Applied Microbiology and Biotechnology

, Volume 102, Issue 5, pp 2363–2377 | Cite as

Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies

  • Arshad Ahmed Padhiar
  • Warren Chanda
  • Thomson Patrick Joseph
  • Xuefang Guo
  • Min Liu
  • Li Sha
  • Samana Batool
  • Yifan Gao
  • Wei Zhang
  • Min Huang
  • Mintao Zhong
Methods and protocols
  • 78 Downloads

Abstract

The formation of inclusion bodies (IBs) is considered as an Achilles heel of heterologous protein expression in bacterial hosts. Wide array of techniques has been developed to recover biochemically challenging proteins from IBs. However, acquiring the active state even from the same protein family was found to be an independent of single established method. Here, we present a new strategy for the recovery of wide sub-classes of recombinant protein from harsh IBs. We found that numerous methods and their combinations for reducing IB formation and producing soluble proteins were not effective, if the inclusion bodies were harsh in nature. On the other hand, different practices with mild solubilization buffers were able to solubilize IBs completely, yet the recovery of active protein requires large screening of refolding buffers. With the integration of previously reported mild solubilization techniques, we proposed an improved method, which comprised low sarkosyl concentration, ranging from 0.05 to 0.1% coupled with slow freezing (− 1 °C/min) and fast thaw (room temperature), resulting in greater solubility and the integrity of solubilized protein. Dilution method was employed with single buffer to restore activity for every sub-class of recombinant protein. Results showed that the recovered protein’s activity was significantly higher compared with traditional solubilization/refolding approach. Solubilization of IBs by the described method was proved milder in nature, which restored native-like conformation of proteins within IBs.

Keywords

Prokaryotic expression system Harsh inclusion bodies Mild solubilization Sarkosyl (SLS) Freeze/thaw Refolding 

Notes

Acknowledgments

Authors would like to thank Dr. Muhammad Kamran Raja (Sun Yat-Sen Cancer Center (SYSCC), Sun Yat-Sen University) for providing excellent technical assistance. This study was supported by grants from The National Natural Science Foundation of China (81301995; 81472836).

Compliance with ethical standards

Competing interest

All authors have seen the content of this manuscript, and have no conflict of interest to disclose.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8754_MOESM1_ESM.pdf (1.8 mb)
ESM 1 (PDF 1890 kb)

