Cloning, expression and characterization of recombinant human Fc receptor like 1, 2 and 4 molecules

ABSTRACT

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 identified for the human FCRL1, 2 and 4 molecules. Cloning, expression, purification and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. 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 fine adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purified by metal affinity chromatography using Ni-NTA resin. Purified FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specific polyclonal antibodies. 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[degree]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. These purified recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specific mAbs for immunotherapeutic interventions.