Abstract
Human epidermal growth factor receptors (EGFR) are an important target in drug discovery in terms of both protein–small-molecule interactions and protein–protein interactions. In this work, the isolation of a stable soluble protein of the tyrosine kinase domain of EGFR in Escherichia coli expression has been accomplished. This successful study presents the expression and purification conditions to obtain a stable soluble protein of the active tyrosine kinase domain of EGFR (EGFR-TK) and ErbB2 (ErbB2-TK) in a bacterial system, albeit in relatively low yields. The recombinant gene was inserted into a pColdI vector and recombinant protein was expressed at low temperature. Purification of EGFR-TK and ErbB2-TK took place under the same conditions by purified supernatant using a diethylaminoethyl sepharose column followed by anion exchange and size-exclusion chromatography columns. The final yields of purified EGFR-TK and ErbB2-TK were 8.4 and 9.5 mg per liter of culture, respectively. Determination of EGFR-TK and ErbB2-TK was performed via enzyme activity with commercial drugs. The IC50 values of erlotinib and afatinib against EGFR-TK were 13.09 nM and 2.36 nM respectively, while the IC50 values of lapatinib and afatinib against ErbB2-TK were 24.69 nM and 1.36 nM, respectively. These results confirmed that soluble proteins of the active intracellular domain of the HERs family were successfully expressed and purified in a bacterial system. The new protein expression and purification protocol will greatly facilitate the enzymatic inhibition and structural studies of this protein for drug discovery.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22(11):1399–1408. https://doi.org/10.1038/nbt1029
Barf T, Kaptein A (2012) Irreversible protein kinase inhibitors: balancing the benefits and risks. J Med Chem 55(14):6243–6262. https://doi.org/10.1021/jm3003203
Baselga J, Cortes J (2005) Epidermal growth factor receptor pathway inhibitors. Cancer Chemother Biol Response Modif 22:205–23. https://doi.org/10.1016/j.uct.2006.08.002
Cha MY, Lee KO, Kang SJ, Jung YH, Song JY, Choi KJ, Byun JY, Lee HJ, Lee GS, Park SB, Kim MS (2012) Synthesis and biological evaluation of pyrimidine-based dual inhibitors of human epidermal growth factor receptor 1 (HER-1) and HER-2 tyrosine kinases. J Med Chem 55(6):2846–2857. https://doi.org/10.1021/jm201758g
Chen R (2012) Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv 30(5):1102–1107. https://doi.org/10.1016/j.biotechadv.2011.09.013
Ciardiello F, Tortora G (2008) EGFR antagonists in cancer treatment. N Engl J Med 358(11):1160–1174. https://doi.org/10.1056/NEJMra0707704
Ciardiello F, Caputo R, Troiani T, Borriello G, Kandimalla ER, Agrawal S, Mendelsohn J, Bianco AR, Tortora G (2001) Antisense oligonucleotides targeting the epidermal growth factor receptor inhibit proliferation, induce apoptosis, and cooperate with cytotoxic drugs in human cancer cell lines. Int J Cancer 93(2):172–178. https://doi.org/10.1002/ijc.1335
Coumar MS, Chu CY, Lin CW, Shiao HY, Ho YL, Reddy R, Lin WH, Chen CH, Peng YH, Leou JS, Lien TW, Huang CT, Fang MY, Wu SH, Wu JS, Chittimalla SK, Song JS, Hsu JT, Wu SY, Liao CC, Chao YS, Hsieh HP (2010) Fast-forwarding hit to lead: aurora and epidermal growth factor receptor kinase inhibitor lead identification. J Med Chem 53(13):4980–4988. https://doi.org/10.1021/jm1000198
Di Massimo AM, Di Loreto M, Pacilli A, Raucci G, D’Alatri L, Mele A, Bolognesi A, Polito L, Stirpe F, De Santis R (1997) Immunoconjugates made of an anti-EGF receptor monoclonal antibody and type 1 ribosome-inactivating proteins from Saponaria ocymoides or Vaccaria pyramidata. Br J Cancer 75(6):822–828. https://doi.org/10.1038/bjc.1997.147
