Purification and properties of a recombinant DNA-derived ovine growth hormone analogue (oGH1) expressed in Escherichia coli.
An Escherichia coli JM109 clone containing a plasmid, pOGHe101, based on pUC8 and the ovine GH (oGH) cDNA sequence, showed very high expression (up to 25% of total cell protein) of an oGH analogue (oGH1) after induction. oGH1 was found in the particulate fraction of induced bacteria, where electron-dense granules could be seen by electron microscopy. A simple method for the purification of oGH1 is described. The particulate fraction isolated from sonicated bacteria was dissolved in 6M guanidinium chloride containing dithiothreitol. After threefold dilution the proteins were reoxidized by gentle stirring overnight in air. Soluble renatured protein, recovered after dialysis, was further purified by ion-exchange and gel-filtration chromatography. Purified oGH1 had an Mr of 22,000, an isoelectric point of about 6.7 and an N-terminal sequence corresponding to that of oGH, with an extension of eight amino acids replacing the N-terminal alanine. oGH1 behaved similarly to authentic bovine GH in a radioimmunoassay, a radioreceptor assay and a weight-gain assay in hypophysectomized rats. Thus the renatured hormone appears to be correctly folded and the N-terminal extension has little or no effect on biological activity.
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Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton.
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D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis* / Q000235:genetics / Q000648:ultrastructure; D015870:Gene Expression; D013006:Growth Hormone / Q000235:genetics / Q000302:isolation & purification*; D019791:Guanidine; D006146:Guanidines; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012756:Sheep
D006146:Guanidines; D011994:Recombinant Proteins; D013006:Growth Hormone; D019791:Guanidine; D004229:Dithiothreitol