tailieunhanh - báo cáo khoa học: " Engineering of an E. coli outer membrane protein FhuA with increased channel diameter"

Tuyển tập báo cáo các nghiên cứu khoa học quốc tế ngành y học dành cho các bạn tham khảo đề tài: Engineering of an E. coli outer membrane protein FhuA with increased channel diameter | Krewinkel et al. Journal of Nanobiotechnology 2011 9 33 http content 9 1 33 JOURNAL OF NANOBIOTECHNOLOGY RESEARCH Open Access Engineering of an E. coli outer membrane protein FhuA with increased channel diameter Manuel Krewinkel Tamara Dworeck and Marco Fioroni 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 . 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 A1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal p-strands resulting in variant FhuA A1-159 Exp. The total number of p-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 p-sheet content suggesting the correct folding of FhuA A1-159 Exp. To further prove the FhuA A1-159 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 A1-159 Exp as compared to FhuA A1-159 well correlated to the expected channel surface area increase of 16 . Conclusion In this study using a simple semi rational approach the FhuA A1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA A1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced broadening the FhuA A1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications. Keywords Channel proteins FhuA liposomes protein engineering HRP TMB-Assay .