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Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

: Gurramkonda, C.; Adnan, A.; Gäbel, T.; Lünsdorf, H.; Ross, A.; Nemani, S.K.; Swaminathan, S.; Khanna, N.; Rinas, U.

Fulltext urn:nbn:de:0011-n-931020 (547 KByte PDF)
MD5 Fingerprint: 3686593e0ac40c5b715ba3dad0cd6ca0
Created on: 8.4.2009

Microbial cell factories. Online journal 8 (2009), Art. 13, 8 pp.
ISSN: 1475-2859
Journal Article, Electronic Publication
Fraunhofer ITEM ()
methylotrophic yeast; operational strategies; expression; culture; purification; stability; virus; fermentation; particles

Background: Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase I locus. Results: Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of similar to 7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. Conclusion: In comparison to the highest yields reported so far, our simple cultivation process resulted in an similar to 7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.