Preparation and characterization of a recombinant DNA-derived ovine growth hormone variant internally labelled with sulphur-35.
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 microCi/micrograms, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Wallis
M
M
Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton, UK.
Gwilliam
D J
DJ
Wallis
O C
OC
D000818:Animals; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D014644:Genetic Variation; D013006:Growth Hormone / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D011247:Pregnancy; D011487:Protein Conformation; D011817:Rabbits; D012756:Sheep; D013462:Sulfur Radioisotopes
D004274:DNA, Recombinant; D013462:Sulfur Radioisotopes; D013006:Growth Hormone