High-throughput Fed-batch Production of Affibody® molecules in a novel Multi-fermentor system
Independent thesis Basic level (professional degree), 20 points / 30 hpStudent thesis
The present Master thesis describes the development and optimization of a fed-batch process for production of recombinant proteins in Escherichia coli BL21(DE3) in a multi-fermentor system. The system consists of six 1-liter fermentors, capable of producing 500-1500 μg/mL with present protocol.
Response surface methodology (RSM) was used for multivariable optimization regarding cultivation time, pH, temperature and feed rate. Optimal protein expression conditions were found out to be 17.8 h cultivation time, 36.7 ºC, pH 6.8 and a feed rate corresponding to specific growth of 0.23 h-1, on glucose substrate. The aggregation of expressed proteins to inclusion bodies, could not be affected by the various growth conditions employed during cultivations.
A study was conducted regarding growth conditions effect on phosphogluconoylation of expressed proteins. In ten fed-batch cultivations on glucose, LC/MS analysis showed a gluconoylated fraction with additional 178 Da mass, but no correlation between growth conditions and gluconoylation could be found. In two fed-batch cultivations on glycerol-feed, a lower feed rate resulted in no gluconoylation, while a higher did. An explanation would be that the lower amount of available intra-cellular carbon limits formation of gluconoylation precursors.
Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi , 2005. , 46 p.
Escherichia coli, fed-batch, Affibody® molecules, phosphogluconoylation
Other Industrial Biotechnology
IdentifiersURN: urn:nbn:se:liu:diva-3490ISRN: LITH-IFM-EX--05/1488--SEOAI: oai:DiVA.org:liu-3490DiVA: diva2:20394