Journal of Isfahan Medical School

Journal of Isfahan Medical School

Optimization of Production and Characterization of a Tandem Single Chain Fragment Variable (taFv) against Human Leptin Receptor and Anti-Human CD4

Document Type : Original Article (s)

Authors
Leila Momajadi 1
Sayyed Hamid Zarkesh-Esfahani 2
Mohammad Rabbani 2
Rahman Emamzadeh 3
Forouzan Safari 1
Arash Babaei 4
1 Department of Biology, School of Science, University of Isfahan, Isfahan, Iran
2 Associate Professor, Department of Biology, School of Science, University of Isfahan, Isfahan, Iran
3 Assistant Professor, Department of Biology, School of Science, University of Isfahan, Isfahan, Iran
4 Assistant Professor, Department of Biology, School of Science, University of Malayer, Malayer, Iran
Abstract
Background: Leptin deficiency or dysfunction in leptin receptor (ObR) signaling may tend to resistance to autoimmune diseases. On the other hand, leptin receptors exist on many cells and therefore blocking all of them will probably result in unfavorable effects. Targeted ObR blocking on specific immune cells with a leptin antagonist such as taFv (Tandem single chain fragment variable or Tandem scFv) may be advantageous for patients with autoimmune diseases. This project aimed to optimize the condition for large scale production of such molecule and to test its effect.Methods: The cloned taFv gene was sub-cloned from pAB1 to pET32a vector. The taFv fragment existence in pET32a vector was confirmed via polymerase chain reaction (PCR) method using T7 primers. Dot blotting was recruited to detect protein expression. Optimization experiments were carried out and assayed using enzyme-linked immunosorbent assay (ELISA). Finally, the functional activity was evaluated via flow cytometry.Findings: The result of PCR confirmed integration of taFv 2300 bp gene fragment in pET32a vector. Dot blotting confirmed taFv higher expression in pET32a vector compared to previous vector. It was found that media containing sorbitol, Escherichia coli BL21 strain, IPTG 0.05 mM and 18˚ C temperatures were resulted in higher production of protein levels. Based on flow cytometry, taFv was able to attach to 20% of lymphocytes.Conclusion: pET32a vector with pel B fragment is suitable for secretory overexpression. Production of taFv could be enhanced via optimizing media and culture conditions.
Keywords
Leptin
Autoimmune disease
Optimization
Bispecific antibody
  1. Zhang F, Basinski MB, Beals JM, Briggs SL, Churgay LM, Clawson DK, et al. Crystal structure of the obese protein leptin-E100. Nature 1997; 387(6629): 206-9.
  2. Babaei A, Zarkesh-Esfahani SH, Bahrami E, Ross RJ. Restricted leptin antagonism as a therapeutic approach to treatment of autoimmune diseases. Hormones (Athens ) 2011; 10(1): 16-26.
  3. Ahima RS, Osei SY. Leptin signaling. Physiol Behav 2004; 81(2): 223-41.
  4. Haghjooy Javanmard Sh, Khorshidi Behzadi M, Amjadi F, Khazaei M, Zarkesh Esfahani H. Leptin enhances melanoma tumor growth by increasing endothelial progenitor cells. J Isfahan Med Sch 2012; 29(170): 2653-61. [In Persian].
  5. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998; 395(6704): 763-70.
  6. Massaer M, Mazzu P, Haumont M, Magi M, Daminet V, Bollen A, et al. High-level expression in mammalian cells of recombinant house dust mite allergen ProDer p 1 with optimized codon usage. Int Arch Allergy Immunol 2001; 125(1): 32-43.
  7. Meier U, Gressner AM. Endocrine regulation of energy metabolism: review of pathobiochemical and clinical chemical aspects of leptin, ghrelin, adiponectin, and resistin. Clin Chem 2004; 50(9): 1511-25.
  8. Fleet JC. Leptin and bone: does the brain control bone biology? Nutr Rev 2000; 58(7): 209-11.
  9. Faggioni R, Jones-Carson J, Reed DA, Dinarello CA, Feingold KR, Grunfeld C, et al. Leptin-deficient (ob/ob) mice are protected from T cell-mediated hepatotoxicity: role of tumor necrosis factor alpha and IL-18. Proc Natl Acad Sci U S A 2000; 97(5): 2367-72.
  10. Chandra RK. Cell-mediated immunity in genetically obese C57BL/6J ob/ob) mice. Am J Clin Nutr 1980; 33(1): 13-6.
  11. Sanna V, Di GA, La CA, Lechler RI, Fontana S, Zappacosta S, et al. Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses. J Clin Invest 2003; 111(2): 241-50.
  12. Li C, Friedman JM. Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction. Proc Natl Acad Sci U S A 1999; 96(17): 9677-82.
  13. Carmen S, Jermutus L. Concepts in antibody phage display. Brief Funct Genomic Proteomic 2002; 1(2): 189-203.
  14. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A 1988; 85(16): 5879-83.
  15. Campbell AM. Monoclonal antibody and immunosensor technology. The production and application of rodent and human monoclonal antibodies. Philadelphia, PA: Elsevier; 1991.
  16. Babaei A. Production and manipulation of a recombinant bispecific antibody which binds simultaneously to leptin receptor and one of the T lymphocytes CD markers in order to modulate the immune response [PhD Thesis]. Isfahan, Iran: University of Isfahan; 2010.
  17. Zarkesh-Esfahani SH, Etemadifar Z. The principle of flow cytometry and its application in biological sciences. 1st ed. Isfahan, Iran: Isfahan University Publications; 2010. p. 179. [In Persian].
  18. Matarese G, Di GA, Sanna V, Lord GM, Howard JK, Di TA, et al. Requirement for leptin in the induction and progression of autoimmune encephalomyelitis. J Immunol 2001; 166(10): 5909-16.
  19. Zwick MB, Labrijn AF, Wang M, Spenlehauer C, Saphire EO, Binley JM, et al. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J Virol 2001; 75(22): 10892-905.
  20. Yang T, Yang L, Chai W, Li R, Xie J, Niu B. A strategy for high-level expression of a single-chain variable fragment against TNFalpha by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220. Protein Expr Purif 2011; 76(1): 109-14.
  21. Arbabi-Ghahroudi M, Tanha J, MacKenzie R. Prokaryotic expression of antibodies. Cancer Metastasis Rev 2005; 24(4): 501-19.
  22. Humphreys DP, Sehdev M, Chapman AP, Ganesh R, Smith BJ, King LM, et al. High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence. Protein Expr Purif 2000; 20(2): 252-64.
  23. Jurado P, Ritz D, Beckwith J, de L, V, Fernandez LA. Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli. J Mol Biol 2002; 320(1): 1-10.
Journal of Isfahan Medical School
Volume 33, Issue 355 - Serial Number 355
September and October 2015
Pages 1740-1751

  • Receive Date 12 May 2015
  • Accept Date 06 April 2022