An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase

Hadi Bakhtiari , Abbas Ali Palizban, Hossein Khanahmad, Mohammad Reza Mofid

Abstract


Background and purpose: Aspartyl/asparaginyl β-hydroxylase (ASPH) is abundantly expressed in malignant neoplastic cells. The establishment of a human cell line overexpressing ASPH could provide the native-like recombinant protein needed for developing theranostic probes. In the process of transfection, the obtained cells normally contain a range of cells expressing the different levels of the target of interest. In this paper, we report on our simple innovative approach in the selection of best-transfected cells with the highest expression of ASPH using subclone selection, quantitative real-time polymerase chain reaction, and gradual increment of hygromycin concentration.

Experimental approach: To achieve this goal, human embryonic kidney (HEK 293T) cells were transfected with an ASPH-bearing pcDNA3.1/Hygro(+) vector. During antibiotic selection, single accumulations of the resistant cells were separately cultured and the ASPH mRNA levels of each flask were evaluated. The best subclones were treated with a gradually increasing amount of hygromycin. The ASPH protein expression of the obtained cells was finally evaluated using flow cytometry and immunocytochemistry.

Findings / Results: The results showed that different selected subclones expressed different levels of ASPH. Furthermore, the gradual increment of hygromycin (up to 400mg/mL) improved the expression of ASPH. The best relative fold change in mRNA levels was 57.59 ± 4.11. Approximately 90.2% of HEKASPH cells overexpressed ASPH on their surface.

Conclusion and implications: The experiments indicated that we have successfully constructed and evaluated a recombinant human cell line overexpressing ASPH on the surface. Moreover, our innovative selection approach provided an effective procedure for enriching highly expressing recombinant cells.

 

 


Keywords


Keywords: Cancer; Aspartyl/asparaginyl β-hydroxylase; Human embryonic kidney (HEK293T) cell line; Transfection; Overexpression.

Full Text:

PDF

References


Suzanne M, Tamaki S, Cantarini MC, Ince N, Wiedmann M, Carter JJ, et al. Aspartyl-(asparaginyl)-β-hydroxylase regulates hepatocellular carcinoma invasiveness. J Hepatol. 2006;44(5): 971-983.

DOI: 10.1016/j.jhep.2006.01.038.

Xian ZH, Zhang SH, Cong WM, Yan HX, Wang K, Wu MC. Expression of aspartyl beta-hydroxylase and its clinicopathological significance in hepatocellular carcinoma. Mod Pathol. 2006;19(2):280-286.

DOI: 10.1038/modpathol.3800530.

Nagaoka K, Bai X, Ogawa K, Dong X, Zhang S, Zhou Y, et al. Anti-tumor activity of antibody drug conjugate targeting aspartate-β-hydroxylase in pancreatic ductal adenocarcinoma. Cancer Lett. 2019;449:87-98.

DOI: 10.1016/j.canlet.2019.02.006.

Jain KK. The handbook of biomarkers. 1st ed. Springer; 2010. pp: 200.

Huyan T, Li Q, Dong DD, Yang H, Xue XP, Huang QS. Development of a novel anti-human aspartyl-(asparaginyl) β-hydroxylase monoclonal antibody with diagnostic and therapeutic potential. Oncol Lett. 2017;13(3):1539-1546.

DOI: 10.3892/ol.2017.5642.

Hou G, Xu B, Bi Y, Wu C, Ru B, Sun B, et al. Recent advances in research on aspartate β-hydroxylase (ASPH) in pancreatic cancer: a brief update. Bosn J Basic Med Sci. 2018;18(4):297-304.

DOI: 10.17305/bjbms.2018.3539.

Yin J, Li G, Ren X, Herrler G. Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol. 2007;127(3):335-347.

DOI: 10.1016/j.jbiotec.2006.07.012.

Dehaghani SA, Babaeipour V, Mofid MR, Divsalar A, Faraji F. An efficient purification method for high recovery of recombinant human granulocyte colony stimulating factor from recombinant E. coli. Int J Environ Sci Dev. 2010;1(2):111-114.

DOI: 10.7763/IJESD.2010.V1.2.

Kubick S, Gerrits M, Merk H, Stiege W, Erdmann VA. Chapter 2 In vitro synthesis of posttranslationally modified membrane proteins. Curr Top Membr. 2009;63:25-49.