References

  1. Ann XH, Lun YZ, Zhang W, Liu B, Li XY, Zhong MT, Wang XL, Cao J, Ning AH, Huang M (2014) Expression and characterization of protein Latcripin-3, an antioxidant and antitumor molecule from Lentinula edodes C91-3. Asian Pac J Cancer Prev 15(12):5055–5061.  https://doi.org/10.7314/APJCP.2014.15.12.5055 CrossRefPubMedGoogle Scholar
  2. Apiyo D, Wittung-Stafshede P (2002) Presence of the cofactor speeds up folding of Desulfovibrio Desulfuricans flavodoxin. Protein Sci 11(5):1129–1135.  https://doi.org/10.1110/ps.3840102 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Arya R, Sabir JSM, Bora RS, Saini KS (2015) Optimization of culture parameters and novel strategies to improve protein solubility. In: García-Fruitós E (ed) Insoluble proteins: methods and protocols. Springer New York, New York, pp 45–63Google Scholar
  4. Bisen PS, Baghel RK, Sanodiya BS, Thakur GS, Prasad GB (2010) Lentinus edodes: a macrofungus with pharmacological activities. Curr Med Chem 17(22):2419–2430.  https://doi.org/10.2174/092986710791698495 CrossRefPubMedGoogle Scholar
  5. Brem B, Seger C, Pacher T, Hartl M, Hadacek F, Hofer O, Vajrodaya S, Greger H (2004) Antioxidant dehydrotocopherols as a new chemical character of Stemona species. Phytochemistry 65(19):2719–2729.  https://doi.org/10.1016/j.phytochem.2004.08.023 CrossRefPubMedGoogle Scholar
  6. Cao E, Chen Y, Cui Z, Foster PR (2003) Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol Bioeng 82(6):684–690.  https://doi.org/10.1002/bit.10612 CrossRefPubMedGoogle Scholar
  7. Carrio MM, Villaverde A (2003) Role of molecular chaperones in inclusion body formation. FEBS Lett 537(1–3):215–221.  https://doi.org/10.1016/S0014-5793(03)00126-1 CrossRefPubMedGoogle Scholar
  8. Chattopadhyay CTaPC (2015) Effect of various osmolytes on the expression and functionality of zebrafish dihydrofolate reductase: An in vivo study. J Protein Proteomic [S.1], May. 2015(ISSN 0975–5151):211–218Google Scholar
  9. Chisnall B, Johnson C, Kulaberoglu Y, Chen YW (2014) Insoluble protein purification with sarkosyl: facts and precautions. Methods Mol Biol 1091:179–186.  https://doi.org/10.1007/978-1-62703-691-7_12 CrossRefPubMedGoogle Scholar
  10. de Marco A (2007) Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli. Nat Protoc 2(10):2632–2639.  https://doi.org/10.1038/nprot.2007.400 CrossRefPubMedGoogle Scholar
  11. de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B (2007) Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol 7(1):32.  https://doi.org/10.1186/1472-6750-7-32 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Desu HR, Narishetty ST (2013) Challenges in freeze–thaw processing of bulk protein solutions. In: Kolhe P, Shah M, Rathore N (eds) Sterile product development: formulation, process, quality and regulatory considerations. Springer New York, New York, pp 167–203.  https://doi.org/10.1007/978-1-4614-7978-9_7 CrossRefGoogle Scholar
  13. Dias CL, Ala-Nissila T, Wong-ekkabut J, Vattulainen I, Grant M, Karttunen M (2010) The hydrophobic effect and its role in cold denaturation. Cryobiology 60(1):91–99.  https://doi.org/10.1016/j.cryobiol.2009.07.005 CrossRefPubMedGoogle Scholar
  14. Dyson MR, Shadbolt SP, Vincent KJ, Perera RL, McCafferty J (2004) Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol 4(1):32.  https://doi.org/10.1186/1472-6750-4-32 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17(4):353–358.  https://doi.org/10.1016/j.copbio.2006.06.003 CrossRefPubMedGoogle Scholar
  16. Frangioni JV, Neel BG (1993) Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 210(1):179–187.  https://doi.org/10.1006/abio.1993.1170 CrossRefPubMedGoogle Scholar
  17. Frankel S, Sohn R, Leinwand L (1991) The use of sarkosyl in generating soluble protein after bacterial expression. Proc Natl Acad Sci U S A 88(4):1192–1196CrossRefPubMedPubMedCentralGoogle Scholar
  18. Gasser B, Prielhofer R, Marx H, Maurer M, Nocon J, Steiger M, Puxbaum V, Sauer M, Mattanovich D (2013) Pichia Pastoris: protein production host and model organism for biomedical research. Future Microbiol 8(2):191–208.  https://doi.org/10.2217/fmb.12.133 CrossRefPubMedGoogle Scholar
  19. Gopal GJ, Kumar A (2013) Strategies for the production of recombinant protein in Escherichia coli. Protein J 32(6):419–425.  https://doi.org/10.1007/s10930-013-9502-5 CrossRefPubMedGoogle Scholar
  20. Jevsevar S, Gaberc-Porekar V, Fonda I, Podobnik B, Grdadolnik J, Menart V (2005) Production of nonclassical inclusion bodies from which correctly folded protein can be extracted. Biotechnol Prog 21(2):632–639.  https://doi.org/10.1021/bp0497839 CrossRefPubMedGoogle Scholar
  21. Johnson IS (1983) Human insulin from recombinant DNA technology. Science 219(4585):632–637.  https://doi.org/10.1126/science.6337396 CrossRefPubMedGoogle Scholar
  22. Kamran M, Long ZJ, Xu D, Lv SS, Liu B, Wang CL, Xu J, Lam EW, Liu Q (2017) Aurora kinase a regulates Survivin stability through targeting FBXL7 in gastric cancer drug resistance and prognosis. Oncogene 6(2):e298.  https://doi.org/10.1038/oncsis.2016.80 CrossRefGoogle Scholar
  23. Kopito RR (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10(12):524–530.  https://doi.org/10.1016/S0962-8924(00)01852-3 CrossRefPubMedGoogle Scholar
