Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

Wang,Fa; Zeng,Liang; Wang,Yue‑Li; Cui,Shi‑Quan; Hu,Liang; Zheng,Jun‑Ming; Huang,Di‑Nan; Hou,Gan

doi:10.3892/mmr.2017.6692

Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

Authors:
- Fa Wang
- Liang Zeng
- Yue‑Li Wang
- Shi‑Quan Cui
- Liang Hu
- Jun‑Ming Zheng
- Di‑Nan Huang
- Gan Hou
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Affiliations: Department of Molecular Biology, Guangdong Medical College, Zhanjiang, Guangdong 524023, P.R. China, Department of Clinical Biochemistry, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China
Published online on: June 6, 2017 https://doi.org/10.3892/mmr.2017.6692
Pages: 1021-1030
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to construct a fast-acting, eukaryotic expression vector in eukaryotic cells based on transmembrane-tumor necrosis factor‑α (TM‑TNF‑α) structure. Two types of recombinant eukaryotic expression vectors were constructed, pcDNA3.1-TM-enterokinase-TNF‑α and pcDNA3.1‑TM‑Factor Xa‑TNF‑α, according to the TNF‑α transmembrane segments. Following the generation of these vectors, mouse embryonic 3T3 fibroblasts were transfected and reverse transcription‑polymerase chain reaction and western blotting analyses were used to analyze mTNF‑α mRNA and protein expression levels, respectively, in total cellular protein extracts and extracellular fluid. The biological activity of TNF-α in the extracellular fluid was then measured using an MTT assay. The vectors were successfully constructed, and mRNA and fusion proteins were detected in the 3T3 cells. Among the fusion proteins, the one observed in pcDNA3.1-TM-FactorXa-TNF-α-transfected 3T3 cells remained as a transmembrane protein. In addition, treatment of L929 cells with TNF‑α derived extracellular fluid samples from pcDNA3.1‑TM‑FactorXa‑TNF‑α‑transfected 3T3 cells was associated with a dose‑dependent reduction in in cell‑specific activity. The results indicate that proteins expressed using pcDNA3.1‑TM‑FactorXa‑TNF‑α vectors form transmembrane proteins. In addition, the results indicate that, only when coupled with FactorXa activity, the extracellular region of TM‑TNF‑α forms s‑TNF‑α, and the controlled expression of the fusion protein is initiated.

