Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies

Javad Salehinia , Hamid Mir Mohammad Sadeghi, Daryoush Abedi, Vajihe Akbari Akbari


Single chain variable fragment antibodies (scFvs) have attracted many attentions due to their small size, faster bio-distribution and better penetration in to the target tissues, and ease of expression in Escherichia coli. Although, scFv expression in E. coli usually leads to formation of inclusion bodies (IBs). The aim of this research was to improve solubilizing and refolding conditions for IBs of scFv version of pertuzumab (anti-human epidermal growth factor receptor 2 (HER2) antibody). After protein overexpression in E. coli BL21 (DE3), bacterial cells were lysed and IBs were extracted via repeated washing and centrifugation. The effect of different types, concentrations, pHs, and additive of denaturing agents on IBs solubility were evaluated. More than 40 refolding additives were screened and combinations of 10 of the best additives were check out using Plackett-Burman design to choose three refolding additives with the most positive effect on refolding of the scFv. Response surface methodology (RSM) was used to optimize the concentration of adopted additives. The most efficient buffer to solubilize IBs was a buffer containing 6 M urea with 6 mM beta mercaptoethanol, pH 11. The optimum concentration of three buffer additives for refolding of the scFv was 23 mM tricine, 0.55 mM arginine, and 14.3 mM imidazole. The bioactivity of the refolded scFv was confirmed by immunohistochemical staining of breast cancer tissue, a specific binding based method. The systematic optimization of refolding buffer developed in the present work will contribute to improve the refolding of other scFv fragments.


Keywords: HER2; Inclusion bodies; Refolding additive; Response surface methodology; Single-chain antibodies; Solubilizing.

Full Text:



Shuptrine CW, Surana R, Weiner LM. Monoclonal antibodies for the treatment of cancer. Semin Cancer Biol. 2012;22(1):3-13.

Adams GP, Schier R, McCall AM, Simmons HH, Horak EM, Alpaugh RK, et al. High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules. Cancer Res. 2001;61(12):4750-4755.

Andersen DC, Reilly DE. Production technologies for monoclonal antibodies and their fragments. Curr Opin Biotechnol. 2004;15(5):456-462.

Chen C, Snedecor B, Nishihara JC, Joly JC, McFarland N, Andersen DC, et al. High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain. Biotechnol Bioeng. 2004;85(5):463-474.

Yuasa N, Koyama T, Fujita-Yamaguchi Y. Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies. Biosci Trends. 2014;8(1):24-31.

Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical considerations in refolding proteins from inclusion bodies. Protein Expr Purif. 2003;28(1):1-8.

Palmer I, Wingfield PT. Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Curr Protoc Protein Sci. 2004;38(1):1-18.

Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nat Biotech. 2004;22(11):1399-1408.

Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Fact. 2015;14. 41-51.

Singh SM, Panda AK. Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng. 2005;99(4):303-310.

Alibolandi M, Mirzahoseini H. Chemical assistance in refolding of bacterial inclusion Bodies. Biochem Res Int. 2011;2011. Article ID 631607.

Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A, Garg L, Panda AK. Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli. Protein Expr Purif. 2000;18(2):182-192.

Sijwali PS, Brinen LS, Rosenthal PJ. Systematic optimization of expression and refolding of the plasmodium falciparum cysteine protease falcipain-2. Protein Expr Purif. 2001;22(1):128-134.

Akbari V, Sadeghi HM, Jafarian-Dehkordi A, Abedi D, Chou CP. Improved biological activity of a single chain antibody fragment against human epidermal growth factor receptor 2 (HER2) expressed in the periplasm of Escherichia coli. Protein Expr Purif. 2015;116:66-74.

Akbari V, Sadeghi HMM, Jafrian-Dehkordi A, Abedi D, Chou CP. Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli. J Ind Microbiol Biotechnol. 2014;41(6):947-956.

Akbari V, Sadeghi HMM, Jafarian-Dehkordi A, Chou CP, Abedi D. Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology. Res Pharm Sci. 2015;10(1):75-83.

Qi X, Sun Y, Xiong S. A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form. Microb Cell Fact. 2015;14:24-35.

Singh SM, Upadhyay AK, Panda AK. Solubilization at high pH results in improved recovery of proteins from inclusion bodies of E. coli. J Chem Technol Biotechnol 2008;83(8):1126-1134.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1):248-254.

Freydell EJ, Ottens M, Eppink M, van Dedem G, van der Wielen L. Efficient solubilization of inclusion bodies. Biotechnol J. 2007;2(6):678-684.

Kudou M, Ejima D, Sato H, Yumioka R, Arakawa T, Tsumoto K. Refolding single-chain antibody (scFv) using lauroyl-L-glutamate as a solubilization detergent and arginine as a refolding additive. Protein Expr Purif. 2011;77(1):68-74.

Gräslund S, Nordlund P, Weigelt J, Oppermann BMH, Arrowsmith C, Hui R, et al. Protein production and purification. Nat Methods. 2008;5(2):135-146.

Khan RH, Rao KB, Eshwari AN, Totey SM, Panda AK. Solubilization of recombinant ovine growth hormone with retention of native-like secondary structure and its refolding from the inclusion bodies of Escherichia coli. Biotechnol Prog. 1998;14(5):722-728.

Zhang Z, Zhang Y, Yang K. Mechanism of enhancement of prochymosin renaturation by solubilization of inclusion bodies at alkaline pH. Sci China Series C Life Sci. 1997;40(2):169-175.

Tsumoto K, Shinoki K, Kondo H, Uchikawa M, Juji T, Kumagai I. Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent-application to a human single-chain Fv fragment. J Immunol Methods. 1998;219(1-2):119-129.

Fischer G, Bang H. The refolding of urea-denatured ribonuclease A is catalyzed by peptidyl-prolyl cis-trans isomerase. Biochim Biophys Acta. 1985;828(1):39-42.

Sakamoto R, Nishikori S, Shiraki K. High temperature increases the refolding yield of reduced lysozyme: implication for the productive process for folding. Biotechnol Prog. 2004;20(4):1128-1133.

Chen J, Liu Y, Wang Y, Ding H, Su Z. Different effects of L-arginine on protein refolding: suppressing aggregates of hydrophobic interaction, not covalent binding. Biotechnol Prog. 2008;24(6):1365-1372.

Shi R, Pan Q, Guan Y, Hua Z, Huang Y, Zhao M, et al. Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein. Arch Biochem Biophys. 2007;459(1):122-128.

Wang Y, Oosterwijk N, Ali AM, Adawy A, Anindya AL, Dömling AS, et al. A systematic protein refolding screen method using the DGR approach reveals that time and secondary TSA are essential variables. Sci Rep. 2017;7(1):9355-9364.

Kashanian F, Masoudi MM, Shamloo A, Habibi-Rezaei M, Moosavi-Movahedi A. Modeling, simulation, and employing dilution-dialysis microfluidic chip (DDMC) for heightening proteins refolding efficiency. Bioprocess Biosyst Eng. 2018;41(5):707-714.

Jain S, Aresu L, Comazzi S, Shi J, Worrall E, Clayton J, et al. The development of a recombinant scFv monoclonal antibody targeting canine CD20 for use in comparative medicine. PloS One. 2016;11(2):e0148366.


  • There are currently no refbacks.

Creative Commons Attribution-NonCommercial 3.0

This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.