Production, characterization and crystallization of the Plasmodium falciparum aquaporin.
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.
Hedfalk
Kristina
K
Department of Chemistry and Bioscience/Molecular Biotechnology, Chalmers University of Technology, Box 462, S-405 30 Göteborg, Sweden. kristina.hedfalk@chem.gu.se
Pettersson
Nina
N
Oberg
Fredrik
F
Hohmann
Stefan
S
Gordon
Euan
E
D000818:Animals; D003062:Codon; D003460:Crystallization; D004926:Escherichia coli / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D018272:Porins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D003062:Codon; C449801:PfAQP protein, Plasmodium falciparum; D018272:Porins; D015800:Protozoan Proteins; D011994:Recombinant Proteins