Dong A, Wodziak D, Lowe AW (2015) Epidermal growth factor receptor (EGFR) signaling requires a specific endoplasmic reticulum thioredoxin for the post-translational control of receptor presentation to the cell surface. J Biol Chem 290(13):8016–8027. https://doi.org/10.1074/jbc.M114.623207
Elloumi-Mseddi J, Jellali K, Aifa S (2013) In vitro activation and inhibition of recombinant EGFR tyrosine kinase expressed in Escherichia coli. ScientificWorldJournal 2013:807284–807285. https://doi.org/10.1155/2013/807284
Elloumi-Mseddi J, Jellali K, Villalobo A, Aifa S (2014) A premature termination of human epidermal growth factor receptor transcription in Escherichia coli. ScientificWorldJournal 2014:830923–830926. https://doi.org/10.1155/2014/830923
Ferguson KM (2008) Structure-based view of epidermal growth factor receptor regulation. Annu Rev Biophys 37:353–373. https://doi.org/10.1146/annurev.biophys.37.032807.125829
Galizia G, Lieto E, De Vita F, Orditura M, Castellano P, Troiani T, Imperatore V, Ciardiello F (2007) Cetuximab, a chimeric human mouse anti-epidermal growth factor receptor monoclonal antibody, in the treatment of human colorectal cancer. Oncogene 26(25):3654–3660. https://doi.org/10.1038/sj.onc.1210381
Garofalo M, Romano G, Di Leva G, Nuovo G, Jeon YJ, Ngankeu A, Sun J, Lovat F, Alder H, Condorelli G, Engelman JA, Ono M, Rho JK, Cascione L, Volinia S, Nephew KP, Croce CM (2011) EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers. Nat Med 18(1):74–82. https://doi.org/10.1038/nm.2577
Gutierrez C, Schiff R (2011) HER2: biology, detection, and clinical implications. Arch Pathol Lab Med 135(1):55–62. https://doi.org/10.1043/2010-0454-RAR.1
Han J, Kaspersen SJ, Nervik S, Norsett KG, Sundby E, Hoff BH (2016) Chiral 6-aryl-furo[2,3-d]pyrimidin-4-amines as EGFR inhibitors. Eur J Med Chem 119:278–299. https://doi.org/10.1016/j.ejmech.2016.04.054
Holbro T, Hynes NE (2004) ErbB receptors: directing key signaling networks throughout life. Annu Rev Pharmacol Toxicol 44:195–217 https://doi.org/10.1146/annurev.pharmtox.44.1018
Ibrahim DA, Abou El Ella DA, El-Motwally AM, Aly RM (2015) Molecular design and synthesis of certain new quinoline derivatives having potential anticancer activity. Eur J Med Chem 102:115–131. https://doi.org/10.1016/j.ejmech.2015.07.030
Jong WS, Vikstrom D, Houben D, van den Berg van Saparoea HB, de Gier JW, Luirink J (2017) Application of an E. coli signal sequence as a versatile inclusion body tag. Microb Cell Fact 16(1):50. https://doi.org/10.1186/s12934-017-0662-4
Knowles JA, Heath CH, Saini R, Umphrey H, Warram J, Hoyt K, Rosenthal EL (2012) Molecular targeting of ultrasonographic contrast agent for detection of head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 138(7):662–668. https://doi.org/10.1001/archoto.2012.1081
Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson M, Johnson BE, Eck MJ, Tenen DG, Halmos B (2005) EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352(8):786–792. https://doi.org/10.1056/NEJMoa044238
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non–small-cell lung cancer to gefitinib. N Engl J Med 350(21):2129–2139. https://doi.org/10.1056/NEJMoa040938
Mahboobi S, Sellmer A, Winkler M, Eichhorn E, Pongratz H, Ciossek T, Baer T, Maier T, Beckers T (2010) Novel chimeric histone deacetylase inhibitors: a series of lapatinib hybrides as potent inhibitors of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and histone deacetylase activity. J Med Chem 53(24):8546–8555. https://doi.org/10.1021/jm100665z
