Effects of operating parameters in in vitro renaturation of a fusion protein of human growth hormone and glutathione S transferase from inclusion body

Using a fusion protein of rhGH and GST fragment as a model protein, the eff ects of the key operating parameters on the recovery yield of in vitro rena turation from inclusion bodies were evaluated. The parameters investigated were type of denaturant and its concentration, protein concentration, pH an d ionic strength of the refolding buffer and refolding-enhancing surfactant s. Of the denaturants tested (8 M urea, 6 M guanidine HCl, 0.5% (w/v) SDS), SDS at alkaline pH (9.5) and ambient temperature was the most effective in dissolving ca. 17.5 mg inclusion body (dry weight) per ml. After ca. one-t hird of the SDS was removed by cryoprecipitation, the denatured protein was successfully air-oxidized at relatively high rhGH-GST concentration range of 0.5-1.0 mg/ml. The effect of ionic strength was not significant. However , a trend was observed where dissolution proceeded better at lower ionic st rength (10 mM) but aggregation was more suppressed at higher ionic strength (> 50 mM). When PEG-4000 and/or Tween were added as a co-solvent or a refo lding-enhancing additive, 1.6-2 times higher yield was realized. The 'maski ng' of the hydrophobic patches located on the surface of the protein with t he surfactant molecules was believed to be responsible for the considerable reduction in aggregation. SDS was completely removed by IRA420 chromatogra phy. The recovery yield from the washed inclusion body through an Amberlite IRA cluate was similar to 28.3%. (C) 2000 Elsevier Science Ltd. All rights reserved.