Volume 25, Issue 6 (11-2021)                   IBJ 2021, 25(6): 390-398 | Back to browse issues page

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Karam S, Raigani M, Hassani Afshar S, Talebkhan Y, Bayat E, Komijani S, et al . Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode. IBJ 2021; 25 (6) :390-398
URL: http://ibj.pasteur.ac.ir/article-1-3400-en.html
Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode
Saba Karam , Mozhgan Raigani , Sahar Hassani Afshar , Yeganeh Talebkhan , Elham Bayat , Samira Komijani , Leila Nematollahi , Farzaneh Barkhordari , Mehdi Shafiee Ardestani , Fatemeh Davami *
Abstract:  
Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is type III secretion system (T3SS). PcrV is an important structural protein of the T3SS. Methods: In the current investigation, a recombinant single-chain fragment variable (scFv) mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results: Increased efficiency was achieved by EnBase® compared to Luria–Bertani broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 μg/mL. Conclusion: Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).
Keywords: Fed Batch, recombinant protein, Pseudomonas aeruginosa, scFv
Full-Text [PDF 1085 kb]      
Type of Study: Full Length/Original Article | Subject: Medical Biotechnology
References
1. Yang L, Jelsbak L, Marvig RL, Damkiaer S, Workman CT, Rau MH, Hansen SK, Folkesson A, Johansen HK, Ciofu O, Hoiby N, Sommer MO, Molin S. Evolutionary dynamics of bacteria in a human host environment. Proceedings of the national academy of sciences of the United States of America 2011; 108(18): 7481-7486. [DOI:10.1073/pnas.1018249108]
2. Botzenhart K, Döring G. Ecology and epidemiology of Pseudomonas aeruginosa. In: Campa M, Friedman H, editors. Pseudomonas aeruginosa as an Opportuistic Pathogen Infectious Agents and Pathogenesis. Boston: Springer; 2018.
3. Steadman R, Heck LW, Abrahamson DR. The Role of Proteases in the Pathogenesis of Pseudomonas aeruginosa Infections. In: Campa M, Bendinelli M, Friedman H., editors. Pseudomonas aeruginosa as an Opportuistic Pathogen Infectious Agents and Pathogenesis. Boston: Springer; 1993. [DOI:10.1007/978-1-4615-3036-7_7]
4. Fuentefria DB, Ferreira AE, Corcao G. Antibiotic-resistant Pseudomonas aeruginosa from hospital wastewater and superficial water: are they genetically related? Journal of environmental management 2011; 92(1): 250-255. [DOI:10.1016/j.jenvman.2010.09.001]
5. Tkaczyk C, Hua L, Varkey R, Shi Y, Dettinger L, Woods R, Barnes A, MacGill RS, Wilson S, Chowdhury P, Stover CK, Sellman BR. Identification of anti-alpha toxin monoclonal antibodies that reduce the severity of Staphylococcus aureus dermonecrosis and exhibit a correlation between affinity and potency. Clinical and vaccine immunology 2012; 19(3): 377-385. [DOI:10.1128/CVI.05589-11]
6. Hueck CJ. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiology and molecular biology reviews 1998; 62(2): 379-433. [DOI:10.1128/MMBR.62.2.379-433.1998]
7. Sawa T, Ito E, Nguyen VH, Haight M. Anti-PcrV antibody strategies against virulent Pseudomonas aeruginosa. Human vaccines and immunotherapeutics 2014; 10(10): 2843-2852. [DOI:10.4161/21645515.2014.971641]
8. Nanao M, Ricard-Blum S, Di Guilmi AM, Lemaire D, Lascoux D, Chabert J, Attree I, Dessen A. Type III secretion proteins PcrV and PcrG from Pseudomonas aeruginosa form a 1:1 complex through high affinity interactions. BMC microbiology 2003; 3: 21. [DOI:10.1186/1471-2180-3-21]
