Cloning, expression, and deletion analysis of large nanH of Clostridium perfringens ATCC 10543

The large sialidase gene (nanH) of Clostridium perfringens ATCC 10543 was cloned in pCRII and subcloned in pQE70 expression vector. The large nanH consists of 2082 bp nucleotides and encodes 694 amino acids; large NanH is with four repeated “Asp-boxes” and one RIP (Arg–Ile–Pro) region formed by amino acids 266–268 in the sequence. It showed 26% of sequence homology with the small NanH from the same species (ATCC 10543). The large nanH, with six His residues adding to the C-terminus of NanH protein, was constructed in pQE70 (recombinant plasmid named pQE70-LSC) and expressed in Escherichia coli M15. The large NanH expressed in the periplasmic space of bacteria M15 showed 430-fold increase in large NanH enzymatic activity upon IPTG induction, as compared to the culture without IPTG added. The recombinant large NanH was purified with Ni–NTA and N-(p-aminophenyl) oxamic acid–agarose column chromatography. The purified enzyme showed specific activity of 136.6 U/mg when assayed with 4-MU-NeuAc as substrate.