Biomedical Research

Bacteriophage lysins are murein hydrolases that act on the cell wall of host bacteria to release progeny phage. Research indicated that lysins can kill bacteria efficiently and specifically in vitro. The streptococcal phage lysin, PlyC, was found to lyse streptococcal species rapidly. The catalytically active PlyC holoenzyme is composed of two components, eight PlyCB subunits for each PlyCA. We cloned the two genes plyCA and plyCB which encoding 465 and 72 amino acids respectively and inserted into prokaryotic expression vector pET-32a (+) to construct the recombinant plasmid pET32a-PlyCA and pET32a-PlyCB, transformed into E. coli BL21 (DE3). The recombinant PlyCA and PlyCB expressed in the form of inclusion body, they were renatured by dilution and dialysis. After separating and preliminary purifying, inclusion body PiyCA and PlyCB over 80% purity were dissolved in 8 mol/L urea separately, then diluted or dialyzed into 0.8 mol/L urea. Research showed that dialysis by gentle removal of urea against 5 mmol/L sodium phosphate buffer (pH 6.1) with redox agents, protein concentration of 50 μg/ml and temperature of 4°C was found to be optimal. The maximum enzyme activity was observed at pH 7.0, 40°C. The results showed that the renatured PlyC could efficiently cleavage Streptococcus pyogenes (group A β-hemolytic streptococci). This study laid the foundation for the further study and achieving an effective treatment for streptococcal infection.

Author(s): Wei-Qing Chen, Jian-Fen Zhang, Hong Chen, Pu Wang
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