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

verfasst von

Chandrasekhar Gurramkonda, Ahmad Adnan, Thomas Gäbel, Heinrich Lünsdorf, Anton Ross, Satish Kumar Nemani, Sathyamangalam Swaminathan, Navin Khanna, Ursula Rinas

Abstract

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 1 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 ∼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 ∼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.

Externe Organisation(en)

International Centre for Genetic Engineering and Biotechnology
Helmholtz-Zentrum für Infektionsforschung GmbH (HZI)
Government College University Lahore
Fraunhofer-Institut für Toxikologie und Experimentelle Medizin (ITEM)

Typ

Artikel

Journal

Microbial cell factories

Band

8

ISSN

1475-2859

Publikationsdatum

10.02.2009

Publikationsstatus

Veröffentlicht

Peer-reviewed

Ja

ASJC Scopus Sachgebiete

Biotechnologie, Bioengineering, Angewandte Mikrobiologie und Biotechnologie

Ziele für nachhaltige Entwicklung

SDG 3 – Gute Gesundheit und Wohlergehen

Elektronische Version(en)

https://doi.org/10.1186/1475-2859-8-13 (Zugang: Offen)