Martinelli E, De Palma R, Orditura M, De Vita F, Ciardiello F (2009) Anti-epidermal growth factor receptor monoclonal antibodies in cancer therapy. Clin Exp Immunol 158(1):1–9. https://doi.org/10.1111/j.1365-2249.2009.03992.x
Maruyama IN (2014) Mechanisms of activation of receptor tyrosine kinases: monomers or dimers. Cells 3(2):304–330. https://doi.org/10.3390/cells3020304
Messersmith WA, Hidalgo M (2007) Panitumumab, a monoclonal anti epidermal growth factor receptor antibody in colorectal cancer: another one or the one? Clin Cancer Res 13(16):4664–4666. https://doi.org/10.1158/1078-0432.CCR-07-0065
Morphy R (2010) Selectively nonselective kinase inhibition: striking the right balance. J Med Chem 53(4):1413–1437. https://doi.org/10.1021/jm901132v
Nakata A, Gotoh N (2012) Recent understanding of the molecular mechanisms for the efficacy and resistance of EGF receptor-specific tyrosine kinase inhibitors in non-small cell lung cancer. Expert Opin Ther Targets 16(8):771–781. https://doi.org/10.1517/14728222.2012.697155
Niculescu-Duvaz I (2010) Trastuzumab emtansine, an antibody-drug conjugate for the treatment of HER2+ metastatic breast cancer. Curr Opin Mol Ther 12(3):350–360
Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE, Meyerson M (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304(5676):1497–1500. https://doi.org/10.1126/science.1099314
Qing G, Ma LC, Khorchid A, Swapna GV, Mal TK, Takayama MM, Xia B, Phadtare S, Ke H, Acton T, Montelione GT, Ikura M, Inouye M (2004) Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol 22(7):877–882. https://doi.org/10.1038/nbt984
Riese DJ 2nd, Stern DF (1998) Specificity within the EGF family/ErbB receptor family signaling network. Bioessays 20(1):41–48. https://doi.org/10.1002/(SICI)1521-1878(199801)20:1<41::AID-BIES7>3.0.CO;2-V
Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. https://doi.org/10.3389/fmicb.2014.00172
Shirano Y, Shibata D (1990) Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level. FEBS Lett 271(1–2):128–130. https://doi.org/10.1016/0014-5793(90)80388-Y
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235(4785):177–182. https://doi.org/10.1126/science.3798106
Smaglo BG, Aldeghaither D, Weiner LM (2014) The development of immunoconjugates for targeted cancer therapy. Nat Rev Clin Oncol 11(11):637–648. https://doi.org/10.1038/nrclinonc.2014.159
Snyder LC, Astsaturov I, Weiner LM (2005) Overview of monoclonal antibodies and small molecules targeting the epidermal growth factor receptor pathway in colorectal cancer. Clin Colorectal Cancer 5(Suppl 2):S71–S80. https://doi.org/10.3816/CCC.2005.s.010
Sun Y, Feng X, Qu J, Han W, Liu Z, Li X, Zou M, Zhen Y, Zhu J (2015) Expression and characterization of the extracellular domain of human HER2 from Escherichia Coli, and production of polyclonal antibodies against the recombinant proteins. Appl Biochem Biotechnol 176(4):1029–1043. https://doi.org/10.1007/s12010-015-1627-x
Suzuki N, Shiota T, Watanabe F, Haga N, Murashi T, Ohara T, Matsuo K, Omori N, Yari H, Dohi K, Inoue M, Iguchi M, Sentou J, Wada T (2012) Discovery of novel 5-alkynyl-4-anilinopyrimidines as potent, orally active dual inhibitors of EGFR and Her-2 tyrosine kinases. Bioorg Med Chem Lett 22(1):456–460. https://doi.org/10.1016/j.bmcl.2011.10.103
Uchihashi T, Kodera N, Ando T (2012) Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy. Nat Protoc 7(6):1193–1206. https://doi.org/10.1038/nprot.2012.047
