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

Journal of Nanobiotechnology

Research

Tamara Dworeck¹  Manuel Krewinkel¹  Marco Fioroni¹ 

[1] Department of Biotechnology (Biology VI), RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany;

关键词: Channel proteins;    FhuA;    liposomes;    protein engineering;    HRP;    TMB-Assay;    nanocontainers;   

DOI  :  10.1186/1477-3155-9-33

 received in 2011-07-14, accepted in 2011-08-19,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundChannel 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.ResultsThe FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-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 β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 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 Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%.ConclusionIn this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

【 授权许可】

Unknown   
© Krewinkel et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.