Substitution of serine for non-disulphide-bond-forming cysteine in grass carp (Ctenopharygodon idellus) growth hormone improves in vitro oxidative renaturation.
Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also nonnative intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cysteine forms are active in radioreceptor binding assay.
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region.
Michael Yiu-Kwong
MY
Leung
Walter Kwok-Keung
WK
Ho
D019943:Amino Acid Substitution; D000818:Animals; D002347:Carps / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D013006:Growth Hormone / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D011994:Recombinant Proteins; D012694:Serine; D013006:Growth Hormone; D019791:Guanidine; D003545:Cysteine