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

The production of recombinant proteins in Escherichia coli oftenleads to the formation of inclusion bodies. Although this hasa number of advantages, a major disadvantage is the need todevelop folding protocols for the renaturing of the proteins.However, the systematic screening of folding conditions is oftenhampered by the lack of convenient assays to detect correctlyfolded proteins. To address this problem we present a simpleprotocol, which combines folding screens and limited proteolysisto rapidly assess and optimize folding conditions. The efficacyof this method, termed FSAP (folding screening assayed by proteolysis),is demonstrated by the large-scale folding, purification andcrystallization of various cystine deletion mutants of the cystineknot family member: vascular endothelial growth factor (VEGF).These mutants are particularly difficult to fold as the cystineknot is believed to make major contributions to the stabilityof the protein and this family of proteins lacks extensive hydrophobiccore regions.