High-level Expression and One-step Purification of Chimeric Antigen Containing HTLV-I-II Diagnostic Epitopes in Escherichia coli
Purification and preparation of three diagnostic antigens used for the detection of human T-lymphotropic virus (HTLV)-I/-II infection in E.coli are different parts of a multi-step method. In this study, our aim was to design a chimeric protein for the simultaneous detection of HTLV-I and HTLV-II antibodies. Immunodominant B cell linear epitopes of envelope and capsid proteins of HTLV-I/-II were selected and linked together; using a suitable amino acid linker and a chimeric antigen (CA). The codon-optimized synthetic DNA encoding the CA was subcloned into the pGS21aexpression vector and CA expressed as His-GST fused protein in E. coli BL21 (DE3) cells. Then the recombinant CA was purified, using the Ni-NTA (Nickle Nitrilotriacetic acid) affinity chromatography under native conditions. The Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric scanning results showed that CA accounted for 15% of the total cellular proteins and approximately 50% of the expressed histidine-glutathione s-transferase-chimeric antigen (His-GST-CA) proteins were soluble. The CA was successfully purified in one step with a purity of greater than 90%, which is suitable for antigenicity evaluations. Enzyme-linked immunosorbent assay (ELISA) results showed that the GST fused CA reacted in a concentration-dependent manner with HTLV-I/-II infected sera and was able to distinguish normal serum from HTLV-I/-II infected one with a proper sensitivity. With further validation, CA, as described in the present study could be introduced as a novel reliable, cost-effective and easy alternative for the three separate HTLV-I/-II diagnostic peptide antigens, which is prepared as a fusion with GST.
2. Verdonck K, Gonzalez E, Van Dooren S, Vandamme AM, Vanham G, Gotuzzo E. Human T-lymphotropic virus 1: recent knowledge about an ancient infection. The Lancet Infect Dis. 2007;7(4):266-81.
3. Thorstensson R, Albert J, Andersson S. Strategies for diagnosis of HTLV-I and -II. Transfusion. 2002;42(6):780-91.
4. Lipka JJ, Miyoshi I, Hadlock KG, Reyes GR, Chow TP, Blattner WA, et al. Segregation of human T cell lymphotropic virus type I and II infections by antibody reactivity to unique viral epitopes. J Infect Dis. 1992;165(2):268-72.
5. Heydari Zarnagh H, Hassanpour K, Rasaee MJ. Constructing Chimeric Antigen for Precise Screening of HTLV-I Infection. Iran J Allergy Asthma Immunol. 2015;14(4):427-36.
6. Singh SM, Panda AK. Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng. 2005;99(4):303-10.
7. Mosadeghi P, Zarnagh HH, Mohammad-Zadeh M, Salehi Moghaddam M. High-Level Soluble Expression and One-step Purification of HTLV-I P19 Protein in Escherichia coli by Fusion Expression. Iran J Allergy Asthma Immunol. 2015;14(6):624-32.
8. Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988;67(1):31-40.
9. Guan KL, Dixon JE. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991;192(2):262-7.
10. Hernandez Marin M, Castellanos Penton P, Marquez Bocalandro Y, Pozo Pena L, et al. Chimeric synthetic peptides containing two immunodominant epitopes from the envelope gp46 and the transmembrane gp21 glycoproteins of HTLV-I virus. Biochem Biophysi Res Commun. 2001;289(1):1-6.
11. Hernandez Marin M, Marquez Bocalandro Y, Vallejo RV, Rodriguez Tanty C, Higginson Clark D, Pozo Pena L, et al. Use of a chimeric synthetic peptide from the core p19 protein and the envelope gp46 glycoprotein in the immunodiagnosis of HTLV-II virus infection. Prepar Biochem Biotech. 2003;33(1):29-38.
12. Mosadeghi P, Heydari-Zarnagh H. Development and Evaluation of a Novel ELISA for Detection of Antibodies against HTLV-I Using Chimeric Peptides. Iran J Allergy Asthma Immunol. 2018;17(2):144-50.
13. Marin MH, Rodríguez-Tanty C, Higginson-Clarke D, Bocalandro YM, Peña LP. Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus. Biochem Biophysi Res Commun. 2005;336(3):983-6.
14. Heydari Zarnagh H, Ravanshad M, Pourfatollah AA, Rasaee MJ. Expression and Purification of a Novel Computationally Designed Antigen for Simultaneously Detection of HTLV-1 and HBV Antibodies. Iran J Allergy Asthma Immunol. 2015;14(2):168-78.
15. Malhotra A. Tagging for protein expression. Methods Enzymol. 2009;463:239-58.
16. Takakura Y, Katayama S, Nagata Y. High-level expression of tamavidin 2 in human cells by codon-usage optimization. Biosci Biotech Biochem. 2015;79(4):610-6.
17. Deml L, Bojak A, Steck S, Graf M, Wild J, Schirmbeck R, et al. Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol. 2001;75(22):10991-1001.
18. Yu K, Ang KS, Lee DY. Synthetic Gene Design Using Codon Optimization On-Line (COOL). Methods Mol Biol. 2017;1472:13-34.
19. Cho HJ, Oh BM, Kim JT, Lim J, Park SY, Hwang YS, et al. Efficient interleukin-21 production by optimization of codon and signal peptide in Chinese hamster ovarian cells. J Microbiol Biotechnol. 2018.
20. San-Miguel T, Perez-Bermudez P, Gavidia I. Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature. Springerplus. 2013;2(1):89.
21. Li J, Zhang WB, McManus DP. Recombinant antigens for immunodiagnosis of cystic echinococcosis. Biol Proced Online. 2004;6:67-77.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.