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Applied Microbiology and Biotechnology

, Volume 93, Issue 3, pp 1065–1074 | Cite as

Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli

  • Xiao Yan
  • Sheng Hu
  • Yi-Xin GuanEmail author
  • Shan-Jing Yao
Biotechnological products and process engineering

Abstract

Recombinant human interferon-gamma (rhIFN-γ) is a protein of great potential for clinical therapy due to its multiple biological activities. However, overexpressing rhIFN-γ in Escherichia coli was found to accumulate as cytoplasmic inclusion bodies. In this work, a system for soluble and active expression of rhIFN-γ was constructed by coexpressing chaperonin GroEL/GroES in E. coli. The rhIFN-γ gene was fused to a pET-28a expression vector, and rhIFN-γ was partially expressed as the soluble form following coexpression with a second vector producing chaperonin GroEL/GroES. The fermentation of recombinant E. coli harboring rhIFN-γ and GroEL/GroES plasmids was investigated, and the optimized conditions were as follows: culture temperature of 25°C, incubation time of 8 h, isopropyl-β-d-thio-galactoside concentration of 0.2 mM, and l-arabinose concentration of 0.5 g/L. As a result, the expression level of rhIFN-γ was improved accordingly by 2.2-fold than the control, while a significantly positive correlation was also found between the ratio of supernatant to precipitate of rhIFN-γ and the amount of chaperonin. Circular dichroism spectra, fluorescence spectra, size exclusion chromatography, and chemical cross-linking method were applied to characterize rhIFN-γ, indicating that the three-dimensional structure of rhIFN-γ was identical to that of the native rhIFN-γ. The enzyme-linked immunosorbent assay for active rhIFN-γ quantification showed that coexpression yielded 72.91 mg rhIFN-γ per liter fermentation broth. Finally, protein–protein interactions between rhIFN-γ and chaperonin were analyzed using the yeast two-hybrid system, which provided the direct evidence that chaperonin GroEL/GroES interacted with rhIFN-γ to increase the soluble expression and presented the potential in producing efficiently recombinant proteins.

Keywords

Recombinant human interferon-gamma GroEL/GroES coexpression system Soluble expression Protein folding Protein–protein interactions 

Notes

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (no. 20876138).

