Bioprocess and Biosystems Engineering

, Volume 35, Issue 1, pp 255–263

Effects of l-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli

Original Paper

DOI: 10.1007/s00449-011-0619-7

Cite this article as:
Choi, S.P., Park, Y., Lee, J. et al. Bioprocess Biosyst Eng (2012) 35: 255. doi:10.1007/s00449-011-0619-7

Abstract

Insulin-like growth factor 1 (IGF1), a therapeutic protein, is highly homologous to proinsulin in 3-dimensional structure. To highly express IGF1 in recombinant Escherichia coli, IGF1 was engineered to be fused with the 6-lysine tag and ubiquitin at its N-terminus (K6Ub-IGF1). Fed-batch fermentation of E. coli TG1/pAPT-K6Ub-IGF1 resulted in 60.8 g/L of dry cell mass, 18% of which was inclusion bodies composed of K6Ub-IGF1. Subsequent refolding processes were conducted using accumulated inclusion bodies. An environment of 50 mM bicine buffer (pH 8.5), 125 mM l-arginine, and 4 °C was chosen to optimize the refolding of K6Ub-IGF1, and 240 mg/L of denatured K6Ub-IGF1 was refolded with a 32% yield. The positive effect of l-arginine on K6Ub-IGF1 refolding might be ascribed to preventing unfolded K6Ub-IGF1 from undergoing self-aggregation and thus increasing its solubility. The simple dilution refolding, followed by cleavage of the fusion protein by site-specific UBP1 and chromatographic purification of IGF1, led production of authentic IGF1 with 97% purity and an 8.5% purification yield, starting from 500 mg of inclusion bodies composed of K6Ub-IGF1, as verified by various analytical tools, such as RP-HPLC, CD spectroscopy, MALDI-TOF mass spectrometry, and Western blotting. Thus, it was confirmed that l-arginine with an aggregation-protecting ability could be applied to the development of refolding processes for other inclusion body-derived proteins.

Keywords

Insulin-like growth factor 1Inclusion bodyRefoldingl-arginine

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Department of Chemical and Biological EngineeringKorea UniversitySeoulKorea
  2. 2.Department of Advanced Fermentation Fusion Science and TechnologyKookmin UniversitySeoulKorea
  3. 3.Department of PediatricsKorea University College of MedicineSeoulKorea
  4. 4.Department of Chemical and Biomolecular EngineeringKAISTDaejeonKorea