Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface

Maryam Boshtam , Seddigheh Asgary , Ilnaz Rahimmanesh, Shirin Kouhpayeh, Jamal Naderi, Zahra Hejazi, Hoda Mohammad-Dezashibi, Ina Laura Pieper, Hossein Khanahmad


Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study was to develop two separate cell lines displaying either human MCP-1 (HMCP-1) or rabbit MCP-1 (RMCP-1) on their surface. A DNA fragment containing HMCP-1- or RMCP-1-encoding sequence was inserted into a pcDNA plasmid. Escherichia coli cells strain TOP 10F' was separately transformed with the pcDNA/RMCP-1 or /HMCP-1 ligation mixture. Following the cloning and construct verification, human embryonic kidney cell line (HEK 293T) was transfected with either of the linearized plasmids. Plasmid integration into the genomic DNA of HEK 293T cells was verified by polymerase chain reaction (PCR). HMCP-1 and RMCP-1 expression was evaluated at RNA and protein levels by real-time PCR and flow cytometry, respectively. PCR products of the expected sizes were amplified from the chromosomal DNA of transfected HEK 293T cells, i.e. 644 bp for H-MCP1 and 737 bp for RMCP-1 constructs. Real-time PCR revealed that the copy numbers of RMCP1 and HMCP1 mRNA per cell were 294 and 500, respectively. Flow cytometry analysis indicated 85% for RMCP-1 and 87% for HMCP-1 expression levels on the surface of transfected cells, when compared with an isotype control. The experiments thus confirmed that the MCP-1 genes were integrated into the HEK 293T genomic DNA and the encoded proteins were stably expressed on the cell surface.


Cell surface display; Chemokine CCL2; Flow cytometry; Transfection; Transformation.

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Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 2009;29(6):313-326.

Nelken NA, Coughlin SR, Gordon D, Wilcox JN. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest. 1991; 88(4):1121-1127.

Rayner K, Van Eersel S, Groot PH, Reape TJ. Localisation of mRNA for JE/MCP-1 and its receptor CCR2 in atherosclerotic lesions of the ApoE knockout mouse. J Vasc Res. 2000;37(2): 93-102.

Yla-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Leonard EJ, et al. Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci U S A. 1991;88(12):5252-5256.

Satonaka H, Suzuki E, Nishimatsu H, Oba S, Takeda R, Goto A, et al. Calcineurin promotes the expression of monocyte chemoattractant protein-1 in vascular myocytes and mediates vascular inflammation. Circ Res. 2004;94(5):693-700.

Song KM, Lee S, Ban C. Aptamers and their biological applications. Sensors (Basel). 2012; 12:612-631.

Mallikaratchy PR, Ruggiero A, Gardner JR, Kuryavyi V, Maguire WF, Heaney ML, et al. A multivalent DNA aptamer specific for the B-cell receptor on human lymphoma and leukemia. Nucleic acids research. 2011;39(6):2458-2469.

Boshtam M, Asgary S, Kouhpayeh S, Shariati L, Khanahmad H. Aptamers against pro- and anti-inflammatory cytokines: A review. Inflammation. 2017;40:340-349.

Mirian M, Khanahmad H, Darzi L, Salehi M, Sadeghi-Aliabadi H. Oligonucleotide aptamers: potential novel molecules against viral hepatitis. Res Pharm Sci. 2017;12(2):88-98.

Parashar A. Aptamers in therapeutics. J Clin Diagn Res. 2016;10(6):BE01-BE06.

Vater A, Sell S, Kaczmarek P, Maasch C, Buchner K, Pruszynska-Oszmalek E, et al. A mixed mirror-image DNA/RNA aptamer inhibits glucagon and acutely improves glucose tolerance in models of type 1 and type 2 diabetes. J Biol Chem. 2013;288(29):21136-21147.

Dua P, Kim S, Lee D-K. Patents on SELEX and therapeutic aptamers. Recent Pat DNA Gene Seq. 2008;2(3):172-186.

