IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system

Biologicals : Journal of the International Association of Biological Standardization
V M Vivek SrinivasMadhusudan Hosamani

Abstract

For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens.

References

Aug 10, 2000·Molecular Therapy : the Journal of the American Society of Gene Therapy·H MizuguchiT Hayakawa
Apr 12, 2001·The Journal of General Virology·E Martínez-SalasS López de Quinto
Nov 11, 2006·The Journal of Biological Chemistry·Christiane JünemannMichael Niepmann

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Citations

Jan 19, 2019·Critical Reviews in Biotechnology·Ana Clara MignaquiAndrés Wigdorovitz

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