Dataset Information

Engineering of an E. coli outer membrane protein FhuA with increased channel diameter.

ABSTRACT:

Background

Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering.

Results

The FhuA ?1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal ?-strands, resulting in variant FhuA ?1-159 Exp. The total number of ?-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high ?-sheet content, suggesting the correct folding of FhuA ?1-159 Exp. To further prove the FhuA ?1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA ?1-159 Exp as compared to FhuA ?1-159, well correlated to the expected channel surface area increase of ~16%.

Conclusion

In this study using a simple "semi rational" approach the FhuA ?1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA ?1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA ?1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

SUBMITTER: Krewinkel M

PROVIDER: S-EPMC3170585 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Publications

Engineering of an E. coli outer membrane protein FhuA with increased channel diameter.

Krewinkel Manuel M Dworeck Tamara T Fioroni Marco M

Journal of nanobiotechnology 20110819

<h4>Background</h4>Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering.<h4>Results</h4>The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid ...[more]

PMID: 21854627

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Project description:BackgroundMembrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved.ResultsWe engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER.ConclusionsWe conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells.

Dataset Information

Engineering of an E. coli outer membrane protein FhuA with increased channel diameter.

Background

Results

Conclusion

Publications

Engineering of an E. coli outer membrane protein FhuA with increased channel diameter.

Similar Datasets

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