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 the formation of inclusion bodies. Although this has a number of advantages, a major disadvantage is the need to develop folding protocols for the renaturing of the proteins. However, the systematic screening of folding conditions is often hampered by the lack of convenient assays to detect correctly folded proteins. To address this problem we present a simple protocol, which combines folding screens and limited proteolysis to rapidly assess and optimize folding conditions. The efficacy of this method, termed FSAP (folding screening assayed by proteolysis), is demonstrated by the large-scale folding, purification and crystallization of various cystine deletion mutants of the cystine knot family member: vascular endothelial growth factor (VEGF). These mutants are particularly difficult to fold as the cystine knot is believed to make major contributions to the stability of the protein and this family of proteins lacks extensive hydrophobic core regions.
Heiring
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Forschungsgruppe Kristallographie, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany.
Muller
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YA
D003001:Cloning, Molecular; D003460:Crystallization; D003553:Cystine / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D016228:Endothelial Growth Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008222:Lymphokines / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D017384:Sequence Deletion / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D003553:Cystine; D010450:Endopeptidases