ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2005) 9 P40

Purifying growth hormone on an affinity column using his-tagged growth hormone receptor

CE Bowles1, IR Wilkinson1, JR Sayers2 & RJM Ross1

1Section of Human Metabolism, Division of Clinical Sciences, The University of Sheffield, Sheffield, UK; 2Section of Functional Genomics, University of Sheffield, Sheffield, UK.

INTRODUCTION: We have been interested to investigate the biological activity of mutant molecules of growth hormone. Purification of growth hormone requires multiple steps. We have investigated a method to purify growth hormone which would be applicable to most protein hormones that bind to cell surface receptors.

AIM: To test whether his-tagged extracellular domain growth hormone receptor could be used to purify recombinant growth hormone.

METHODS: The extracellular domain of the growth hormone receptor was cloned into an expression vector with an N-terminal his-tag and a long linker between the his-tag and the N-terminus of the growth hormone receptor. The protein was expressed in E. coli, large quantities of cell paste was produced using fermentation techniques and soluble protein generated by re-folding from 4.5M urea followed by exhaustive dialysis. The protein was purified using immobilised metal affinity chromatography in a cobalt column to 90% purity. The purified extracellular domain growth hormone receptor was concentrated to 1mg/ml. This purified his-tag growth hormone receptor was then used for column purification. 1mg of his-tag growth hormone receptor was bound to a cobalt column. Recombinant growth hormone was then added to the column followed by washing steps. The growth hormone receptor and growth hormone were then eluted from the column.

RESULTS: Growth hormone bound with a 1:1 ratio to immobilised growth hormone receptor and could be eluted from the column.

CONCLUSION: His-tag soluble receptor can be used to purify ligand using metal affinity chromatography.

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