Selection of single-chain variable fragments specific for Mycobacterium tuberculosis ESAT-6 antigen using ribosome display

Document Type: Original Article


1 Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran


Objective(s): Tuberculosis (TB) is still one of the problematic infectious diseases in developing countries, especially in Iran. In the present study, we applied ribosome display technique to select single chain variable fragments (scFvs) specific for the 6-kDa early secretory antigenic target (ESAT-6) antigen of Mycobacterium tuberculosis from a mouse scFv library.
Materials and Methods: The gene encoding ESAT-6 was cloned into pET22b(+) plasmid and expressed in Escherichia coli BL21 (DE3). The purified recombinant ESAT-6 protein was injected into female BALB/c mice for immunization, and then m-RNA was extracted from the spleen of immunized mice. The anti-ESAT-6 VH/k chain library was assembled by joining of VH and k into the VH/k chain with a 72-bp DNA linker by SOE (splicing by overlap extension) PCR. The scFv library was panned against ESAT-6 using a single round of ribosome display via a rabbit reticulocyte lysate system.
Results: ELISA assay showed that one of the selected scFvs had higher affinity against the recombinant ESAT-6 protein. The affinity of the candidate scFv was ̴ 3.74×108 M-1.
Conclusion: It could be proposed that the isolated scFv in this study may be useful for the diagnosis of TB.


1. Zumla A, George A, Sharma V, Herbert RH, Baroness Masham of I, Oxley A, et al. The WHO 2014 global tuberculosis report--further to go. Lancet Glob Health. 2015; 3:e10-2.

2. Getahun H, Harrington M, O'Brien R, Nunn P. Diagnosis of smear-negative pulmonary tuberculosis in people with HIV infection or AIDS in resource-constrained settings: informing urgent policy changes. Lancet 2007; 369:2042-2049.

3. Mukundan H, Kumar S, Price DN, Ray SM, Lee YJ, Min S, et al. Rapid detection of Mycobacterium tuberculosis biomarkers in a sandwich immunoassay format using a waveguide-based optical biosensor. Tuberculosis (Edinb)  2012; 92:407-416.

4. van Ingen J, de Zwaan R, Dekhuijzen R, Boeree M, van Soolingen D. Region of difference 1 in nontuberculous Mycobacterium species adds a phylogenetic and taxonomical character. J Bacteriol 2009; 191:5865-5867.

5. Ganguly N, Giang PH, Gupta C, Basu SK, Siddiqui I, Salunke DM, et al. Mycobacterium tuberculosis secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex inhibit lipopolysaccharide-induced NF-kappaB transactivation by downregulation of reactive oxidative species (ROS) production. Immunol Cell Biol 2008; 86:98-106.

6. van Pinxteren LA, Ravn P, Agger EM, Pollock J, Andersen P. Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10. Clin Diagn Lab Immunol 2000; 7:155-160.

7. Brodin P, de Jonge MI, Majlessi L, Leclerc C, Nilges M, Cole ST, et al. Functional analysis of early secreted antigenic target-6, the dominant T-cell antigen of Mycobacterium tuberculosis, reveals key residues involved in secretion, complex formation, virulence, and immunogenicity. J Biol Chem 2005; 280:33953-33959.

8. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol 2012; 2012:980250.

9. Nelson AL. Antibody fragments: hope and hype. MAbs 2010; 277-83.

10. Gan R, Jewett MC. Evolution of translation initiation sequences using in vitro yeast ribosome display. Biotechnol Bioeng 2016; 113:1777-1786.

11. Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol 2005; 23:1126-1136.

12. He M, Taussig MJ. Ribosome display of antibodies: expression, specificity and recovery in a eukaryotic system. J Immunol Methods 2005; 297:73-82.

13. Weichhart T, Horky M, Sollner J, Gangl S, Henics T, Nagy E, et al. Functional selection of vaccine candidate peptides from Staphylococcus aureus whole-genome expression libraries in vitro. Infect Immun 2003; 71:4633-4641.

14. Hanes J, Pluckthun A. In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci U S A 1997; 94:4937-4942.

15. Mattheakis LC, Bhatt RR, Dower WJ. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci U S A 1994; 91:9022-9026.

16. Pluckthun A. Ribosome display: a perspective. Methods Mol Biol 2012; 805:3-28.

17. Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, et al. High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol 2004; 22:575-582.

