Restriction-free cloning for molecular manipulation and augmented expression of banana bunchy top viral coat protein.

Banana bunchy top virus (BBTV) causing bunchy top disease, is one of the most devastating diseases of banana and plantain. All the six genomic components of isolates from different parts of the world have been well characterised, with most of the studies focusing on replicase gene and coat protein gene. Overexpression of coat protein (CP) in Escherichia coli system can contribute significantly in structural as well as immunological studies. In the present investigation, the full length BBTV CP was cloned to pGEX-4T-2 expression vector and overexpressed in various Escherichia coli strains to obtain high quality and quantity of the CP. An augmented overexpression and stability of recombinant coat protein was achieved by molecular manipulation of the clone by restriction-free (RF) cloning platform. The RF cloning was employed to replace the thrombin cleavage site in the vector backbone, which was also present in the protein of interest, and to incorporate TEV protease site to cleave fusion protein at this specific site, and separate the affinity tag. The RF method allows direct transformation of the PCR product to undergo ligation in vivo and obtain the transformants thereby avoiding the restriction digestion and ligation of the product to the linearized plasmid. From a litre culture, 1.084 mg/ml of fusion protein with GST tag was obtained after GSH sepharose affinity column chromatography. The fluorescence spectra indicated partial disordered tertiary structure of the fusion protein. Cleavage of tag was attempted using TEV protease overexpressed and purified in the laboratory. [ABSTRACT FROM AUTHOR]