Optimization of buffer additives for efficient recovery of hGM-CSF from inclusion bodies using response surface methodology

Document Type : Research article

Authors

1 Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.

2 Department of Pharmaceutical Biotechnology, School of Pharmacy, Isfahan University of Medical Sciences

Abstract

Overexpression of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by Escherichia coli leads to formation of insoluble and inactive proteins, inclusion bodies. The aim of this study was to improve recovery of biologically active hGM-CSF from inclusion bodies. The effect of types, concentrations and pHs of denaturing agents and addition of reducing agents on the yield of inclusion bodies solubilization was evaluated. Next, various conditions were evaluated for refolding of hGM-CSF using a two-step design of experiment (DOE) including primary screening by factorial design, and then optimization by response surface design. It was found that hGM-CSF inclusion bodies can be efficiently solubilized with 4 M urea and 4 mM β-mercaptoethanol, pH 9. A response surface quadratic model was employed to predict the optimum refolding conditions and the accuracy of this model was confirmed by high value of R2 (0.99) and F-value of 0.64. DOE results revealed that sorbitol (0.235 M), imidazole (97 mM) and SDS (0.09 %) would be the optimum buffer additives for refolding of hGM-CSF. Following refolding studies, the obtained protein was subjected to circular dichroism which confirmed correct secondary structure of the refolded hGM-CSF. The refolded hGM-CSF exhibited reasonable biological activity compared with standard protein. The approach developed in this work can be important to improve the refolding of other proteins with similar structural features.

Graphical Abstract

Optimization of buffer additives for efficient recovery of hGM-CSF from inclusion bodies using response surface methodology