18. He M, Taussig MJ. Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res 1997; 25:5132-5134.

19. Zhou L, Mao WP, Fen J, Liu HY, Wei CJ, Li WX, et al. Selection of scFvs specific for the HepG2 cell line using ribosome display. J Biosci 2009; 34:221-226.

20. Hosseini ES, Moniri R, Goli YD, Kashani HH. Purification of antibacterial CHAPK protein using a self-cleaving fusion tag and its activity against methicillin-resistant staphylococcus aureus. Probiotics Antimicrob Proteins 2016; 8:202-210.

21. Kashani HH, Moniri R. Expression of recombinant pET22b-LysK-cysteine/histidine-dependent amidohydrolase/peptidase bacteriophage thera-peutic protein in escherichia coli BL21 (DE3). Osong Public Health Res Perspect 2015; 6:256-260.

22. Yarian F, Bandehpour M, Seyed N, Kazemi B. Cloning, expression and purification of the factor H binding protein and its interaction with factor H. Iran J Microbiol 2016; 8:29-35.

23. Schmelcher M, Powell AM, Becker SC, Camp MJ, Donovan DM. Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands. Appl Environ Microbiol 2012; 78:2297-2305.

24. Lee MS, Kwon MH, Kim KH, Shin HJ, Park S, Kim HI. Selection of scFvs specific for HBV DNA polymerase using ribosome display. J Immunol Methods 2004; 284:147-157.

25. Azizi A, Arora A, Markiv A, Lampe DJ, Miller TA, Kang AS. Ribosome display of combinatorial antibody libraries derived from mice immunized with heat-killed Xylella fastidiosa and the selection of MopB-specific single-chain antibodies. Appl Environ Microbiol 2012; 78:2638-26347.

26. Luo Y, Xia Y. Selection of single-chain variable fragment antibodies against fenitrothion by ribosome display. Anal Biochem 2012; 421:130-137.

27. Sankian M, Yousefi M, Pazouki N, Varasteh A. One-step purification of histidine-tagged profilin with high purity and yield by using metal precipitation. Biotechnol Appl Biochem 2007; 47:185-189.

28. Beatty JD, Beatty BG, Vlahos WG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods 1987; 100:173-179.

29. Ireton GC, Greenwald R, Liang H, Esfandiari J, Lyashchenko KP, Reed SG. Identification of Mycobacterium tuberculosis antigens of high serodiagnostic value. Clin Vaccine Immunol 2010; 17:1539-1547.

30. Sreejit G, Ahmed A, Parveen N, Jha V, Valluri VL, Ghosh S, et al. The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (beta2M) affecting antigen presentation function of macrophage. PLoS Pathog 2014; 10:e1004446.

31. Ohashi H, Shimizu Y, Ying BW, Ueda T. Efficient protein selection based on ribosome display system with purified components. Biochem Biophys Res Commun 2007; 352:270-276.

32. Qing Yuan YX, Siji Nian, Youping Yin, Yueqing Cao, Wang Z. Selection of single chain fragments against the phytopathogen Xanthomonas axonopodis pv. citri by ribosome display. Enzyme and Microbial Technol 2007; 41:383–389.

33. Zhao XL, Chen WQ, Yang ZH, Li JM, Zhang SJ, Tian LF. Selection and affinity maturation of human antibodies against rabies virus from a scFv gene library using ribosome display. J Biotechnol 2009; 144:253-258.

34. Feng F, Zhang H, Zhu Z, Li C, Shi Y, Zhang Z. The application of anti-ESAT-6 monoclonal antibody fluorescent probe in ex vivo near-infrared fluorescence imaging in mice with pulmonary tuberculosis. Luminescence 2014; 29:614-620.

35. Feng TT, Shou CM, Shen L, Qian Y, Wu ZG, Fan J,   et al. Novel monoclonal antibodies to ESAT-6 and CFP-10 antigens for ELISA-based diagnosis of pleural tuberculosis. Int J Tuberc Lung Dis 2011; 15:804-810.

36. Leng J, Ding Y, Shou C, Wu Z, Zhuo G, Wang K, et al. Development of a novel anti ESAT-6 monoclonal antibody for screening of Mycobacterium tuberculosis. Int J Clin Exp Med 2014;7:4238-4243.