Sefah K, Shangguan D, Xiong X, O'Donoghue MB, Tan W. Development of DNA aptamers using cell-SELEX. Nat Protoc. 2010;5(6):1169-1185.

Ho M, Pastan I. Mammalian cell display for antibody engineering. Methods Mol Biol. 2009;525:337-352.

White R, Rusconi C, Scardino E, Wolberg A, Lawson J, Hoffman M, et al. Generation of species cross-reactive aptamers using" toggle" SELEX. Mol Ther. 2001;4(6):567-574.

Kim YS, Gu MB. Advances in aptamer screening and small molecule aptasensors. Adv Biochem Eng Biotechnol. 2014;140:29-67.

Kapourchali FR, Surendiran G, Chen L, Uitz E, Bahadori B, Moghadasian MH. Animal models of atherosclerosis. World J Clin Cases. 2014;2(5): 126-132.

Li X, Sui X, Zhang Y, Sun Y, Zhao Y, Zhai Y, et al. An improved calcium chloride method preparation and transformation of competent cells. Afr J Biotechnol.2010;9(50):8549-8554.

Dalton AC, Barton WA. Over‐expression of secreted proteins from mammalian cell lines. Protein Sci. 2014;23(5):517-525.

Ho M, Pastan I. Display and selection of scFv antibodies on HEK-293T cells. Methods Mol Biol. 2009;562:99-113.

Apfel J, Reischmann P, Muller O. A new fluorescence-based reporter gene vector as a tool for analyzing and fishing cells with activated wnt signaling pathway. ISRN Oncol. 2013;2013:603129.

Li XL, Zhao YX, Sun LR, Yang J, Xu HJ. The preparation of HL-60 cells vaccine expressing BCG heat shock protein 70 and detection of its immunogenicity in vitro. Hum Vaccin Immunother. 2012;8(10):1376-81.

Chen L, Li G, Tang L, Wang J, Ge XR. The inhibition of lung cancer cell growth by intracellular immunization with LC-1 ScFv. Cell Res. 2002;12:47-54.

Rahimmanesh I, Khanahmad H, Boshtam M, Kouhpayeh S, Hejazi Z. Cell surface display of rabbit MCP1 on human embryonic kidney 293T cell line. IJBT. 2017;16(3):284-288.

Mirian M, Taghizadeh R, Khanahmad H, Salehi M, Jahanian-Najafabadi A, Sadeghi-Aliabadi H, et al. Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression. Res Pharm Sci. 2016;11(5):366-373.

Naderi Beni S, Kouhpayeh S, Hejazi Z, Heidari Hafshejani N, Khanahmad H. Construction and characterization of recombinant HEK cell over expressing alpha4 Integrin. Adv Pharm Bull. 2015;5(3):429-434.

Ebrahimi M, Kazemi T, Ganjalikhani-Hakemi M, Majidi J, Khanahmad H, Rahimmanesh I, et al. Development of a stable cell line, overexpressing human T-cell immunoglobulin mucin 1. Iran J Biotechnol. 2015;13(4):25-31.

Moballegh M, Khanahmad H, Homayouni V, Ganjalikhani H, Salehi M, Rahimmanesh I, et al. Producing recombinant HEK293 T-cells with high expression of T-cell immunoglobulin and mucin domain-3 (TIM3) protein. JIMS. 2015;32(317):2312-2323.

Bruun TH, Grassmann V, Zimmer B, Asbach B, Peterhoff D, Kliche A, et al. Mammalian cell surface display for monoclonal antibody-based FACS selection of viral envelope proteins. MAbs. 2017;9(7):1052-1064.

Bowers PM, Horlick RA, Kehry MR, Neben TY, Tomlinson GL, Altobell L, et al. Mammalian cell display for the discovery and optimization of antibody therapeutics. Methods. 2014;65:44-56.

King DJ, Bowers PM, Kehry MR, Horlick RA. Mammalian cell display and somatic hypermutation in vitro for human antibody discovery. Curr Drug Discov Technol. 2014;11:56-64.


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