Effect of gene copy number, methanol and time period on production of CVS rabies glycoprotein expressed in Pichia pastoris

Abstract

Rabies is a viral disease that causes acute encephalitis in warm blooded animals. Rabies is almost invariably fatal if post-exposure prophylaxis is not administered prior to the onset of severe symptoms. Glycoprotein is the major surface protein of rabies virus, responsible for the production of neutralizing antibodies. Currently, available mammalian cell culture based rabies vaccine is highly expensive because of cell line maintenance and downstream processes. Large quantity of this vaccine is required to meet the clinical requirements at economical level. Therefore, the present investigation was conducted to study the effect of multicopy gene insertion, methanol concentration and incubation time interval on the protein expression of challenge virus standard (CVS) rabies glycoprotein (RGP) in Pichia pastoris. The optimal methanol concentration and incubation time for induced CVS rabies glycoprotein were 1 % (w/v) and 96 h respectively. Clone 13 (single copy number) produced 0.4g/l whereas Clone 14 (5 copies) produced 1.5 g/L of recombinant protein. The clone with five copy number expressed CVS_RGP at a higher level than single copy number clone. Therefore, the gene copy number is one of the major factor involved in heterologous protein expression in P. pastoris.