Loading...

Summer Research Fellowship Programme of India's Science Academies

Cloning, Expression and Purification of Respiratory Syncitial Virus Fusion Protein in Bacterial Expression System

Ayan Das

Department of Biotechnology, Techno India University EM-4, Salt lake, Sector 5, West Bengal, India.

Dr. Guruprasad R. Medigeshi

Clinical and Cellular Virology Lab, Vaccine and Infectious Disease Research Center, Translational Health Science and Technology Institute, NCR-Biotech Science Cluster, Faridabad, India

Abstract

Respiratory Syncytial Virus (RSV) is a common respiratory virus that infects the upper and lower respiratory tracts and affects mostly infants less than two years of age. During RSV infection, the glycoprotein (G) functions in attachment to host cell and fusion (F) protein helps in fusion between viral envelope and plasma membrane. The broad objective of this work was to generate antibody against the RSV-F protein to study RSV biology in cell culture models of infection. It is reported that the amount of glycosylation in the G protein is high, which makes it an undesirable candidate for antibody generation. The F protein on the other hand has low glycosylation. It is synthesized as an inactive precursor F0, which is later cleaved into three polypeptides F2, p27 and F1. The F1 polypeptide sequence is conserved among the Paramyxoviridae family and is a suitable candidate for antibody generation. In this study, I cloned and expressed F1 gene in a bacterial expression system. I standardized the protein expression in different E. coli expression systems BL21(DE3)pLysS, Rosetta, Codon+, pGro7 and DE3. Only Codon+ strain showed expression of RSV-F1 protein which was confirmed by western blot analysis. I also checked the expression in Codon+ at different temperatures, but did not find any significant change in protein expression. Upon analysis of the protein localization in bacterial cells, I found that the protein was present in the bacterial pellet fraction and not in the supernatant, suggesting that it was either present in the membrane fraction or packed in inclusion bodies. Finally, protein expression was assessed by using denaturing conditions to retrieve protein from bacterial cell pellet but the expression of protein was very less. Different strategies to increase the concentration of protein by cloning F1-gene into another vector is being explored further.

More
Written, reviewed, revised, proofed and published with