confirm the presence of the cleavage site of a signal peptidase. To address this and prepare sufficient amount of the protein for biochemical and structural characterization, we present a strategy for efficient expression and purification of IspC and analyze the purified protein by N-terminal sequencing and mass spectrometry. Expression of IspC in Escherichia coli using a pET30a-based expression construct was efficiently improved by incubating the culture at 37 degrees C for 2h followed by 4 degrees C for 16-18 h. The recombinant product rIspC remained as a soluble form in the cellular extract and was purified to electrophorectic homogeneity by the combination of metal chelate affinity chromatography with cation-exchange chromatography. The IspC was shown to contain a 23-residue N-terminal signal peptide being processed between Thr 23 and Thr 24 in E. coli, resulting in an 84-kDa mature protein. The highly purified form of rIspC from this study, exhibiting both peptidoglycan hydrolase activity and immunogenicity as previously reported, would facilitate further biochemical, structural, and functional studies of this autolysin.
Wang
Linru
L
Canadian Food Inspection Agency, Animal Diseases Research Institute, 3851 Fallowfield Road, Ottawa, Ont., Canada K2H 8P9.
Walrond
Lisa
L
Cyr
Terry D
TD
Lin
Min
M
D019747:Aldose-Ketose Isomerases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001433:Bacteriolysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D008089:Listeria monocytogenes / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology*; D012703:Serotyping
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011498:Protein Precursors; D021382:Protein Sorting Signals; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D009238:N-Acetylmuramoyl-L-alanine Amidase; D019747:Aldose-Ketose Isomerases