A generic expression system to produce proteins that co-assemble with alkane thiol SAM

Abstract

Benjamin T Chaffey1, Elizabeth Mitchell1, Mark A Birch2, Jeremy H Lakey11The Institute for Cell and Molecular Biosciences; 2The School of Surgical and Reproductive Sciences, The Medical School, Framlington Place, The University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, Great BritainAbstract: Surface biology aims to observe and control biological processes by combining bio-, surface, and physical chemistry. Self-assembled monolayers (SAM) on gold surfaces have provided excellent methods for nanoscale surface preparation for such studies. However, extension of this work requires the specific immobilization of whole protein domains and the direct incorporation of recombinant proteins into SAM is still problematic. In this study a short random coil peptide has been designed to insert into thioalkane layers by formation of a hydrophobic helix. Surface plasmon resonance (SPR) studies show that specific immobilization via the internal cysteine is achieved. Addition of the peptide sequence to the terminus of a protein at the genetic level enables the production of a range of recombinant fusion-proteins with good yield. SPR shows that the proteins display the same gold-binding behavior as the peptide. It is shown that cell growth control can be achieved by printing the proteins using soft lithography with subsequent infilling with thio-alkanes The expression plasmid is constructed so that any stable protein domain can be easily cloned, expressed, purified and immobilized.Keywords: self-assembling monolayer, protein immobilization, nanotechnology, circular dichroism spectroscopy, surface plasmon resonance, osteoblast