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. 2022 May 13:35:e00736.
doi: 10.1016/j.btre.2022.e00736. eCollection 2022 Sep.

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

Hana Askri  1 , Ines Akrouti  1 , Samia Rourou  1 , Hela Kallèl  1   2
Affiliations

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

Hana Askri et al. Biotechnol Rep (Amst). .

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 (YP/X) of 0.032 and 0.06 µg/g(DCW) respectively. The use of semi-defined feeding solution enhanced the production and the YP/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 YP/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.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Schematic representation of the expression vectors construction and expression Strains transformation.
Fig. 2
Fig. 2
Influence of growth temperature and carbon source on recombinant RABV-G production in shake flask culture. Growth profile (cell density measured at OD 600 nm) and Western blot analyses of the productivity of the recombinant clone S4LC cultured in shake flask using seven different carbon sources (Glucose (Glc), Glycerol (Gly), Fructose (Frc), Lactose (Lac), Sorbitol (Sor), Methanol (Meth)) at 180 rpm agitation rate, in filling volume of 10:1 flask/culture volume ratio and at incubation temperature of (a) 20 °C, (b) 25 °C, (c) 30 °C, and (d) 32 °C. the secretion of the recombinant RABV-G was investigated by western blot analyses of 20 µl of pelleted culture broth samples from different culture time-points (24 h, 48 h, and 72 h). PC (= positive control): native Rabies virus glycoprotein contained in Rabies reference vaccine consisting of purified inactivated rabies virus particles of PV vaccine strain.
Fig. 3
Fig. 3
Relative transcription level of gene encoding the recombinant RABV-G in diffrent carbon source growth condition at 30 °C and 180 rpm shake flask cultures. Yeast cells total RNA was extracted at the 24-h time point, and the mRNA level of RABV-G was quantifiedgene by real time PCR, relative to β-actin endogenous gene and then the transcript levels were normalized relative to cells grown on glucose the commonly used carbon source in GAP promoter expression systems .
Fig. 4
Fig. 4
Influence of oxygen availability and carbon source on RABV-G production in Shake flask culture. The production of RABV-G was investigated by western blot analysis of 20 µl of pelleted culture broth samples from different culture time-points (24 h, 48 h, and 72 h) of cultures grown on seven different carbon sources (Glucose (Glc), Glycerol (Gly), Fructose (Frc), Lactose (Lac), Sorbitol (Sor), Methanol (Meth)) in hypoxic condition at30 °C, 180 rpm in filling volume of (a) 5:1 and (b) 3:1 flask/culture volume ratio and cultures grown in ameliorated oxygen transfer condition (c) at 30 °C, 250 rpm and in filling volume of 10:1 flask/culture volume ratio. PC (= positive control): native Rabies virus glycoprotein contained in Rabies reference vaccine consisting of purified inactivated rabies virus particles of PV vaccine strain.
Fig. 5
Fig. 5
Time courses of fed-batch fermentation production of the recombinant RABV-G in 5 l bioreactor. Curves (upper part) of Biomass production (Dry cell weight g/l), Residual Substrate (carbon source g/l), Dissolved oxygen level (DO%), Stirrer rate (rpm), Temperature ( °C) and pH variation in addition to western blot analysis (lower part) of time-course secretion of the produced RABV-G during cultures grown, in the batch phase, on glycerol as carbon source and fed with glycerol (a) or glucose (b) or glucose + casamino acid (c) in the fed batch phase; and culture grown on sucrose as carbon source in both the batch and fed batch phases (d).The secretion of the recombinant RABV-G was investigated by western blot analyses of 20 µl of pelleted culture broth samples from different culture time-points. The arrow on western blot figures indicates the monomeric correctly glycosylated form of RABV-G and the higher bands represent aggregated and oligomerized forms while the lower bands are non-glycosylated forms. PC (= positive control): native Rabies virus glycoprotein contained in Rabies reference vaccine consisting of purified inactivated rabies virus particles of PV vaccine strain.
Fig. 6
Fig. 6
Purification and characterization of recombinant RABV-G (a) 10% SDS-PAGE silver-stained gel (lower part) and western blotting (upper part) analysis of collected fraction during recombinant RABV-G purification using SP Sepharose cation exchange chromatography column. (b) western blotting analysis of (1) purified recombinant RABV-G and (2) native Rabies virus glycoprotein trimerization state under native PAGE condition. (c) western blot analysis of PNGase treated (1), (3) and untreated (2), (4) purified recombinant RABV-G and native Rabies virus glycoprotein respectively, to characterize the glycosylation pattern of the purified RABV-G compared to the native Rabies virus glycoprotein. (d) competitive inhibition of Rabies virus cell binding in presence of varying concentrations of purified recombinant RABV-G as demonstrated by Fluorescence inhibition assay compared to controls: cell control with no fluorescence and virus control with typical fluorescence of 100% infectivity. Scale bar 200 µm.
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