Solubilization and Refolding of Inclusion Body Proteins

Expression of heterologous proteins in E. coli often leads to the formation of protein aggregates known as inclusion bodies (IBs). Inclusion body aggregates pose a major hurdle in the recovery of bioactive proteins from E. coli. Usage of strong denaturing buffers for solubilization of bacterial IBs results in poor recovery of bioactive protein. Structure–function understanding of IBs in the last two decades have led to the development of several mild solubilization buffers, which improve the recovery of bioactive from IBs. Recently, combinatorial mild solubilization methods have paved the way for solubilization of wide range of inclusion bodies with appreciable refolding yield. Here, we describe a simple protocol for solubilization and refolding of an inclusion body protein with appreciable recovery.

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Solubilization and Refolding of Inclusion Body Proteins

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Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process

Article Open access 25 March 2015

References

  1. Slouka C, Kopp J, Hutwimmer S et al (2018) Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes. Microb Cell Factories 17:148 ArticleCASGoogle Scholar
  2. Jürgen B, Breitenstein A, Urlacher V et al (2010) Quality control of inclusion bodies in Escherichia coli. Microb Cell Factories 9:41 ArticleGoogle Scholar
  3. Singh A, Upadhyay V, Upadhyay AK et al (2015) Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Factories 14:41 ArticleGoogle Scholar
  4. Singh SM, Sharma A, Upadhyay AK et al (2012) Solubilization of inclusion body proteins using n-propanol and its refolding into bioactive form. Protein Expr Purif 81:75–82 ArticleCASPubMedGoogle Scholar
  5. Dill KA, Shortle D (1991) Denatured states of proteins. Annu Rev Biochem 60:795–825 ArticleCASPubMedGoogle Scholar
  6. Rudolph R, Lilie H (1996) In vitro folding of inclusion body proteins. FASEB J 10:49–56 ArticleCASPubMedGoogle Scholar
  7. Anfinsen CB (1973) Principles that govern the folding of protein chains. Science 181:223–230 ArticleCASPubMedGoogle Scholar
  8. Singhvi P, Saneja A, Srichandan S et al (2020) Bacterial inclusion bodies: a treasure trove of bioactive proteins. Trends Biotechnol 38:474–486 ArticleCASPubMedGoogle Scholar
  9. Chung WJ, Huang CL, Gong HY et al (2015) Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture. Protein Expr Purif 110:79–88 ArticleCASPubMedGoogle Scholar
  10. Malavasi NV, Foguel D, Bonafe CFS et al (2011) Protein refolding at high pressure: optimization using eGFP as a model. Process Biochem 46:512–518 ArticleCASGoogle Scholar
  11. Berini F, Presti I, Beltrametti F et al (2017) Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome. Microb Cell Factories 16:16 ArticleGoogle Scholar
  12. Mohammadian A, Kaghazian H, Kavianpour A et al (2018) Solubilization of inclusion body proteins using low and very low concentrations of chemicals: implications of novel combined chemical treatment designs in enhancement of post-solubilization target protein purity and biological activity. J Chem Technol Biotechnol 93:1579–1587 ArticleCASGoogle Scholar
  13. Panda AK, Seikh GB Eshwari ANS et al (2007) A process for solubilization of recombinant protein expressed as inclusion body. US patent number US7189811B2 Google Scholar
  14. Upadhyay V, Singh A, Jha D et al (2016) Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent. Microb Cell Factories 15:100 ArticleGoogle Scholar
  15. Park AR, Jang SW, Kim JS et al (2018) Efficient recovery of recombinant CRM197 expressed as inclusion bodies in E. coli. PLoS One 13:e0201060 ArticlePubMedPubMed CentralGoogle Scholar
  16. Qi X, Sun Y, Xiong S (2015) A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form. Microb Cell Factories 14:24 ArticleGoogle Scholar
  17. Chura-Chambi RM, Rosa Da Silva CM, Pereira LR et al (2019) Protein refolding based on high hydrostatic pressure and alkaline pH: application on a recombinant dengue virus NS1 protein. PLoS One 14:e0211162 ArticleCASPubMedPubMed CentralGoogle Scholar
  18. Padhiar AA, Chanda W, Joseph TP et al (2018) Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies. Appl Microbiol Biotechnol 102:2363–2377 ArticleCASPubMedGoogle Scholar
  19. Wang Q, Liu Y, Zhang C et al (2017) High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration. Protein Expr Purif 133:152–159 ArticleCASPubMedGoogle Scholar
  20. Mirzadeh A, Saadatnia G, Golkar M et al (2017) Production of refolded Toxoplasma gondii recombinant SAG1-related sequence 3 (SRS3) and its use for serodiagnosis of human toxoplasmosis. Protein Expr Purif 133:66–74 ArticleCASPubMedGoogle Scholar
  21. Pan S, Odabas N, Sissolak B et al (2015) Engineering batch and pulse refolding with transition of aggregation kinetics: an investigation using green fluorescent protein (GFP). Chem Eng Sci 131:91–100 ArticleCASGoogle Scholar
