Production and characterization of recombinant light chain and carboxyterminal heavy chain fragments of tetanus toxin

Light chain [LC] and heavy chain Carboxyterminal subdomain [Hoc] fragments are the most important parts of tetanus neurotoxin [TeNT] which play hey roles in toxicity and binding of TeNT, respectively. In the present study, these two fragments were cloned and expressed in a proharyotic system and their identity was confirmed using anti-TeNT specific polyclonal and monoclonal antibodies. LC and HCc gene segments were amplified from Clostridium tetani genomic DMA by PCR, cloned into pET28b[+] cloning vector and transformed in Escherichia coli [E colt] BL21[DE3] expression host. Recombinant proteins were then purified through His-tag using Nickel-based chromatography and characterized by SDS-PAGE, Western blotting and ELISA techniques. Recombinant light chain and HCc fragments were successfully cloned and expressed in [E coli] BL21 [DE3]. Optimization of the induction protocol resulted in production of high levels of Hcc [-35% of total bacterial protein] and to lesser extends of LC [-5%]. Reactivity of the His-tag purified proteins with specific polyclonal and monoclonal antibodies confirmed their renatured structure and identity. Our results indicate successful cloning and production of recom-binant LC and H[cc] fragments of TeNT. These two recombinant proteins are potentially useful tools for screening and monitoring of anti-TeNT antibody response and vaccine production

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

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.