Analysis of disulphide bridge function in recombinant bovine prolactin using site-specific mutagenesis and renaturation under mild alkaline conditions: a crucial role for the central disulphide bridge in the mitogenic activity of the hormone.
We have previously described a method for isolating Escherichia coli-produced methionyl bovine prolactin (Met-bPRL) and its renaturation using thioredoxin. This report describes an alternative renaturation procedure in which extracted Met-bPRL is incubated in air at pH 10 and 20 degrees C. Within 1 h of such treatment essentially all of the reduced Met-bPRL was converted to the oxidized form; this was accompanied by an increase to full mitogenic activity in the Nb2 cell bioassay. It was also found that, to minimize contamination by high mol. wt Met-bPRL derivatives, it is essential to have a reducing agent (dithiothreitol) present during disruption of the bacteria and to extract the protein at neutral pH. The contribution of each of the three disulphide bridges in bPRL to its bioactivity was studied with Met-bPRL variants, prepared via site-specific mutagenesis, in which cysteines were replaced by serines to prevent disulphide bond formation. Variants lacking the C4-C11 bridge, the C191-C199 bridge or both these terminal bridges were as mitogenic as authentic bPRL. (Variants lacking the C191-C199 bridge had markedly increased solubility in the presence of deoxycholate.) In contrast, variants lacking the C58-C174 bridge had greatly reduced bioactivity, indicating that integrity of the large disulphide loop is crucial to the hormone's mitogenic activity.
Luck
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Department of Biochemistry, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
Gout
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Sutherland
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Fox
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Huyer
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Smith
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D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003553:Cystine; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011388:Prolactin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D013696:Temperature; D013879:Thioredoxins
D011993:Recombinant Fusion Proteins; D003553:Cystine; D013879:Thioredoxins; D011388:Prolactin