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 69-78

Ämneskategorier

Biokemi och molekylärbiologi

Kemi

DOI

10.1016/j.pep.2008.01.004

PubMed

18295508