  24. Lee N, Zhang SQ, Cozzitorto J, Yang JS, Testa D (1987) Modification of mRNA secondary structure and alteration of the expression of human interferon alpha 1 in Escherichia coli. Gene 58(1):77–86.  https://doi.org/10.1016/0378-1119(87)90031-X CrossRefPubMedGoogle Scholar
  25. Massiah MA, Wright KM, Du H (2016) Obtaining soluble folded proteins from inclusion bodies using Sarkosyl, triton X-100, and CHAPS: application to LB and M9 minimal media. Curr Protoc Protein Sci 84:6.13.1–6.13.24.  https://doi.org/10.1002/0471140864.ps0613s84 CrossRefGoogle Scholar
  26. Palomares LA, Estrada-Mondaca S, Ramirez OT (2004) Production of recombinant proteins: challenges and solutions. Methods Mol Biol 267:15–52.  https://doi.org/10.1385/1-59259-774-2:015 PubMedGoogle Scholar
  27. Pan SH, Malcolm BA (2000) Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3). BioTechniques 29(6):1234–1238PubMedGoogle Scholar
  28. Park DW, Kim SS, Nam MK, Kim GY, Kim J, Rhim H (2011) Improved recovery of active GST-fusion proteins from insoluble aggregates: solubilization and purification conditions using PKM2 and HtrA2 as model proteins. BMB Rep 44(4):279–284.  https://doi.org/10.5483/BMBRep.2011.44.4.279 CrossRefPubMedGoogle Scholar
  29. Peternel Š, Bele M, Gaberc-Porekar V, Menart V (2006) Nonclassical inclusion bodies in Escherichia coli. Microb Cell Factories 5(1):P23.  https://doi.org/10.1186/1475-2859-5-s1-p23 CrossRefGoogle Scholar
  30. Phan J, Yamout N, Schmidberger J, Bottomley SP, Buckle AM (2011) Refolding your protein with a little help from REFOLD. Methods Mol Biol 752:45–57.  https://doi.org/10.1007/978-1-60327-223-0_4 CrossRefPubMedGoogle Scholar
  31. 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(1):24.  https://doi.org/10.1186/s12934-015-0208-6 CrossRefGoogle Scholar
  32. Schwartz PL, Batt M (1973) The aggregation of (125-I) human growth hormone in response to freezing and thawing. Endocrinology 92(6):1795–1798.  https://doi.org/10.1210/endo-92-6-1795 CrossRefPubMedGoogle Scholar
  33. Schwegman JJ, Carpenter JF, Nail SL (2009) Evidence of partial unfolding of proteins at the ice/freeze-concentrate interface by infrared microscopy. J Pharm Sci 98(9):3239–3246.  https://doi.org/10.1002/jps.21843 CrossRefPubMedGoogle Scholar
  34. Shikama K, Yamazaki T (1961) Denaturation of catalase by freezing and thawing. Nature 190(4770):83–84.  https://doi.org/10.1038/190083a0 CrossRefGoogle Scholar
  35. Sieracki NA, Hwang HJ, Lee MK, Garner DK, Lu Y (2008) A temperature independent pH (TIP) buffer for biomedical biophysical applications at low temperatures. Chem Commun (Camb) 7:823–825.  https://doi.org/10.1039/b714446f CrossRefGoogle Scholar
  36. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99(4):303–310.  https://doi.org/10.1263/jbb.99.303 CrossRefPubMedGoogle Scholar
  37. Strambini GB, Gonnelli M (2007) Protein stability in ice. Biophys J 92(6):2131–2138.  https://doi.org/10.1529/biophysj.106.099531 CrossRefPubMedGoogle Scholar
  38. Tao H, Liu W, Simmons BN, Harris HK, Cox TC, Massiah MA (2010) Purifying natively folded proteins from inclusion bodies using sarkosyl, triton X-100, and CHAPS. BioTechniques 48(1):61–64.  https://doi.org/10.2144/000113304 CrossRefPubMedGoogle Scholar
  39. Tsumoto K, Umetsu M, Kumagai I, Ejima D, Arakawa T (2003) Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine. Biochem Biophys Res Commun 312(4):1383–1386CrossRefPubMedGoogle Scholar
  40. Villaverde A, Garcia-Fruitos E, Rinas U, Seras-Franzoso J, Kosoy A, Corchero JL, Vazquez E (2012) Packaging protein drugs as bacterial inclusion bodies for therapeutic applications. Microb Cell Factories 11(1):76.  https://doi.org/10.1186/1475-2859-11-76 CrossRefGoogle Scholar
  41. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398.  https://doi.org/10.1038/nbt1026 CrossRefPubMedGoogle Scholar
  42. Yang Z, Zhang L, Zhang Y, Zhang T, Feng Y, Lu X, Lan W, Wang J, Wu H, Cao C, Wang X (2011) Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method. PLoS One 6(7):e22981.  https://doi.org/10.1371/journal.pone.0022981 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Zhao Q, Liu L, Xie Q (2012) In vitro protein ubiquitination assay. Methods Mol Biol 876:163–172.  https://doi.org/10.1007/978-1-61779-809-2_13 CrossRefPubMedGoogle Scholar
  44. Zhong M, Liu B, Wang X, Liu L, Lun Y, Li X, Ning A, Cao J, Huang M (2013) De novo characterization of Lentinula edodes C91-3 transcriptome by deep Solexa sequencing. Biochem Biophys Res Commun 431(1):111–115.  https://doi.org/10.1016/j.bbrc.2012.12.065 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Arshad Ahmed Padhiar
    • 1
    • 2
  • Warren Chanda
    • 1
  • Thomson Patrick Joseph
    • 1
  • Xuefang Guo
    • 1
  • Min Liu
    • 1
  • Li Sha
    • 1
  • Samana Batool
    • 1
  • Yifan Gao
    • 1
  • Wei Zhang
    • 1
  • Min Huang
    • 1
  • Mintao Zhong
    • 1
  1. 1.Department of Microbiology, Basic Medical SciencesDalian Medical UniversityDalianChina
  2. 2.Department of Biosciences, Faculty of ScienceBarrett Hodgson UniversityKarachiPakistan

Personalised recommendations