References

  1. Bendixen C, Gangloff S, Rothstein R (1994) A yeast mating-selection scheme for detection of protein–protein interactions. Nucleic Acids Res 22:1778–1779CrossRefGoogle Scholar
  2. Blalock JE, Georgiades JA, Langford MP, Johnson HM (1980) Purified human immune interferon has more potent anticellular activity than fibroblast or leukocyte interferon. Cell Immunol 49:390–394CrossRefGoogle Scholar
  3. Brusehaber E, Schwiebs A, Schmidt M, Bottcher D, Bornscheuer UT (2010) Production of pig liver esterase in batch fermentation of E. coli Origami. Appl Microbiol Biotechnol 86:1337–1344CrossRefGoogle Scholar
  4. Contreras JAR, Pedraza-Reyes M, Ordonez LG, Estrada NU, de la Rosa APB, De Leon-Rodriguez A (2010) Replicative and integrative plasmids for production of human interferon gamma in Bacillus subtilis. Plasmid 64:170–176CrossRefGoogle Scholar
  5. Ealick SE, Cook WJ, Vijay-Kumar S, Carson M, Nagabhushan TL, Trotta PP, Bugg CE (1991) Three-dimensional structure of recombinant human interferon-gamma. Science 252:698–702CrossRefGoogle Scholar
  6. Ellerhorst JA, Kilbourn RG, Amato RJ, Zukiwski AA, Jones E, Logothetis CJ (1994) Phase-II trial of low-dose gamma-interferon in metastatic renal-cell carcinoma. J Urol 152:841–845Google Scholar
  7. Fenton WA, Horwich AL (1997) GroEL-mediated protein folding. Protein Sci 6(4):743–760CrossRefGoogle Scholar
  8. Gao YG, Guan YX, Yao SJ, Cho MG (2003) On-column refolding of recombinant human interferon-gamma with an immobilized chaperone fragment. Biotechnol Prog 19:915–920CrossRefGoogle Scholar
  9. Giannopoulos A, Constantinides C, Fokaeas E, Stravodimos C, Giannopoulou M, Kyroudi A, Gounaris A (2003) The immunomodulating effect of interferon-γ intravesical instillations in preventing bladder cancer recurrence. Clin Cancer Res 9:5550–5558Google Scholar
  10. Gietz D, St. Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425CrossRefGoogle Scholar
  11. Gnoth S, Simutis R, Lubbert A (2010) Selective expression of the soluble product fraction in Escherichia coli cultures employed in recombinant protein production processes. Appl Microbiol Biotechnol 87:2047–2058CrossRefGoogle Scholar
  12. Goloubinoff P, Diamant S, Weiss C, Azem A (1997) GroES binding regulates GroEL chaperonin activity under heat shock. FEBS Lett 407:215–219CrossRefGoogle Scholar
  13. Gray PW, Leung DW, Pennica D, Yelverton E, Najarian R, Simonsen CC, Derynck R, Sherwood PJ, Wallace DM, Berger SL (1982) Expression of human immune interferon cDNA in E. coli and monkey cells. Nature 295:503–508CrossRefGoogle Scholar
  14. Gupta P, Aggarwal N, Batra P, Mishra S, Chaudhuri TK (2006) Co-expression of chaperonin GroEL/GroES enhances in vivo folding of yeast mitochondrial aconitase and alters the growth characteristics of Escherichia coli. Int J Biochem Cell Biol 38:1975–1985CrossRefGoogle Scholar
  15. Hartl FU, Hayer-Hartl M (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295:1852–1858CrossRefGoogle Scholar
  16. Hernandez VEB, Maldonado L, Rivero EM, de la Rosa APB, Jimenez-Bremont JF, Acevedo LGO, Rodriguez ADL (2008) Periplasmic expression and recovery of human interferon gamma in Escherichia coli. Protein Expression Purif 59:169–174Google Scholar
  17. Houry WA, Frishman D, Eckerskorn C, Lottspeich F, Hartl FU (1999) Identification of in vivo substrates of the chaperonin GroEL. Nature 402:147–154CrossRefGoogle Scholar
  18. Hsieh CA (2010) Expression and purification of recombinant human interferon-γ and interleukin-12 employing starch binding domain. Dissertation, National Tsing Hua University, Hsinchu City, TaiwanGoogle Scholar
  19. Jin T, Guan YX, Yao SJ, Lin DQ, Cho MG (2006) On-column refolding of recombinant human interferon-γ inclusion bodies by expanded bed adsorption chromatography. Biotechnol Bioeng 93:755–760CrossRefGoogle Scholar
  20. Kerner MJ, Naylor DJ, Ishihama Y, Maier T, Chang HC, Stines AP, Georgopoulos C, Frishman D, Hayer-Hartl M, Mann M (2005) Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122:209–220CrossRefGoogle Scholar
  21. Kim SY, Heo MA, Jeong YJ (2009) Improving the productivity of recombinant protein in Escherichia coli under thermal stress by coexpressing GroELS chaperone system. J Microbiol Biotechnol 19:72–77Google Scholar
  22. Levitt M, Chothia C (1976) Structural patterns in globular proteins. Nature 261:552–558CrossRefGoogle Scholar
  23. Liberek K, Lewandowska A, Zietkiewicz S (2008) Chaperones in control of protein disaggregation. EMBO J 27:328–335CrossRefGoogle Scholar
