The High-throughput production of recombinant proteins for structural and functional characterization by NMR spectroscopy

With the identification of >35000 genes in the human genome, there is an increasing demand for high-throughput expression of recombinant proteins. An immediate challenge of the post genomic era is to assign biological function to all the proteins encoded by the genome1.This work is part of an high-throughput project faced to the production of recombinant proteins belonging to the calcium-binding family and their targets, selected for the high interest in human cellular function. The aim is to obtain pure recombinant protein samples correctly folded for structural determination by NMR. This information is essential to understand the mechanism of protein function and for the general purpose to identify new drug targets. To increase the number of folded samples, many different cloning and expression conditions were tested, applying Gateway technology. The genes coding for selected proteins were recombined into five different expression vectors (pETG-20A, pETG-30A, pETG-60A, pDEST17, pDEST15) carrying specific protein fusion tags. Three different E.coli host strains were used for proteins expression (BL21 Gold, pLys, Codon plus). The proteins mainly expressed in a soluble form were produced on a large-scale and purified for spectroscopic analysis. Results so far obtained on one of the protein selected, S100A16, are here reported and discussed.