DOI: 10.1016/S1063-5823(09)63002-7.

DeMaria CT, Cairns V, Schwarz C, Zhang J, Guerin M, Zuena E, et al. Accelerated clone selection for recombinant CHO cells using a FACS‐based high‐throughput screen. Biotechnol Prog. 2007;23(2):465-472.

DOI: 10.1021/bp060298i.

Brezinsky SCG, Chiang GG, Szilvasi A, Mohan S, Shapiro RI, MacLean A, et al. A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J Immunol Methods. 2003;277(1-2):141-155.

DOI: 10.1016/s0022-1759(03)00108-x.

Carroll S, Al-Rubeai M. The selection of high-producing cell lines using flow cytometry and cell sorting. Expert Opin Biol Ther. 2004;4(11):1821-1829.

DOI: 10.1517/14712598.4.11.1821.

Yoshikawa T, Nakanishi F, Ogura Y, Oi D, Omasa T, Katakura Y, et al. Flow cytometry: an improved method for the selection of highly productive gene‐amplified CHO cells using flow cytometry. Biotechnol Bioeng. 2001;74(5):435-442.

DOI: 10.1002/bit.1134.

Bailey CG, Tait AS, Sunstrom NA. High‐throughput clonal selection of recombinant CHO cells using a dominant selectable and amplifiable metallothionein‐GFP fusion protein. Biotechnol Bioeng. 2002;80(6):670-676.

DOI: 10.1002/bit.10424.

Meng YG, Liang J, Wong WL, Chisholm V. Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene. 2000;242(1-2):201-207.

DOI: 10.1016/s0378-1119(99)00524-7.

Fatahi A, Rahimmanesh I, Mirian M, Rohani F, Boshtam M, Gheibi A, et al. Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin. Res Pharm Sci. 2018;13(4):353-359.

DOI: 10.4103/1735-5362.235162.

Gheysarzadeh A, Bakhtiari H, Ansari A, Emami Razavi A, Emami MH, Mofid MR. The insulin‐like growth factor binding protein‐3 and its death receptor in pancreatic ductal adenocarcinoma poor prognosis. J Cell Physiol. 2019;234(12):23537-23546.

DOI: 10.1002/jcp.28922.

Dinarvand N, Khanahmad H, Hakimian SM, Sheikhi A, Rashidi B, Bakhtiari H, et al. Expression and clinicopathological significance of lipin‐1 in human breast cancer and its association with p53 tumor suppressor gene. J Cell Physiol. 2020;235(7-8):5835-5846.

DOI: 10.1002/jcp.29523.

Jafari S, Babaeipour V, Eslampanah Seyedi HA, Rahaie M, Mofid M, Haddad L, et al. Recombinant production of mecasermin in E. coli expression system. Res Pharm Sci. 2014;9(6):453-461.

Lin Q, Chen X, Meng F, Ogawa K, Li M, Song R, et al. ASPH-notch axis guided exosomal delivery of prometastatic secretome renders breast cancer multi-organ metastasis. Mol Cancer. 2019;18(156):1-17.

DOI: 10.1186/s12943-019-1077-0.

Bakhtiari H, Palizban AA, Khanahmad H, Mofid MR. Aptamer-based approaches for in vitro molecular detection of cancer. Res Pharm Sci. 2020;15(2):107-122.

Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Anal Bioanal Chem. 2010;397(8):3173-3178.

DOI: 10.1007/s00216-010-3821-6.

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.

DOI: 10.4103/1735-5362.192485.

Boshtam M, Asgary S, Rahimmanesh I, Kouhpayeh S, Naderi J, Hejazi Z, et al. Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface. Res Pharm Sci. 2018;13(5):430-439.

DOI: 10.4103/1735-5362.236836.

Chen D, Sun D, Wang Z, Qin W, Chen L, Zhou L, et al. A DNA nanostructured aptasensor for the sensitive electrochemical detection of HepG2 cells based on multibranched hybridization chain reaction amplification strategy. Biosens Bioelectron. 2018;117:416-421.

DOI: 10.1016/j.bios.2018.06.041.

Tan W, Fang X. Aptamers selected by cell-SELEX for theranostics. 1st ed. Springer; 2015. pp: 31-33.

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

DOI: 10.1002/pro.2439.


Refbacks

  • 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.