  24. Marth C, Windbichler GH, Hausmaninger H, Petru E, Estermann K, Pelzer A, Mueller-Holzner E (2006) Interferon-gamma in combination with carboplatin and paclitaxel as a safe and effective first-line treatment option for advanced ovarian cancer: results of a phase I/II study. Int J Gynecol Cancer 16:1522–1528CrossRefGoogle Scholar
  25. Matsushita K, Takenouchi T, Shimada H, Tomonaga T, Hayashi H, Shioya A, Komatsu A, Matsubara H, Ochiai T (2006) Strong HLA-DR antigen expression on cancer cells relates to better prognosis of colorectal cancer patients: Possible involvement of c-myc suppression by interferon-gamma in situ. Cancer Sci 97:57–63CrossRefGoogle Scholar
  26. Miller CHT, Maher SG, Young HA (2009) Clinical use of interferon-γ. Ann NY Acad Sci 1182:69–79CrossRefGoogle Scholar
  27. Moon HJ, Jeya M, Yu IS, Ji JH, Oh DK, Lee JK (2009) Chaperone-aided expression of LipA and LplA followed by the increase in alpha-lipoic acid production. Appl Microbiol Biotechnol 83:329–337CrossRefGoogle Scholar
  28. Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK–DnaJ–GrpE and GroEL–GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2 in Escherichia coli. Appl Environ Microbiol 64:1694–1699Google Scholar
  29. Parvez MK, Sehgal D, Sarin SK, Basir SF, Jameel S (2006) Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-gamma. World J Gastroenterol 12:3006–3014Google Scholar
  30. Reddy PK, Reddy SG, Narala VR, Majee SS, Konda S, Gunwar S, Reddy RC (2007) Increased yield of high purity recombinant human interferon-gamma utilizing reversed phase column chromatography. Protein Expression Purif 52:123–130CrossRefGoogle Scholar
  31. Rubin BY, Gupta SL (1980) Differential efficacies of human type I and type II interferons as antiviral and antiproliferative agents. Proc Natl Acad Sci U S A 77:5928–5932CrossRefGoogle Scholar
  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  33. Schoenborn JR, Wilson CB (2007) Regulation of interferon-gamma during innate and adaptive immune responses. Adv Immunol 96:41–101CrossRefGoogle Scholar
  34. Sharon MK, Thomas JJ, Nicholas CP (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751:119–139Google Scholar
  35. Sigidin YA, Loukina GV, Skurkovich B, Skurkovich S (2001) Randomized, double-blind trial of anti-interferon-gamma antibodies in rheumatoid arthritis. Scand J Rheumatol Suppl 30:203–207CrossRefGoogle Scholar
  36. Stefani M, Dobson CM (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med 81:678–699CrossRefGoogle Scholar
  37. Suárez-Méndez R, García-García I, Fernández-Olivera N, Valdés-Quintana M, Milanés-Virelles MT, Carbonell D, Machado-Molina D, Valenzuela-Silva CM, López-Saura PA (2004) Adjuvant interferon gamma in patients with drug-resistant pulmonary tuberculosis: a pilot study. BMC Infect Dis 4:44CrossRefGoogle Scholar
  38. Tsaprailis G, Chan DWS, English AM (1998) Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism. Biochemistry 37:2004–2016CrossRefGoogle Scholar
  39. Vandenbroeck K, Billiau A (1998) Interferon-gamma is a target for binding and folding by both Escherichia coli chaperone model systems GroEL/GroES and DnaK/DnaJ/GrpE. Biochimie 80:729–737CrossRefGoogle Scholar
  40. Vandenbroeck K, Martens E, Dandrea S, Billiau A (1993) Refolding and single-step purification of porcine interferon-gamma from Escherichia coli inclusion bodies. Eur J Biochem 215:481–486CrossRefGoogle Scholar
  41. Vandenbroeck K, Martens E, Billiau A (1998) GroEL/ES chaperonins protect interferon-gamma against physicochemical stress–study of tertiary structure formation by alpha-casein quenching and ELISA. Eur J Biochem 251:181–188CrossRefGoogle Scholar
  42. Vanstokkum IHM, Spoelder HJW, Bloemendal M, Vangrondelle R, Groen FCA (1990) Estimation of protein secondary structure and error analysis from circular-dichroism spectra. Anal Biochem 191:110–118CrossRefGoogle Scholar
  43. Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89:392–400CrossRefGoogle Scholar
  44. Zhang Z, Tong KT, Belew M, Pettersson T, Janson JC (1992) Production, purification and characterization of recombinant human interferon-gamma. J Chromatogr A 604:143–155CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Xiao Yan
    • 1
  • Sheng Hu
    • 2
  • Yi-Xin Guan
    • 1
    Email author
  • Shan-Jing Yao
    • 1
  1. 1.Department of Chemical and Biological EngineeringZhejiang UniversityHangzhouPeople’s Republic of China
  2. 2.School of Biotechnology and Chemical Engineering, Ningbo Institute of TechnologyZhejiang UniversityNingboPeople’s Republic of China

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