Cet ouvrage et des milliers d'autres font partie de la bibliothèque YouScribe
Obtenez un accès à la bibliothèque pour les lire en ligne
En savoir plus

Partagez cette publication

Microbial Cell Factories
Open Access Research Simple high-cell density fed-batch technique for high-level recombinant protein production withPichia pastoris: Application to intracellular production of Hepatitis B surface antigen 1,2 2,3 2,4 Chandrasekhar Gurramkonda , Ahmad Adnan , Thomas Gäbel , 2 2,4 1 Heinrich Lünsdorf , Anton Ross , Satish Kumar Nemani , 1 1 2 Sathyamangalam Swaminathan , Navin Khanna and Ursula Rinas*
1 2 Address: International Centre for Genetic Engineering & Biotechnology, New Delhi, India, Helmholtz Centre for Infection Research, 3 4 Braunschweig, Germany, Department of Chemistry, Government College University Lahore, Lahore, Pakistan and Fraunhofer ITEM, Hannover/ Braunschweig, Germany Email: Chandrasekhar Gurramkonda  chskrg@icgeb.res.in; Ahmad Adnan  adnan_biochem@yahoo.com; Thomas Gäbel  thomas.gaebel@item.fraunhofer.de; Heinrich Lünsdorf  heinrich.luensdorf@helmholtzhzi.de; Anton Ross  anton.ross@item.fraunhofer.de; Satish Kumar Nemani  nemani@icgeb.res.in; Sathyamangalam Swaminathan  swami@icgeb.res.in; Navin Khanna  navin@icgeb.res.in; Ursula Rinas*  ursula.rinas@helmholtzhzi.de * Corresponding author
Published: 10 February 2009 Received: 3 December 2008 Accepted: 10 February 2009 Microbial Cell Factories2009,8:13 doi:10.1186/1475-2859-8-13 This article is available from: http://www.microbialcellfactories.com/content/8/1/13 © 2009 Gurramkonda et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 yeastPichia pastorisGS115 by inserting theHBsAggene 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 -1 addition of methanol to a final concentration of 6 g L . This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-linesignal 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.
Page 1 of 8 (page number not for citation purposes)
Un pour Un
Permettre à tous d'accéder à la lecture
Pour chaque accès à la bibliothèque, YouScribe donne un accès à une personne dans le besoin