About the Webinar
Continued process enhancement aids optimal output from protein production facilities. Assessing potential efficiencies in both expression and purification can alleviate bottlenecks and accelerate associated research programs. The synthesis and application of codon-optimized cDNAs is a well-established method for increasing recombinant protein yields. However, we often note that a substantial amount of this uplift partitions to the insoluble fraction in Escherichia coli, and is not available for purification under native conditions. Using gene synthesis and custom molecular biology, we assessed a number of molecular approaches that aimed to improve soluble recovery. In particular, our case study using small GTPases demonstrates a benefit from incorporating a modest number of non-optimal synonymous codons back into otherwise optimized constructs. We hypothesize that a reduction in the rate of translation and nascent polypeptide extension enhances the efficiency of co-translational folding, resulting in a larger proportion of recombinant product partitioning to the soluble fraction. An additional valued methodology in our laboratory is the automated multidimensional purification of proteins on FPLC systems. The development of automated production on the ÄKTA purification platform has allowed for complex purifications of several proteins in parallel, with minimal need for operator intervention. We have extended this process to include protein conditioning steps, for example the exchange of the active site nucleotide in our small GTPase targets.
About the Presenter
Chris Gray is a Team Leader in the Drug Discovery Program at the CRUK Beatson Institute in Glasgow, UK. Chris gained his Ph.D. in Microbial Biochemistry from University of Glasgow in 2001. He then trained as a protein crystallographer at the Institute of Cancer Research, London. He currently oversees the structural and biophysical science that supports the Beatson Institute’s drug discovery activities, and this includes a substantial protein production effort. In this context, Dr Gray’s team actively develops process optimizations that enhance the rates of expression, recovery and throughput towards the efficient generation of high-quality recombinant proteins.