Effects of operating parameters in in vitro renaturation of fusion protein of human growth hormone and glutahione S transferase from inclusion body
- Title
- Effects of operating parameters in in vitro renaturation of fusion protein of human growth hormone and glutahione S transferase from inclusion body
- Author
- 이은규
- Keywords
- Recombinant hGH; Refolding; Renaturation; Inclusion bodies
- Issue Date
- 2000-04
- Publisher
- ELSEVIER
- Citation
- PROCESS BIOCHEMISTRY, v. 36, no. 1-2, page. 111-117
- Abstract
- Using a fusion protein of rhGH and GST fragment as a model protein, the effects of the key operating parameters on the recovery yield of in vitro renaturation from inclusion bodies were evaluated. The parameters investigated were type of denaturant and its concentration, protein concentration, pH and ionic strength of the refolding buffer and refolding–enhancing surfactants. 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-third 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 strength (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 refolding–enhancing additive, 1.6–2 times higher yield was realized. The ‘masking’ of the hydrophobic patches located on the surface of the protein with the surfactant molecules was believed to be responsible for the considerable reduction in aggregation. SDS was completely removed by IRA420 chromatography. The recovery yield from the washed inclusion body through an Amberlite IRA cluate was ∼28.3%.
- URI
- https://www.sciencedirect.com/science/article/pii/S0032959200001850https://repository.hanyang.ac.kr/handle/20.500.11754/161773
- ISSN
- 1359-5113
- DOI
- 10.1016/S0032-9592(00)00185-0
- Appears in Collections:
- COLLEGE OF ENGINEERING SCIENCES[E](공학대학) > BIONANO ENGINEERING(생명나노공학과) > Articles
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