Two-phase fed-batch modification for 48 hour peak expression of hepatitis B surface antigen in Pichia pastoris shake flask system

Abstract

A study of the Mut + phenotype for the expression of recombinant hepatitis B surface antigen (HBsAg) in Pichia pastoris strain GS115 using the pPIC3.5K vector with a two-phase fed-batch protocol in a shake flask system is described. Expression levels of HBsAg protein of 6.74 g L -1 Dry Cell Weight (DCW) and 26.07 mg L -1 of HBsAg concentration were achieved 48 h from the induction point which added to a 120 h reduction in production period in comparison with Mut S expression (168 h). The use of the pPIC3.5K-HBsAg plasmid in the Mut + phenotype enhanced the expression of HBsAg by a nearly 13 times higher volumetric productivity in the first 24 h and 35 times higher at peak production concentration. Comparison of AOX expression cassettes relative to the HBsAg gene in the role of mRNA secondary structure during translation initiation revealed that HBsAg possesses lower folding stability with AOX1 Mut + phenotype. The results from this study demonstrated that expression of HBsAg with Mut + AOX1 promoter is adequate as an alternative for the production of HBsAg. In addition, the AOX promoter of the Mut + phenotype was observed to be better suited for HBsAg expression, which correlates with the ease of translation initiation under shake flask conditions.

Accurate and cost‐effective prediction of HBsAg titer in industrial scale fermentation process of recombinant Pichia pastoris by using neural network based soft sensor

“…As illustrated in Fig. , after these initial upstream stages, the inoculum was transferred to the 30‐L fermenter to perform glycerol batch fermentation in the presence of Basal salts media (15 L) . Reaching the dry cell weight (DCW) of about 3.36 g/L, the culture broth was transferred to 600‐L fermenter containing culture media (180 L Basal salts + glycerol) .…”

“…For recombinant protein expression in P. pastoris , the AOX1 promoter is usually used due to its strong and tightly regulated feature . For Mut + phenotype of P. pastoris , having both AOX genes, the addition of methanol in the fermentation process as a carbon source not only induces protein expression but also leads to biomass growth .…”

Wide dynamic range of surface‐plasmon‐resonance‐based assay for hepatitis B surface antigen antibody optimal detection in comparison with ELISA

“…Recombinant HBsAg was expressed from P. pastoris according to the methods described previously . Briefly, production of intracellular HBsAg recombinant protein from P. pastoris (GS115 strain, methanol utilization plus [Mut + ]) was cultivated using fed‐batch fermentation.…”

“…Hepatitis B is a liver infection caused by the hepatitis B virus (HBV) . Worldwide, it is estimated that more than 240 million people have chronic hepatitis B infections , and it is responsible for approximately 600,000 deaths per year from acute infection and hepatitis‐related liver cancer and cirrhosis .…”