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Production, characterization and crystallization of the Plasmodium falciparum aquaporin.

Kristina Hedfalk ; Nina Pettersson ; Fredrik Oberg ; Stefan Hohmann ; Euan Gordon (Institutionen för kemi- och bioteknik)
Protein expression and purification (1096-0279). Vol. 59 (2008), 1, p. 69-78.
[Artikel, refereegranskad vetenskaplig]

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.

Nyckelord: Animals, Codon, Crystallization, Escherichia coli, metabolism, Pichia, metabolism, Porins, biosynthesis, chemistry, isolation & purification, Protozoan Proteins, biosynthesis, chemistry, isolation & purification, Recombinant Proteins, biosynthesis, Saccharomyces cerevisiae, metabolism



Denna post skapades 2008-10-09. Senast ändrad 2011-01-20.
CPL Pubid: 75035

 

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Institutioner (Chalmers)

Institutionen för kemi (2001-2011)
Institutionen för cell- och molekylärbiologi (1994-2011)
Institutionen för kemi- och bioteknik (2005-2014)

Ämnesområden

Kemi
Cell- och molekylärbiologi

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