title: Identification of in vitro folding conditions for procathepsin S and cathepsin S using fractional factorial screens.
abstract: Human procathepsin S and cathepsin S were expressed as inclusion bodies in Escherichia coli. Following solubilization of the inclusion body proteins, fractional factorial protein folding screens were used to identify folding conditions for procathepsin S and cathepsin S. A primary folding screen, including eight factors each at two levels, identified pH and arginine as the main factors affecting procathepsin S folding. In a second simple screen, the yields were further improved. The in vitro folding of mature cathepsin S has never been reported previously. In this study we used a series of fractional factorial screens to identify conditions that enabled the active enzyme to be generated without the prodomain although the yields were much lower than achieved with procathepsin S. Our data show the power of fractional factorial screens to rapidly identify folding conditions even for a protein that does not easily fold into its active conformation.
authors.affiliation: Department of Enabling Science and Technology, AstraZeneca, Mereside, Alderley Park, Macclesfield, SK10 4TG, United Kingdom.
authors.forename: Dominique A
authors.initials: DA
authors.lastname: Tobbell
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Middleton
authors.forename: Stephanie
authors.initials: S
authors.lastname: Raines
authors.forename: Maurice R C
authors.initials: MR
authors.lastname: Needham
authors.forename: Ian W F
authors.initials: IW
authors.lastname: Taylor
authors.forename: John Y
authors.initials: JY
authors.lastname: Beveridge
authors.forename: W Mark
authors.initials: WM
authors.lastname: Abbott
mesh_terms: D001120:Arginine; D002403:Cathepsins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies; D017510:Protein Folding
chemical_list: D004792:Enzyme Precursors; D001120:Arginine; D002403:Cathepsins; C028217:cathepsin S