Expression of the non-structural protein 4 (nsp4) of rotavirus in Escherichia coli based expression systems

Viral gastroenteritis infecting young children and babies remains a major medical problem around the world and rotavirus is the major pathogen of viral gastroenteritis. Diarrhoea resulting from viral gastroenteritis can cause severe dehydration leading to death. The non structural protein NSP4 was discovered as an enterotoxin that accounts for the toxicity of the rotavirus acting against the intestinal lining of the infected hosts. Thus, the production of abundant NSP4 proteins at the laboratory scale is required for NSP4-orientated experiments to examine the pathology of rotavirus infection. So far, molecular biology techniques have been successful in producing NSP4 proteins at the laboratory scale. This was achieved by inserting the NSP4 gene into an expression vector, such as a bacterial plasmid, and later introducing the recombinant plasmid into an expression host like bacteria, insect cells or mammalian cells for the expression of NSP4 proteins. The expression of NSP4 proteins in bacteria, particularly Escherichia coli, is of particular interest as the expression system offers advantages compared with other expression systems. First, E. coli is easily cultivated in laboratory with inexpensive reagents and media. Second, the well-characterised genetics and biology of E. coli enable the flexible manipulation of the bacterial cell to express the toxic NSP4 proteins. Therefore, in this study, the expression of full-length NSP4 proteins of human RV4 and RV5 strains was attempted in two E. coli-based systems: (i) M15 strain carrying recombinant pQE60 containing NSP4 genes and (ii) recombinant pET28a expression vector containing NSP4 genes in the Rosetta-gami 2 (DE3) pLysS strain. Construction of appropriate recombinant plasmids was successful. However, after inducing the expression of NSP4 in E. coli host cells, the optical densities of the bacterial cultures declined, indicating that the expressed NSP4 proteins were toxic to the bacterial cells. The declining bacterial cell numbers also accounted for very low expression levels of NSP4 proteins and made the purification of the expressed proteins difficult. The results of this study suggested that expression of full-length human NSP4 proteins in E. coli cells is problematic and that other expression systems (e.g. baculovirus/insect cell-based systems) are more reliable options.