Bioprocess and Biosystems Engineering

, Volume 35, Issue 1–2, pp 255–263

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

Original Paper

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 1 Inclusion body Refolding l-arginine 

References

  1. 1.
    Rinderknecht E, Humbel RE (1978) The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J Biol Chem 253:2769–2776Google Scholar
  2. 2.
    Blundell TL, Bedarkar S, Rinderknecht E, Humbel RE (1978) Insulin-like growth factor: a model for tertiary structure accounting for immunoreactivity and receptor binding. Proc Natl Acad Sci USA 75:180–184CrossRefGoogle Scholar
  3. 3.
    Arlan LR (2009) Mecasermin (Recombinant Human Insulin-like Growth Factor I). Adv Ther 26(1):40–54CrossRefGoogle Scholar
  4. 4.
    Forsberg G, Palm G, Ekebacke A, Josephson S, Hartmanis M (1990) Separation and characterization of modified variants of recombinant human insulin-like growth factor-I derived from a fusion protein secreted from Escherichia coli. Biochem J 271:357–363Google Scholar
  5. 5.
    Moks T, Abrahmsen L, Osterlof B, Josephson S, Ostling M, Enfors SO, Persson I, Nilsson B, Uhlen M (1987) Large-scale affinity purification of human insulin-like growth factor-I from culture-medium of Escherichia coli. Bio/Technol 5:379–382CrossRefGoogle Scholar
  6. 6.
    Chelius D, Baldwin MA, Lu X, Spencer EM (2001) Expression, purification and characterization of the structure and disulfide linkages of insulin-like growth factor binding protein-4. J Endocrinol 168:283–296CrossRefGoogle Scholar
  7. 7.
    Elliott S, Fagin KD, Narhi LO, Miller JA, Jones M, Koski R, Peters M, Hsieh P, Sachdev R, Rosenfeld RD, Rohde MF, Arakawa T (1990) Yeast-derived recombinant human insulin-like growth factor-I–production, purification, and structural characterization. J Protein Chem 9:95–104CrossRefGoogle Scholar
  8. 8.
    Gellerfors P, Axelsson K, Helander A, Johansson S, Kenne L, Lindqvist S, Pavlu B, Skottner A, Fryklund L (1989) Isolation and characterization of a glycosylated form of human insulin-like growth factor-I produced in Saccharomyces cerevisiae. J Biol Chem 264:11444–11449Google Scholar
  9. 9.
    Schulz MF, Buell G, Schmid E, Movva R, Selzer G (1987) Increased expression in Escherichia coli of a synthetic gene encoding human somatomedin C after gene duplication and fusion. J Bacteriol 169:5385–5392Google Scholar
  10. 10.
    Tikhonov RV, Pechenov SE, Belacheu IA, Yakimov SA, Klyushnichenko VE, Tunes H, Thiemann JE, Vilela L, Wulfson AN (2002) Recombinant human insulin IX. Investigation of factors, influencing the folding of fusion protein-S-sulfonates, biotechnological precursors of human insulin. Protein Expr Purif 26:187–193CrossRefGoogle Scholar
  11. 11.
    Kotlarski N, O’Neill BK, Francis GL, Middelberg APJ (1997) Design analysis for refolding monomeric protein. AICHE J 43:2123–2132CrossRefGoogle Scholar
  12. 12.
    Clark EDB (2001) Protein refolding for industrial processes. Curr Opin Biotechnol 12:202–207CrossRefGoogle Scholar
  13. 13.
    Jungbauer A, Kaar W (2007) Current status of technical protein refolding. J Biotechnol 128:587–596CrossRefGoogle Scholar
  14. 14.
    Carrio MM, Villaverde A (2002) Construction and deconstruction of bacterial inclusion bodies. J Biotechnol 96:3–12CrossRefGoogle Scholar
  15. 15.
    Speed MA, Wang DIC, King J (1996) Specific aggregation of partially folded polypeptide chains: the molecular basis of inclusion body composition. Nat Biotechnol 14:1283–1287CrossRefGoogle Scholar
  16. 16.
    John WT, Alexander V (1991) Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae. J Biol Chem 266:12021–12028Google Scholar
  17. 17.
    Park YC, Kim SJ, Choi JH, Lee WH, Park KM, Kawamukai M, Ryu YW, Seo JH (2005) Batch and fed-batch production of coenzyme Q10 in recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Gluconobacter suboxydans. Appl Microbiol Biotechnol 67:192–196CrossRefGoogle Scholar
  18. 18.
    Kim SG, Kweon DH, Lee DH, Park YC, Seo JH (2005) Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli. Protein Expr Purif 41:426–432CrossRefGoogle Scholar
  19. 19.
    Xu X, Jin F, Yu X, Ren S, Hu J, Zhang W (2007) High-level expression of the recombinant hybrid peptide cecropinA(1–8)-magainin2(1–12) with an ubiquitin fusion partner in Escherichia coli. Protein Expr Purif 55:175–182CrossRefGoogle Scholar
  20. 20.
    Kweon DH, Lee DH, Han NS, Seo JH (2004) Solid-phase refolding of cyclodextrin glycosyltransferase adsorbed on cation-exchange resin. Biotechnol Prog 20:277–283CrossRefGoogle Scholar
  21. 21.
    Huang Y, Shi R, Zhong X, Wang D, Zhao M, Li Y (2007) Enzyme-linked immunosorbent assays for insulin-like growth factor-I using six-histidine tag fused proteins. Anal Chim Acta 596:116–123CrossRefGoogle Scholar
  22. 22.
    Zhang T, Xu X, Shen L, Feng Y, Yang Z, Shen Y, Wang J, Jin W, Wang X (2009) Modeling of protein refolding from inclusion bodies. Acta Biochim Biophys Sin (Shanghai) 41:1044–1052CrossRefGoogle Scholar
  23. 23.
    Umetsu M, Tsumoto K, Hara M, Ashish K, Goda S, Adschiri T, Kumagai I (2003) How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system. J Biol Chem 278:8979–8987CrossRefGoogle Scholar
  24. 24.
    Tischer A, Lilie H, Rudolph R, Lange C (2010) l-arginine hydrochloride increases the solubility of folded and unfolded recombinant plasminogen activator rPA. Protein Sci 19:1783–1795CrossRefGoogle Scholar
  25. 25.
    Arakawa T, Ejima D, Tsumoto K, Obeyama N, Tanaka Y, Kita Y, Timasheff SN (2007) Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects. Biophys Chem 127:1–8CrossRefGoogle Scholar
  26. 26.
    Wang L (2009) Towards revealing the structure of bacterial inclusion bodies. Prion 3:139–145CrossRefGoogle Scholar

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

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