Production, characterization and crystallization of the Plasmodium falciparum aquaporin.
Journal article, 2008
The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the first bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to different genomic A+T content and different codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be confirmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64mg PfAQP per liter cell culture) PfAQP was purified to homogeneity (18mg/L) and initial attempts at crystallization of the protein yielded several different forms.
Porins
metabolism
Protozoan Proteins
Escherichia coli
Animals
Codon
isolation & purification
Crystallization
isolation & purification
biosynthesis
biosynthesis
metabolism
biosynthesis
Pichia
chemistry
metabolism
Recombinant Proteins
Saccharomyces cerevisiae
chemistry
Author
Kristina Hedfalk
University of Gothenburg
Nina Pettersson
University of Gothenburg
Fredrik Öberg
University of Gothenburg
Stefan Hohmann
University of Gothenburg
Euan Gordon
Chalmers, Chemical and Biological Engineering, Molecular Imaging
Protein Expression and Purification
1046-5928 (ISSN) 1096-0279 (eISSN)
Vol. 59 1 69-78Subject Categories
Biochemistry and Molecular Biology
Chemical Sciences
DOI
10.1016/j.pep.2008.01.004
PubMed
18295508