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

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