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ABSTRACT

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 6 M 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 gelfiltration chromatography. Purified oGH1 had an M _r 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.

ABSTRACT

¹²⁵I-Labelled polypeptide hormones have been extremely valuable for radioimmunoassays, receptor-binding studies and investigation of the processing and metabolism of hormones. However, such externally labelled material has the disadvantage that addition of one or more iodine atoms may alter the properties of the polypeptide. Furthermore, for studies on hormone metabolism and processing, the label may become separated from the hormone or its main breakdown products. Use of internally labelled polypeptides produced by biosynthesis can avoid such problems, but previously such material has usually been of low specific radioactivity, and unsuitable for many purposes. Here we describe the development of a procedure for the production of an internally labelled ovine GH analogue (oGH1) using a plasmid produced by recombinant DNA methods and expression in Escherichia coli.

Bacteria were grown in medium containing a low sulphate concentration, and then incubated in medium containing ³⁵SO₄ ²⁻ as the sole sulphur source. Under these conditions, the bacteria incorporated ³⁵S into proteins including GH. Purification of such material required considerable modification of previously described methods, because of the need to handle very small amounts of highly radioactive material. The bacteria were lysed using lysozyme, and inclusion bodies were solubilized using 6 m guanidinium chloride. [³⁵S]oGH1 was renatured and then purified by gel filtration on Sephacryl S-100, followed by immunoaffinity chromatography and a second gel filtration step. Material prepared in this way had a specific radioactivity of 6–27 μCi/μg, and showed high 'bind-ability' to polyclonal and monoclonal antibodies and to receptors. ³⁵S-Labelled material bound to receptors more effectively than ¹²⁵I-labelled GH and showed improved stability. Such material appears to be well suited to receptor-binding studies and studies on the processing and metabolism of GH. The procedure developed should be applicable to other polypeptide hormones.