Construction of a system for heterologous production of carbonic anhydrase from Plasmodium falciparum in Pichia pastoris

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.


1 Introduction
1.1 Aim of this project
2 Background
2.1 Malaria
2.1.1 Epidemiology and pathogenesis
2.1.2 The Plasmodium genome and proteome
2.1.3 Drug resistance development
2.2 Carbonic anhydrase
2.2.1 α-carbonic anhydrases
2.2.2 The role of carbonic anhydrase in Plasmodium
2.3 Codon Bias
2.4 Escherichia coli as expression system
2.5 Pichia pastoris as expression system
2.5.1 Controlling gene expression in Pichia pastoris
2.5.2 The pPICZαA vector
2.6 Immobilized metal ion affinity chromatography (IMAC)
3 Materials and methods
3.1 Expression in Pichia pastoris
3.1.1 Design of the synthetic PfCA gene
3.1.2 Construction of expression plasmid
3.1.3 Transformation
3.1.4 Verification of insert
3.1.5 Expression
3.1.6 Detection of protein
3.1.7 Purification of protein
4 Results
4.1 PfCA gene length and vector design
4.2 Transformation
4.3 Verification of insert
4.4 Expression
4.4.1 SDS-PAGE analysis
4.4.2 Dot blot analysis
4.4.3 Western blot analysis
4.5 Purification
5 Discussion
5.1 Future research
6 Conclusions
7 Acknowledgements
8 References

Author: Gullberg, Erik

Source: Linkoping University

Download URL 2: Visit Now

Leave a Comment