Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein

Farideh Rasooli, Atieh Hashemi


Recombinant epithelial cell adhesion molecule extracellular domain (EpEX) has a high potential as a candidate for passive and active immunotherapy as well as cancer vaccination. In the present study, EpEX was expressed as a thioredoxin fusion protein in Escherichia coli (E. coli). The effect of different hosts and expression conditions on the expression level of the fusion protein was also evaluated.Moreover, the effect of temperature and isopropyl-β-d-thiogalactopyranoside (IPTG) concentration on protein solubility was assessed. The codon optimized-synthetic gene was cloned into pET32a (+) expression vector and transformed into E. coli BL21 (DE3), RosettaTM (DE3), and OrigamiTM (DE3).The protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Lowering the expression temperature to 16 °C and IPTG concentration to 0.5 mM also dramatically increased the volumetric productivity of the fusion protein. In optimum culture condition, high-level expression of the target fusion protein was detected in RosettaTM (DE3) and OrigamiTM (DE3) (207 and 334 μg/mL, respectively), though they were expressed as inclusion bodies. No improvement was observed in the solubility of the fusion protein by reducing the temperature or IPTG concentration even when expressed in a TrxB/gor mutant strain. Results showed that Trx tag combined with other strategies utilized here could be effective to achieve high level of protein production but not effective in solubility improvement. However, new approaches might be necessary to enhance the solubility of EpEX in the E. coli system.


EpCAM; EpEX; Escherichia coli; Expression; Thioredoxin.

Full Text:



Karabulut S, Tas F, Tastekin D, Karabulut M, Yasasever C, Ciftci R, et al. The diagnostic, predictive, and prognostic role of serum epithelial cell adhesion molecule (EpCAM) and vascular cell adhesion molecule-1 (VCAM-1) levels in breast cancer. Tumor Biol. 2014;35(9):8849-8860.

Schmetzer O, Moldenhauer G, Nicolaou A,Schlag P, Riesenberg R, Pezzutto A. Detection of circulating tumor-associated antigen depends on the domains recognized by the monoclonal antibodies used: N-terminal trimmed EpCAM-levels are much higher than untrimmed forms. Immunol Lett. 2012;143(2):184-192.

Staib L, Birebent B, Somasundaram R, Purev E, Braumüller H, Leeser C, et al. Immunogenicity of recombinant GA733‐2E antigen (CO17‐1A, EGP, KS1‐4, KSA, Ep‐CAM) in gastro‐intestinal carcinoma patients. Int J Cancer. 2001;92(1):79-87.

Verch T, Hooper DC, Kiyatkin A, Steplewski Z, Koprowski H. Immunization with a plant-produced colorectal cancer antigen. Cancer Immunol Immunother. 2004;53(2):92-99.

Salehinia J, Sadeghi HMM, Abedi D, Akbari V. Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies. Res Pharm Sci. 2018;13(6):566-574.

Malekian R, Jahanian-Najafabadi A, Moazen F, Ghavimi R, Mohammadi E, Akbari V. High-yield production of granulocyte-macrophage colony-stimulating factor in E. coli BL21 (DE3) by an auto-induction strategy. Iran J Pharm Res. 2019;18(1):469-478.

Malekian R, Sima S, Jahanian-Najafabadi A, Moazen F, Akbari V. Improvement of soluble expression of GM-CSF in the cytoplasm of Escherichia coli using chemical and molecular chaperones. Protein Expr Purif. 2019;160:66-72.

Pourhadi M, Jamalzade F, Jahanian-Najafabadi A, Shafiee F. Expression, purification, and cytotoxic evaluation of IL24-BR2 fusion protein. Res Pharm Sci. 2019;14(4):320-328.

Francis DM, Page R. Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci. 2010;5:1-29.

Young CL, Britton ZT, Robinson AS. Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J. 2012;7(5):620-634.

Gopal GJ, Kumar A. Strategies for the production of recombinant protein in Escherichia coli. Protein J. 2013;32(6):419-425.

Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989. pp. 339-367.

Schnell U, Cirulli V, Giepmans BN. EpCAM: structure and function in health and disease. Biochim Biophys Acta. 2013;1828(8):1989-2001.

Baghbani-Arani F, Roohvandv F, Aghasadeghi M, Eidi A, Amini S, Motevalli F, et al. Expression and characterization of Escherichia coli derived hepatitis C virus ARFP/F protein. Mol Biol (Mosk). 2012;46(2):251-259.

Jurado P, de Lorenzo V, Fernández LA. Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli: evidence that chaperone activity is the prime effect of thioredoxin. J Mol Biol. 2006;357(1):49-61.

Sonoda H, Kumada Y, Katsuda T, Yamaji H. Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones. Protein Expr Purif. 2010;70(2):248-253.

Zheng P, Sun X, Guo L, Shen J. Cloning, expression, and characterization of an acetolactate synthase (ALS) gene from Anabaena azotica. Process Biochem. 2015;50(9):1349-1356.

Tsai WC, Wu TC, Chiang BL, Wen HW. Cloning, expression, and purification of recombinant major mango allergen Man i 1 in Escherichia coli. Protein Expr Purif. 2017;130:35-43.

Lim KP, Li H, Nathan S. Expression and purification of a recombinant scFv towards the exotoxin of the pathogen, Burkholderia pseudomallei. J Microbiol. 2004;42(2):126-132.

Dyson MR, Shadbolt SP, Vincent KJ, Perera RL, McCafferty J. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol. 2004;4(1):32-48.

Sun W, Xie J, Lin H, Mi S, Li Z, Hua F, et al. A combined strategy improves the solubility of aggregation-prone single-chain variable fragment antibodies. Protein Expr Purif. 2012;83(1):21-29.

Piubelli L, Campa M, Temporini C, Binda E, Mangione F, Amicosante M, et al. Optimizing Escherichia coli as a protein expression platform to produce Mycobacterium tuberculosis immunogenic proteins. Microb Cell Fact. 2013;12(1):115-128.

Liu Y, Zhao TJ, Yan YB, Zhou HM. Increase of soluble expression in Escherichia coli cytoplasm by a protein disulfide isomerase gene fusion system. Protein Expr Purif. 2005;44(2):155-161.

Klock HE, Koesema EJ, Knuth MW, Lesley SA. Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins. 2008;71(2):982-994.

Niiranen L, Espelid S, Karlsen CR, Mustonen M, Paulsen SM, Heikinheimo P, et al. Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli. Protein Expr Purif. 2007;52(1):210-218.


  • There are currently no refbacks.

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International 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.