Production, characterization and crystallization of the Plasmodium falciparum aquaporin.
Artikel i vetenskaplig tidskrift, 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
Författare
Kristina Hedfalk
Göteborgs universitet
Nina Pettersson
Göteborgs universitet
Fredrik Öberg
Göteborgs universitet
Stefan Hohmann
Göteborgs universitet
Euan Gordon
Chalmers, Kemi- och bioteknik, Molekylär mikroskopi
Protein Expression and Purification
1046-5928 (ISSN) 1096-0279 (eISSN)
Vol. 59 1 69-78Ämneskategorier
Biokemi och molekylärbiologi
Kemi
DOI
10.1016/j.pep.2008.01.004
PubMed
18295508