Construction of a system for heterologous production of carbonic anhydrase from Plasmodium falciparum in Pichia pastoris
Independent thesis Advanced level (degree of Master), 20 points / 30 hpStudent thesis
Malaria is one of the biggest current global health problems, and with the increasing occurance of drug resistant Plasmodium falciparum strains, there is an urgent need for new antimalarial drugs. Given the important role of carbonic anhydrase in Plasmodium falciparum (PfCA), it is a potential novel drug target. Heterologous expression of malaria proteins is problematic due to the unusual codon usage of the Plasmodium genome, so to overcome this problem a synthetic PfCA gene was designed, optimized for expression in Pichia pastoris. This gene was also modified to avoid glycosylation, and cloned into the vector pPICZαA under the control of the methanol inducible promoter AOX1. To facilitate export of the protein into the growth medium, the gene was fused in-frame with the α-factor secretion signal from Saccharomyces cerevisiae. The construct was successfully integrated in the genome of P. pastoris GS115, and attempts were made to express the protein and purify it using immobilized metal ion affinity chromatography.In this work, no expression of the PfCA protein could be detected, so further research should focus on optimization of expression conditions, or redesign of the expression vector.
Place, publisher, year, edition, pages
2008. , 42 p.
Carbonic anhydrase, Plasmodium, Pichia pastoris, expression system
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:liu:diva-12454ISRN: LITH-IFM-EX-08/2009-SEOAI: oai:DiVA.org:liu-12454DiVA: diva2:139
Höst, Gunnar, Dr
Jonsson, Bengt-Harald, Professor