  22. García-Fruitós E, Vázquez E, Díez-Gil C et al (2012) Bacterial inclusion bodies: making gold from waste. Trends Biotechnol 30:65–70 ArticlePubMedGoogle Scholar
  23. Ramón A, Señorale-Pose M, Marín M (2014) Inclusion bodies: not that bad. Front Microbiol 5:56 ArticlePubMedPubMed CentralGoogle Scholar
  24. Jevševar S, Gaberc-Porekar V, Fonda I et al (2005) Production of nonclassical inclusion bodies from which correctly folded protein can be extracted. Biotechnol Prog 21:632–639 ArticlePubMedGoogle Scholar
  25. Pesarrodona M, Fernández Y, Foradada L et al (2016) Conformational and functional variants of CD44-targeted protein nanoparticles bio-produced in bacteria. Biofabrication 8:025001 ArticlePubMedGoogle Scholar
  26. Kumar L, Colomb W, Czerski J et al (2018) Efficient protease based purification of recombinant matrix metalloprotease-1 in E. coli. Protein Expr Purif 148:59–67 ArticleCASPubMedGoogle Scholar
  27. Stark GR, Stein WH, Moore S (1960) Reactions of the cyanate present in aqueous urea with amino acids and proteins. J Biol Chem 235:3177–3181 ArticleCASGoogle Scholar
  28. Yang Z, Zhang L, Zhang Y et al (2011) Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method. PLoS One 6:e22981 ArticleCASPubMedPubMed CentralGoogle Scholar
  29. Feng Y, Liu L, Wang J et al (2012) Integrated refolding techniques for Schistosoma japonicum MTH1 overexpressed as inclusion bodies in Escherichia coli. Protein Expr Purif 84:181–187 ArticleCASPubMedGoogle Scholar
  30. Gifre-Renom L, Cano-Garrido O, Fàbregas F et al (2018) A new approach to obtain pure and active proteins from Lactococcus lactis protein aggregates. Sci Rep 8:13917 ArticleCASPubMedPubMed CentralGoogle Scholar
  31. Hagel P, Gerding JJT, Fieggen W et al (1971) Cyanate formation in solutions of urea. I. Calculation of cyanate concentrations at different temperature and pH. Biochim Biophys Acta 243:366–373 ArticleCASPubMedGoogle Scholar
  32. Noji M, So M, Yamaguchi K et al (2018) Heat-induced aggregation of hen ovalbumin suggests a key factor responsible for serpin polymerization. Biochemistry 57:5415–5426 ArticleCASPubMedGoogle Scholar
  33. Alam MT, Rizvi A, Rauf MA et al (2018) Thermal unfolding of human lysozyme induces aggregation: recognition of the aggregates by antisera against the native protein. Int J Biol Macromol 113:976–982 ArticleCASPubMedGoogle Scholar
  34. Datta I, Gautam S, Gupta MN (2013) Microwave assisted solubilization of inclusion bodies. Sustain Chem Process 1:2 ArticleGoogle Scholar
  35. Singh A, Upadhyay V, Panda AK (2013) N-propanol based solubilization buffer enhances refolding yield of inclusion body protein by populating intermediates to the folding pathway. Biophys J 104:396a ArticleGoogle Scholar
  36. Yu Y, Wang J, Shao Q et al (2016) The effects of organic solvents on the folding pathway and associated thermodynamics of proteins: a microscopic view. Sci Rep 6:19500 ArticleCASPubMedPubMed CentralGoogle Scholar
  37. Perham M, Liao J, Wittung-Stafshede P (2006) Differential effects of alcohols on conformational switchovers in α-helical and β-sheet protein models. Biochemistry 45:7740–7749 ArticleCASPubMedGoogle Scholar
  38. Li A, Sowder RC, Henderson LE et al (2001) Chemical cleavage at aspartyl residues for protein identification. Anal Chem 73:5395–5402 ArticleCASPubMedGoogle Scholar
  39. Hauptmann A, Podgoršek K, Kuzman D et al (2018) Impact of buffer, protein concentration and sucrose addition on the aggregation and particle formation during freezing and thawing. Pharm Res 35:101 ArticlePubMedPubMed CentralGoogle Scholar
  40. Quaas B, Burmeister L, Li Z et al (2018) Properties of dimeric, disulfide-linked rhBMP-2 recovered from E. coli derived inclusion bodies by mild extraction or chaotropic solubilization and subsequent refolding. Process Biochem 67:80–87 ArticleCASGoogle Scholar
  41. Upadhyay AK, Singh A, Mukherjee KJ et al (2014) Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein. Front Microbiol 5:486 ArticlePubMedPubMed CentralGoogle Scholar
  42. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99:303–310 ArticleCASPubMedGoogle Scholar
  43. Upadhyay AK, Murmu A, Singh A et al (2012) Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli. PLoS One 7:e33951 ArticleCASPubMedPubMed CentralGoogle Scholar
  44. Alibolandi M, Mirzahoseini H (2011) Chemical assistance in refolding of bacterial inclusion bodies. Biochem Res Int 2011:631607 ArticlePubMedPubMed CentralGoogle Scholar
  45. Burgess RR (2009) Chapter 17 refolding solubilized inclusion body proteins. In: Methods of enzymology. Academic, pp 259–282 Google Scholar

Acknowledgments

This work is supported by the core grant of the National Institute of Immunology, received from the Department of Biotechnology, Government of India.