van der Geer P, Hunter T, Lindberg RA (1994) Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol 10:251–337. https://doi.org/10.1146/annurev.cb.10.110194.001343
Vasina JA, Baneyx F (1996) Recombinant protein expression at low temperatures under the transcriptional control of the major Escherichia coli cold shock promoter cspA. Appl Environ Microbiol 62(4):1444–1447 https://www.ncbi.nlm.nih.gov/pubmed/8919809
Wang GT, Mantei RA, Hubbard RD, Wilsbacher JL, Zhang Q, Tucker L, Hu X, Kovar P, Johnson EF, Osterling DJ, Bouska J, Wang J, Davidsen SK, Bell RL, Sheppard GS (2010) Substituted 4-amino-1H-pyrazolo[3,4-d]pyrimidines as multi-targeted inhibitors of insulin-like growth factor-1 receptor (IGF1R) and members of ErbB-family receptor kinases. Bioorg Med Chem Lett 20(20):6067–6071. https://doi.org/10.1016/j.bmcl.2010.08.052
Wang L, Yan J, Yan J, Xu H, Zhang D, Wang X, Sheng J (2018) Expression and purification of the human epidermal growth factor receptor extracellular domain. Protein Expr Purif 144:33–38. https://doi.org/10.1016/j.pep.2017.11.009
Ward RA, Anderton MJ, Ashton S, Bethel PA, Box M, Butterworth S, Colclough N, Chorley CG, Chuaqui C, Cross DA, Dakin LA, Debreczeni JE, Eberlein C, Finlay MR, Hill GB, Grist M, Klinowska TC, Lane C, Martin S, Orme JP, Smith P, Wang F, Waring MJ (2013) Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR). J Med Chem 56(17):7025–7048. https://doi.org/10.1021/jm400822z
Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125(6):1137–1149. https://doi.org/10.1016/j.cell.2006.05.013
Zhang F, Wang S, Yin L, Yang Y, Guan Y, Wang W, Xu H, Tao N (2015a) Quantification of epidermal growth factor receptor expression level and binding kinetics on cell surfaces by surface plasmon resonance imaging. Anal Chem 87(19):9960–9965. https://doi.org/10.1021/acs.analchem.5b02572
Zhang L, Yang Y, Zhou H, Zheng Q, Li Y, Zheng S, Zhao S, Chen D, Fan C (2015b) Structure-activity study of quinazoline derivatives leading to the discovery of potent EGFR-T790M inhibitors. Eur J Med Chem 102:445–463. https://doi.org/10.1016/j.ejmech.2015.08.026
Zhang JH, Chung TD, Oldenburg KR (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4(2):67–73. https://doi.org/10.1177/108705719900400206
Zheng W, Thorne N, McKew JC (2013) Phenotypic screens as a renewed approach for drug discovery. Drug Discov Today 18(21–22):1067–1073. https://doi.org/10.1016/j.drudis.2013.07.001
Acknowledgments
The authors would like to sincerely thank the National Omics Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand for LC-MS/MS Technology.
Funding
This work was supported by the Thailand Research Fund (RSA6080078 and DBG6080007) and the Center for Advanced Studies in Nanotechnology for Chemical, Food and Agricultural Industries, KU Institute for Advanced Studies, Kasetsart University and Development Institute. This research was also supported by Joint Research and by Exploratory Research Center on Life and Living Systems (ExCELLS), Japan (ExCELLS program No. 18-101 to H.W. and No. 19-310 to K.C.).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 1065 kb)
Rights and permissions
About this article
Cite this article
Seetaha, S., Ratanabanyong, S. & Choowongkomon, K. Expression, purification, and characterization of the native intracellular domain of human epidermal growth factor receptors 1 and 2 in Escherichia coli. Appl Microbiol Biotechnol 103, 8427–8438 (2019). https://doi.org/10.1007/s00253-019-10116-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-019-10116-6