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1	Itai T, Tanaka M and Nagata S: Processing of tumor necrosis factor by the membrane-bound TNF-alpha-converting enzyme, but not its truncated soluble form. Eur J Biochem. 268:2074–2082. 2001. View Article : Google Scholar : PubMed/NCBI
2	Wang AM, Creasey AA, Ladner MB, Lin LS, Strickler J, van Arsdell JN, Yamamoto R and Mark DF: Molecular cloning of the complementary DNA for human tumor necrosis factor. Science. 228:149–154. 1985. View Article : Google Scholar : PubMed/NCBI
3	Kriegler M, Perez C, DeFay K, Albert I and Lu SD: A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: Ramifications for the complex physiology of TNF. Cell. 53:45–53. 1988. View Article : Google Scholar : PubMed/NCBI
4	Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, et al: A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 385:729–733. 1997. View Article : Google Scholar : PubMed/NCBI
5	Abu-Amer Y, Erdmann J, Alexopoulou L, Kollias G, Ross FP and Teitelbaum SL: Tumor necrosis factor receptors types 1 and 2 differentially regulate osteoclastogenesis. J Biol Chem. 275:27307–27310. 2000.PubMed/NCBI
6	Nagano K, Alles N, Mian AH, Shimoda A, Morimoto N, Tamura Y, Shimokawa H, Akiyoshi K, Ohya K and Aoki K: The tumor necrosis factor type 2 receptor plays a protective role in tumor necrosis factor-alpha-induced bone resorption lacunae on mouse calvariae. J Bone Miner Metab. 29:671–681. 2011. View Article : Google Scholar : PubMed/NCBI
7	Horiuchi T, Mitoma H, Harashima S, Tsukamoto H and Shimoda T: Transmembrane TNF-alpha: Structure, function and interaction with anti-TNF agents. Rheumatology (Oxford). 49:1215–1228. 2010. View Article : Google Scholar : PubMed/NCBI
8	Bjornberg F, Lantz M, Olsson I and Gullberg U: Mechanisms involved in the processing of the p55 and the p75 tumor necrosis factor (TNF) receptors to soluble receptor forms. Lymphokine Cytokine Res. 13:203–211. 1994.PubMed/NCBI
9	Jonasson P, Liljeqvist S, Nygren PA and Ståhl S: Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol Appl Biochem. 35:91–105. 2002. View Article : Google Scholar : PubMed/NCBI
10	Blobel G and Dobberstein B: Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 67:835–851. 1975. View Article : Google Scholar : PubMed/NCBI
11	Pennica D, Nedwin GE, Hayflick JS, Seeburg PH, Derynck R, Palladino MA, Kohr WJ, Aggarwal BB and Goeddel DV: Human tumour necrosis factor: Precursor structure, expression and homology to lymphotoxin. Nature. 312:724–729. 1984. View Article : Google Scholar : PubMed/NCBI
12	Hehlgans T and Pfeffer K: The intriguing biology of the tumour necrosis factor/tumour necrosis factor receptor superfamily: Players, rules and the games. Immunology. 115:1–20. 2005. View Article : Google Scholar : PubMed/NCBI
13	Qin YW, Cheng C, Wang HB, Gao YJ, Shao XY and Shen AG: Role of P38 in LPS induced TNF-α expression in Schwann cells. Chin J Biochemistry Mol Biol. 05:382–387. 2007.(In Chinese).
14	Li YP, Pei YY, Ding J, Shen ZM, Zhang XY, Gu ZH and Zhou JJ: PEGylated recombinant human tumor necrosis factor alpha: Preparation and anti-tumor potency. Acta Pharmacol Sin. 22:549–555. 2001.PubMed/NCBI
15	Mao H: A self-cleavable sortase fusion for one-step purification of free recombinant proteins. Protein Expr Purif. 37:253–263. 2004. View Article : Google Scholar : PubMed/NCBI
16	Kim SO and Lee YI: High-level expression and simple purification of recombinant human insulin-like growth factor I. J Biotechnol. 48:97–105. 1996. View Article : Google Scholar : PubMed/NCBI
17	Singh SM and Panda AK: Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng. 99:303–310. 2005. View Article : Google Scholar : PubMed/NCBI
18	Daly R and Hearn MT: Expression of heterologous proteins in Pichia pastoris: A useful experimental tool in protein engineering and production. J Mol Recognit. 18:119–138. 2005. View Article : Google Scholar : PubMed/NCBI
19	Chaudhuri TK, Horii K, Yoda T, Arai M, Nagata S, Terada TP, Uchiyama H, Ikura T, Tsumoto K, Kataoka H, et al: Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli. J Mol Biol. 285:1179–1194. 1999. View Article : Google Scholar : PubMed/NCBI
20	Chen H, Shaffer PL, Huang X and Rose PE: Rapid screening of membrane protein expression in transiently transfected insect cells. Protein Expr Purif. 88:134–142. 2013. View Article : Google Scholar : PubMed/NCBI
21	Zheng J, Ren F, Yu X, Lai BC and Wang Y: Expression of HPV16E6 protein with insect-baculovirus expression system. J Northwest Univ (Natural Sci Ed). 05:775–778. 2008.(In Chinese).
22	Makadiya N, Brownlie R, van den Hurk J, Berube N, Allan B, Gerdts V and Zakhartchouk A: S1 domain of the procine epidemic diarrhea virus spike protein as a vaccine antigen. Virol J. 13:572016. View Article : Google Scholar : PubMed/NCBI
23	Stock J, Sarkari P, Kreibich S, Brefort T, Feldbrügge M and Schipper K: Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis. J Biotechnol. 161:80–91. 2012. View Article : Google Scholar : PubMed/NCBI
24	Arnau J, Lauritzen C, Petersen GE and Pedersen J: Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif. 48:1–13. 2006. View Article : Google Scholar : PubMed/NCBI
25	Hu J, Qin H, Gao FP and Cross TA: A systematic assessment of mature MBP in membrane protein production: Overexpression, membrane targeting and purification. Protein Expr Purif. 80:34–40. 2011. View Article : Google Scholar : PubMed/NCBI
26	Klein S, Geiger T, Linchevski I, Lebendiker M, Itkin A, Assayag K and Levitzki A: Expression and purification of active PKB kinase from Escherichia coli. Protein Expr Purif. 41:162–169. 2005. View Article : Google Scholar : PubMed/NCBI
27	Leviatan S, Sawada K, Moriyama Y and Nelson N: Combinatorial method for overexpression of membrane proteins in Escherichia coli. J Biol Chem. 285:23548–23556. 2010. View Article : Google Scholar : PubMed/NCBI
28	Kim DJ, Jang HJ, Pyun YR and Kim YS: Cloning, expression, and characterization of thermostable DNA polymerase from Thermoanaerobacter yonseiensis. J Biochem Mol Biol. 35:320–329. 2002.PubMed/NCBI
29	Magliery TJ, Wilson CG, Pan W, Mishler D, Ghosh I, Hamilton AD and Regan L: Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: Scope and mechanism. J Am Chem Soc. 127:146–157. 2005. View Article : Google Scholar : PubMed/NCBI
30	Han S, Li H, Jin Z, Huang D, Ren C and Lin Y: Yeast cell surface display and its application of enzymatic synthesis in non-aqueous phase. Sheng Wu Gong Cheng Xue Bao. 25:1784–1788. 2009.(In Chinese). PubMed/NCBI
31	Jia D, Yang H, Wan L, Cheng J and Lu X: Production of bioactive, SUMO-modified, and native-like TNF-α of the rhesus monkey, Macaca mulatta, in Escherichia coli. Appl Microbiol Biotechnol. 93:2345–2355. 2012. View Article : Google Scholar : PubMed/NCBI
32	Alizadeh AA, Hamzeh-Mivehroud M, Farajzadeh M, Moosavi-Movahedi AA and Dastmalchi S: A simple and rapid method for expression and purification of functional TNF-α using GST fusion system. Curr Pharm Biotechnol. 16:707–715. 2015. View Article : Google Scholar : PubMed/NCBI
33	Evans DH, Willer DO and Yao XD: DNA joining method US Patent 7575860 B2. March 7–2001, issued August 18, 2009.