9. Cordes FS, Komoriya K, Larquet E, Yang S, Egelman EH, Blocker A, Lea SM. Helical structure of the needle of the type III secretion system of Shigella flexneri. The journal of biological chemistry 2003; 278(19): 17103-17107. [DOI:10.1074/jbc.M300091200]
10. Dacheux D, Goure J, Chabert J, Usson Y, Attree I. Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages. Molecular microbiology 2001; 40(1): 76-85. [DOI:10.1046/j.1365-2958.2001.02368.x]
11. Faure K, Fujimoto J, Shimabukuro DW, Ajayi T, Shime N, Moriyama K, Spack EG, Wiener-Kronish JP, Sawa T. Effects of monoclonal anti-PcrV antibody on Pseudomonas aeruginosa-induced acute lung injury in a rat model. Journal of immune based therapies and vaccines 2003; 1(1): 2. [DOI:10.1186/1476-8518-1-2]
12. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clinical and developmental immunology 2012; 2012: 980250. [DOI:10.1155/2012/980250]
13. Baer M, Sawa T, Flynn P, Luehrsen K, Martinez D, Wiener-Kronish JP, Yarranton G, Bebbington C. An engineered human antibody fab fragment specific for Pseudomonas aeruginosa PcrV antigen has potent antibacterial activity. Infection and immunity 2009; 77(3): 1083-1090. [DOI:10.1128/IAI.00815-08]
14. Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of molecular biology 1986; 189(1): 113-130. [DOI:10.1016/0022-2836(86)90385-2]
15. Ikura K, Kokubu T, Natsuka S, Ichikawa A, Adachi M, Nishihara K, Yanagi H, Utsumi S. Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli. Preparative biochemistry and biotechnology 2002; 32(2): 189-205. [DOI:10.1081/PB-120004130]
16. Berrow NS, Bussow K, Coutard B, Diprose J, Ekberg M, Folkers GE, Levy N, Lieu V, Owens RJ, Peleg Y, Pinaglia C, Quevillon-Cheruel S, Salim L, Scheich C, Vincentelli R, Busso D. Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta crystallographica section D biological crystallography 2006; 62(Pt 10): 1218-1226. [DOI:10.1107/S0907444906031337]
17. Pryor KD, Leiting B. High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding-protein double-affinity fusion system. Protein expression and purification 1997; 10(3): 309-319. [DOI:10.1006/prep.1997.0759]
18. Namvar S, Barkhordari F, Raigani M, Jahandar H, Nematollahi L, Davami F. Cloning and soluble expression of mature α-luffin from Luffa cylindrica in E. coli using SUMO fusion protein. Turkish journal of biology 2018; 42: 23-32. [DOI:10.3906/biy-1708-12]
19. Bird LE. High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli. Methods 2011; 55(1): 29-37. [DOI:10.1016/j.ymeth.2011.08.002]
20. Lee CD, Sun HC, Hu SM, Chiu CF, Homhuan A, Liang S-M, Leng C-H, Wang T-F. An improved SUMO fusion protein system for effective production of native proteins. Protein science 2008; 17(7): 1241-1248. [DOI:10.1110/ps.035188.108]
21. Panula-Perälä J, Šiurkus J, Vasala A, Wilmanowski R, Casteleijn MG, Neubauer P. Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks. Microbial cell factories 2008; 7(1): 31. [DOI:10.1186/1475-2859-7-31]
22. Krause M, Ukkonen K, Haataja T, Ruottinen M, Glumoff T, Neubauer A, Neubauer P, Vasala A. A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microbial cell factories 2010; 9: 11. [DOI:10.1186/1475-2859-9-11]
23. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259): 680-685. [DOI:10.1038/227680a0]
24. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. (second ed.). N.Y.: Cold Spring Harbor, 1989.
