Production, purification, and characterization of recombinant rabies virus glycoprotein expressed in PichiaPink™ yeast

Hana Askri; Ines Akrouti; Samia Rourou; Hela Kallèl

doi:10.1016/j.btre.2022.e00736

Production, purification, and characterization of recombinant rabies virus glycoprotein expressed in PichiaPink™ yeast

Biotechnol Rep (Amst). 2022 May 13:35:e00736. doi: 10.1016/j.btre.2022.e00736. eCollection 2022 Sep.

Authors

Hana Askri¹, Ines Akrouti¹, Samia Rourou¹, Hela Kallèl^{1

2}

Affiliations

¹ Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Group of Biotechnology Development, Institut Pasteur de Tunis, Université Tunis El Manar, 13, place Pasteur. BP.74., Tunis 1002, Tunisia.
² UnivercellsVaccines, Nivelles, Belgium.

Abstract

The commonly used host for industrial production of recombinant proteins Pichia pastoris, has been used in this work to produce the rabies virus glycoprotein (RABV-G). To allow a constitutive expression and the secretion of the expressed recombinant RABV-G, the PichiaPink™ commercialized expression vectors were modified to contain the constitutive GAP promoter and the α secretion signal sequences. Recombinant PichiaPink™ strains co-expressing the RABV-G and the protein chaperone PDI, have been then generated and screened for the best producer clone. The influence of seven carbon sources on the expression of the RABV-G, has been studied under different culture conditions in shake flask culture. An incubation temperature of 30°C under an agitation rate of 250 rpm in a filling volume of 10:1 flask/culture volume ratio were the optimal conditions for the RABV-G production in shake flask for all screened carbon sources. A bioreactor Fed batch culture has been then carried using glycerol and glucose as they were good carbon sources for cell growth and RABV-G production in shake flask scale. Cells were grown on glycerol during the batch phase then fed with glycerol or glucose defined solutions, a final RABV-G concentration of 2.7 µg/l was obtained with a specific product yield (Y_P/X) of 0.032 and 0.06 µg/g(_DCW) respectively. The use of semi-defined feeding solution enhanced the production and the Y_P/X to 12.9 µg/l and 0.135 µg/g(_DCW) respectively. However, the high cell density favored by these carbon sources resulted in oxygen limitation which influenced the glycosylation pattern of the secreted RABV-G. Alternatively, the use of sucrose as substrate for RABV-G production in large scale culture, resulted in less biomass production and a Y_P/X of 0.310 µg/g_(DCW) was obtained. A cation exchange chromatography was then used for RABV-G purification as one step method. The purified protein was correctly folded and glycosylated and able to adopt trimeric conformation. The knowledges gained through this work offer a valuable insight into the bioprocess design of RABV-G production in Pichia pastoris to obtain a correctly folded protein which can be used during an immunization proposal for subunit Rabies vaccine development.

Keywords: Carbon source; Fed-batch fermentation; Pichia pastoris constitutive expression; Rabies virus glycoprotein.