Keywords


(1) Shi Y, Liu CH, Roberts AI, Das J, Xu G and Ren G.Granulocyte-macrophage colony-stimulating factor(GM-CSF) and T-cell responses: what we do anddon’t know. Cel Res. (2006) 16: 126-33.
(2) Srinivasa Babu K, Pulicherla KK, Antony A andMeenakshisundaram S. Cloning and expression ofrecombinant human GMCSF from Pichia pastorisGS115--a progressive strategy for economicproduction. Am. J. Ther. (2014) 21: 462-9.
(3) Hussein AM, Ross M, Vredenburgh J, MeisenbergB, Hars V and Gilbert C. Effects of granulocytemacrophage colony stimulating factor producedin Chinese hamster ovary cells(regramostim),Escherichia coli (molgramostim) and yeast(sargramostim) on priming peripheral bloodprogenitor cells for use with autologous bone marrowafter high-dose chemotherapy. Eur .J. Haematol.(1995) 55: 348-56.
(4) Belew M, Zhou Y, Wang S, Nystrom LE andJanson JC. Purification of recombinant humangranulocyte-macrophage colony-stimulating factorfrom the inclusion bodies produced by transformedEscherichia coli cells. J. Chromatogr.A. (1994) 679:67-83.
(5) Schwanke RC, Renard G, Chies JM, Campos MM,Batista EL, Jr and Santos DS. Molecular cloning,expression in Escherichia coli and productionof bioactive homogeneous recombinant humangranulocyte and macrophage colony stimulatingfactor. Int. J. Biol. Macromol. (2009) 45:97-102.
(6) Mayer M and Buchner J. Refolding of inclusion bodyproteins. Methods Mol. Med. (2004) 94: 239-54.
(7) Singh SM and Panda AK. Solubilization andrefolding of bacterial inclusion body proteins. J.Biosci. Bioeng. (2005) 99: 303-10.
(8) Singh A, Upadhyay V, Upadhyay AK, Singh SM andPanda AK. Protein recovery from inclusion bodies ofEscherichia coli using mild solubilization process.Microb. Cell Fact. (2015) 14: 41-51.
(9) Alibolandi M and Mirzahoseini H. Chemicalassistance in refolding of bacterial inclusion bodies.Biochem. Res. Int. (2011) 2011: 631607.
(10) Jungbauer A and Kaar W. Current status of technicalprotein refolding. J. Biotechnol. (2007) 128: 587-96.
(11) Quinn GP and Keough MJ. In Experimental Designand Data Analysis for Biologists. CambridgeUniversity Press, Cambridge (2002) I-IV.
(12) Studier FW. Protein production by auto-inductionin high-density shaking cultures. Protein Expr. Purif.
(2005) 41: 207-34.(13) Esmaili I, Sadeghi HMM and Akbari V. Effect ofbuffer additives on solubilization and refolding ofreteplase inclusion bodies. Res. Pharm. Sci. (2018)13: 413-21.
(14) Qi X, Sun Y and Xiong S. A single freeze-thawingcycle for highly efficient solubilization of inclusionbody proteins and its refolding into bioactive form.Microb. Cell Fact. (2015) 14: 24-36.
(15) Akbari V, Sadeghi HMM, Jafarian-Dehkordi A,Chou CP and Abedi D. Optimization of a single-chainantibody fragment overexpression in Escherichia
coli using response surface methodology. Res.Pharm. Sci. (2015) 10: 81-9.
(16) Kosobokova E, Skrypnik K, Pinyugina M,Shcherbakov A and Kosorukov V. Optimization ofthe refolding of recombinant human granulocytemacrophage colony-stimulating factor immobilizedon affinity sorbent. Appl. Biochem. Microbiol.(2014) 50: 773-9.
(17) Palmer I and Wingfield PT. Palmer I, Wingfield PT.Preparation and extraction of insoluble (inclusionbody) proteins from Escherichia coli. Curr ProtocProtein Sci. (2012) Chapter 6:Unit6.3.1-25.
(18) Burgess AW, Begley CG, Johnson GR, LopezAF, Williamson DJ and Mermod JJ. Purificationand properties of bacterially synthesized humangranulocyte-macrophage colony stimulating factor.Blood (1987) 69: 43-51.
(19) Upadhyay AK, Murmu A, Singh A and PandaAK. Kinetics of inclusion body formation andits correlation with the characteristics of proteinaggregates in Escherichia coli. PloS One. (2012) 7:e33951
(20) Upadhyay AK, Singh A, Mukherjee KJ and PandaAK. Refolding and purification of recombinantL-asparaginase from inclusion bodies of E. coli intoactive tetrameric protein. Front. Microbiol. (2014) 5:486.
(21) Marston FAO and Hartley DL. Solubilization ofprotein aggregates. Methods Enzymol. (1990) 182:264-76.309Optimization of Additives for GM-CSF Refolding Buffer
(22) Estapé D and Rinas U. Optimized procedures forpurification and solubilization of basic fibroblastgrowth factor inclusion bodies. Biotechnol. Tech.
(1996)10: 481-4.
(23) Okumura S, Saitoh H, Wasano N, Katayama H,Higuchi K and Mizuki E. Efficient solubilization,activation, and purification of recombinant Cry45Aaof Bacillus thuringiensis expressed as inclusionbodies in Escherichia coli. Protein Expr. Purif.
(2006) 47: 144-51.
(24) Schwanke RC, Renard G, Chies JM, Campos MM,Junior ELB and Santos DS. Molecular cloning,expression in Escherichia coli and productionof bioactive homogeneous recombinant humangranulocyte and macrophage colony stimulatingfactor. Int. J. Biol. Macromol. (2009) 45: 97-102.
(25) Clark ED. Protein refolding for industrial processes.Curr. Opin. Biotechnol. (2001) 12: 202-7.
(26) Singh SM and Panda AK. Solubilization andrefolding of bacterial inclusion body proteins. J.Biosci. Bioeng. (2005) 99: 303-10.
(27) Thomson CA, Olson M, Jackson LM and SchraderJW. A simplified method for the efficient refoldingand purification of recombinant human GM-CSF.PloS One. (2012) 7: e49891.
(28) Protein production and purification. Nat. Methods
(2008) 5: 135-46.
(29) Biswas A and Das KP. SDS induced structuralchanges in alpha-crystallin and it’s effect onrefolding. Protein J. (2004) 23: 529-38.
(30) Michaux C, Roussel G, Lopes-Rodrigues M,Matagne A and Perpète EA. Unravelling themechanisms of a protein refolding process basedon the association of detergents and co-solvents. J.Pept. Sci. (2016) 22: 485-91.
(31) Xie G and Timasheff SN. Mechanism of thestabilization of ribonuclease A by sorbitol:preferential hydration is greater for the denaturedthan for the native protein. Protein Sci. (1997) 6:
211-21.
(32) Wingfield PT. Overview of the purification ofrecombinant proteins. Curr. Protoc. Protein Sci.(2015) 80: 6.1.1-6.1.35.
(33) Akbari V, Sadeghi HMM, Jafrian-Dehkordi A, AbediD and Chou CP. Functional expression of a singlechain antibody fragment against human epidermalgrowth factor receptor 2 (HER2) in Escherichia coli.J. Ind. Microbiol. Biotechnol. (2014) 41: 947-56.
(34) Gu Z, Weidenhaupt M, Ivanova N, Pavlov M,Xu B and Su Z-G. Chromatographic methods forthe isolation of, and refolding of proteins from,Escherichia coli inclusion bodies. Protein Expr.Purif. (2002) 25: 174-9.
(35) van Mierlo CPM and Steensma E. Protein foldingand stability investigated by fluorescence, circulardichroism (CD), and nuclear magnetic resonance(NMR) spectroscopy: the flavodoxin story. J.Biotechnol. (2000) 79: 281-98.
(36) Vedadi M, Arrowsmith CH, Allali-HassaniA, Senisterra G and Wasney GA. Biophysicalcharacterization of recombinant proteins: a key tohigher structural genomics success. J. Struct. Biol.(2010) 172: 107-19.
(37) Waldo GS, Standish BM, Berendzen J andTerwilliger TC. Rapid protein-folding assay usinggreen fluorescent protein. Nat. Biotechnol. (1999)17: 691-5.
(38) Walter MR, Cook WJ, Ealick SE, Nagabhushan TL,Trotta PP and Bugg CE. Three-dimensional structureof recombinant human granulocyte-macrophagecolony-stimulating factor. J. Mol. Biol. (1992) 224:1075-85.
(39) Cannistra SA, Rambaldi A, Spriggs DR, HerrmannF, Kufe D and Griffin JD. Human granulocytemacrophage colony-stimulating factor inducesexpression of the tumor necrosis factor gene by theU937 cell line and by normal human monocytes. J.Clin. Invest. (1987) 79: 1720.
(40) DeLamarter JF, Mermod JJ, Liang CM, Eliason JFand Thatcher DR. Recombinant murine GM-CSFfrom E. coli has biological activity and is neutralizedby a specific antiserum. EMBO J. (1985) 4: 2575-81.
(41) Das KMP, Banerjee S, Shekhar N, Damodaran K,Nair R and Somani S. Cloning, Soluble Expressionand Purification of High Yield RecombinanthGMCSF in Escherichia coli. Int. J. Mol. Sci. (2011)12: 2064-76.
(42) Lebendiker M and Danieli T. Production of proneto-aggregate proteins. FEBS Lett. (2014) 588: 236-46.