An effective and simplified DO-stat control strategy for production of rabies glycoprotein in Pichia pastoris

Abstract

The glycoprotein (G-protein) of rabies virus is responsible for viral attachment to the host cell surface and induces virus neutralization antibodies. In the present study, the G-protein gene of rabies virus CVS strain was cloned, sequenced and expressed in the yeast, Pichia pastoris, as a secreted protein, using a simplified DO-stat control feeding strategy. This strategy involves the addition of methanol when the dissolved oxygen (DO) level rises above the setpoint avoiding methanol accumulation and oxygen limitation. The G-protein expression was evaluated by SDS-PAGE, ELISA, and western blot assays. Like native G-protein, the recombinant G-protein was found reactive when it was challenged against specific antibodies. The data indicate that the recombinant G-protein can be easily expressed and isolated, and may be useful as a safe source in the production of diagnostic kits and subunit vaccines to prevent rabies.

Highly efficient production of rabies virus glycoprotein G ectodomain in Sf9 insect cells

“…The G E has been expressed in several expression systems: mammalian cells (Gupta et al 2005), insect Sf21 cells (Sissoëff et al 2005), and yeast cells (Qian et al 2013;Picotto et al 2017). In all cases, the soluble protein expressed was antigenic and immunogenic, and could be used to detect the immunological status of vaccinated hosts.…”

Dissolved-oxygen feedback control fermentation for enhancing β-carotene in engineered Yarrowia lipolytica

“…Subsequently, a constant DO level can be maintained by continuous feeding and keep a balance between oxygen consumption and supply. The DO-stat strategy typically works well in a defined media where nutrient depletion results in cell death and a rapid elevating DO 33 . Many authors have used the DO-stat feeding strategy to achieve high yield.…”

The cloning of the virus envelope glycoprotein F of canine distemper virus expressed in Pichia pastoris

“…The expression levels obtained for the F 0 protein were low (35 µg/mL); however, the expression system in P. pastoris allowed the production of the recombinant polypeptide without the need for as many steps of optimization of expression as those required for other expression systems [24]. Besides, the metabolism of P. pastoris is mainly aerobic and unlike Sacharomyces cereviceae it can grow in fermenters in large-scale cultures, reaching very high cell densities.…”