25. Warrener P, Varkey R, Bonnell JC, DiGiandomenico A, Camara M, Cook K, Peng L, Zha J, Chowdury P, Sellman B, Stover CK. A novel anti-PcrV antibody providing enhanced protection against Pseudomonas aeruginosa in multiple animal infection models. Antimicrobial agents and chemotherapy 2014; 58(8): 4384-4391. [DOI:10.1128/AAC.02643-14]
26. KÖHler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256(5517): 495-497. [DOI:10.1038/256495a0]
27. Ying T, Chen W, Feng Y, Wang Y, Gong R, Dimitrov DS. Engineered soluble monomeric IgG1 CH3 domain: generation, mechanisms of function, and implications for design of biological therapeutics. The journal of biological chemistry 2013; 288(35): 25154-25164. [DOI:10.1074/jbc.M113.484154]
28. Hua Q, Yang C, Oshima T, Mori H, Shimizu K. Analysis of gene expression in Escherichia coli in response to changes of growth-limiting nutrient in chemostat cultures. Applied and environmental microbiology 2004; 70(4): 2354-2366. [DOI:10.1128/AEM.70.4.2354-2366.2004]
29. Fernandez LA, Sola I, Enjuanes L, de Lorenzo V. Specific secretion of active single-chain Fv antibodies into the supernatants of Escherichia coli cultures by use of the hemolysin system. Applied and environmental microbiology 2000; 66(11): 5024. [DOI:10.1128/AEM.66.11.5024-5029.2000]
30. Barkhordari F, Raigani M, Garoosi YT, Mahboudi F, Davami F. Optimization of EnBase fed-batch cultivation to improve soluble fraction ratio of alpha-luffin ribosome inactivating protein. Iranian journal of biotechnology 2018; 16(1): e1482. [DOI:10.21859/ijb.1482]
31. Yun HS, Hong J, Lim HC. Regulation of ribosome synthesis in Escherichia coli: effects of temperature and dilution rate changes. Biotechnology bioengineering 1996; 52(5): 615-624. https://doi.org/10.1002/(SICI)1097-0290(19961205)52:5<615::AID-BIT9>3.0.CO;2-M [DOI:10.1002/(SICI)1097-0290(19961205)52:53.0.CO;2-M]
32. Secher T, Fas S, Fauconnier L, Mathieu M, Rutschi O, Ryffel B, Rudolf M. The anti-Pseudomonas aeruginosa antibody Panobacumab is efficacious on acute pneumonia in neutropenic mice and has additive effects with meropenem. PloS one 2013; 8(9): e73396. [DOI:10.1371/journal.pone.0073396]
33. Ali SO, Yu XQ, Robbie GJ, Wu Y, Shoemaker K, Yu L, DiGiandomenico A, Keller AE, Anude C, Hernandez-Illas M. Phase 1 study of MEDI3902, an investigational anti-Pseudomonas aeruginosa PcrV and Psl bispecific human monoclonal antibody, in healthy adults. Clinical microbiology and infection 2019; 25(5): 629. [DOI:10.1016/j.cmi.2018.08.004]
34. Goure J, Broz P, Attree O, Cornelis GR, Attree I. Protective anti-V antibodies inhibit Pseudomonas and Yersinia translocon assembly within host membranes. Journal of infectious diseases 2005; 192(2): 218-225. [DOI:10.1086/430932]
35. Frank DW, Vallis A, Wiener-Kronish JP, Roy-Burman A, Spack EG, Mullaney BP, Megdoud M, Marks JD, Fritz R, Sawa T. Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV. The Journal of infectious diseases 2002; 186(1): 64-73. [DOI:10.1086/341069]
36. Rothlisberger D, Honegger A, Pluckthun A. Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. Journal of molecular biology 2005; 347(4): 773-789. [DOI:10.1016/j.jmb.2005.01.053]
37. Singh P. Surface plasmon resonance (SPR) based binding studies of refolded single chain antibody fragments. Biochemistry and biophysics reports 2018; 14: 83-88. [DOI:10.1016/j.bbrep.2018.04.005]
38. Maruta Y, Kuwata T, Tanaka K, Alam M, Valdez KP, Egami Y, Suwa Y, Morioka H, Matsushita S. Cross-neutralization activity of single-chain variable fragment (scFv) derived from anti-V3 monoclonal antibodies mediated by post-attachment binding. Japanese journal of infectious diseases 2016; 69(5): 395-404. [DOI:10.7883/yoken.JJID.2015.667]
39. Qi JY, Ye XL, Ren GP, Kan FM, Zhang Y, Guo M, Zhang ZY, Li DS. Pharmacological efficacy of anti-IL-1 beta scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis. Molecular immunology 2014; 57(2): 59-65. [DOI:10.1016/j.molimm.2013.08.002]
Add your comments about this article : Your username or Email:
CAPTCHA
Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2024 CC BY-NC 4.0 | Iranian Biomedical Journal

Designed & Developed by : Yektaweb