Cloning, Expression and Characterization of Recombinant Human Fc Receptor Like 1, 2 and 4 Molecules

نویسندگان

Azam Hemmati Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Fazel Shokri Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Hodjatallah Rabbani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Jalal Khoshnoodi Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran

Mahdi Shabani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran and Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran

Mahdi Zandemami Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Zahra Amirghofran Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran

چکیده مقاله:

Background: The Fc receptor like (FCRL) molecules belong to the immunoglobulin (Ig) superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identifid for the human FCRL1, 2 and 4 molecules. Objectives: Cloning, expression, purifiation and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. Materials and Methods: In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b (+) and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fie adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purifid by metal affity chromatography using Ni-NTA resin. Purifid FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specifi polyclonal antibodies. Results: Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b (+) vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37˚C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% (FCRL1) to 25% (FCRL2 and 4) of the total bacterial lysate proteins. Conclusions: These purifid recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specifi mAbs for immunotherapeutic interventions.

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