The conformation and stability of recombinant-derived human and murine granulocyte-macrophage colony stimulating factors produced in Escherichia coli have been investigated by analytical ultracentrifugation, urea-gradient polyacrylamide gel electrophoresis and several spectroscopic methods. The proteins were demonstrated to be physically homogeneous monomeric proteins with compact globular shapes and shown to have similar secondary structures containing both alpha-helix and beta-sheet structure. The intramolecular disulphide linkages of both proteins were shown to be essential for maintaining native conformation as reduction with dithiothreitol resulted in protein unfolding. Comparison of the human E. coli-derived (non-glycosylated) and mammalian cell culture-derived (glycosylated) proteins by urea-gradient electrophoresis indicated that glycosylation had no major effect on the conformational stability and kinetics of urea induced unfolding and refolding. Study holds ProTherm entries: 7550, 7551, 7552, 7553 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:34 p.m.
|Number of data points||6|
|Proteins||Granulocyte colony-stimulating factor ; Granulocyte-macrophage colony-stimulating factor ; Granulocyte-macrophage colony-stimulating factor|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O buffers:Tris-glycine: 50 mM, ionic:: , pH:9.0, temp:4.0 C ; Experimental Assay: Cm ; Experimental Assay: dG_H2O pH:7.7, ionic:NaCl: 50 mM, temp:25.0 C, buffers:Tris-HCl: 50 mM|
|Libraries||Mutations for sequence TPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP ; Mutations for sequence MWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK|