Folding screening assayed by proteolysis: application to various cystine deletion mutants of vascular endothelial growth factor

The production of recombinant proteins in Escherichia coli often leads to t he formation of inclusion bodies. Although this has a number of advantages, a major disadvantage is the need to develop folding protocols for the rena turing of the proteins. However, the systematic screening of folding condit ions is often hampered by the lack of convenient assays to detect correctly folded proteins. To address this problem we present a simple protocol, whi ch combines folding screens and limited proteolysis to rapidly assess and o ptimize folding conditions. The efficacy of this method, termed FSAP (foldi ng screening assayed by proteolysis), is demonstrated by the large-scale fo lding, purification and crystallization of various cystine deletion mutants of the cystine knot family member: vascular endothelial growth factor (VEG F). These mutants are particularly difficult to fold as the cystine knot is believed to make major contributions to the stability of the protein and t his family of proteins lacks extensive hydrophobic core regions.