Porin-based Biohybrids - Modification and Characterisation


Simon Reitz, Wolfgang Große, Menekse Cebi, Ulrich Koert, and Lars-Oliver Essen

Faculty of Chemistry, University of Marburg, Hans-Meerwein-Str., 35032 Marburg, Germany

e-mail: reitz@chemie.uni-marburg.de

URL: http://www.uni-marburg.de/fb15/ag-essen

 

OmpF and OmpG are outer membrane porins from the gram-negative bacterium E. coli. These porins form very stable β-barrel proteins and mediate the uptake of molecules up to 600 Da. The relatively wide pore diameter allows us to place molecular filters inside the pore, resulting in artificial biohybrids that exhibit new properties concerning conductivity and open/closure characteristics. In order to achieve this we split the protein into two parts. One half is produced by heterologous overexpression while the other one bearing an artificial amino acid is produced by solid phase peptide synthesis. These two halves are combined by native chemical ligation before covalent attachment of the filter via copper mediated click-chemistry. The ligated protein is refolded to afford the native form and subsequently purified and measured via black lipid membrane experiments. Hereby the porin is placed in an artificial lipid bilayer across which different voltages can be imposed. Another way to obtain porin-based biohybrids is to prepare cysteine mutants within the conductory channel of the porin and attach filter molecules bearing an iodo-acetamide-group by nucleophilic substitution. Structural characterisation is carried out by X-ray crystallography, which enables us to gain insights into the three dimensional architecture of modified porins.