Cloning and expression of a recombinant tumor necrosis factor-α (TNF-α) protein

Abstract

Introduction: Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease and in many patients can lead to joint destruction and loss of function. The expression of the proinflammatory cytokine (TNF-a) is widely related to the pathogenesis of RA. Recent evidence suggests that providing exogenous CO suppresses the inflammatory response associated with various disease states this is one of the mechanisms involved in the regulation of inflammation based on the inhibition of pro-inflammatory cytokine production such as TNF-α, CO might be a novel and important molecule in the treatment of RA. However, inhalation of gaseous CO presents serious intrinsic limitations as a drug due to the potent scavenging effect of hemoglobin, to circumvent these problems, CO Releasing Molecules (CORMs) have been designed to act as pro-drugs, releasing CO in vivo and eliciting its therapeutic action. In order to developed ligands (monoclonal and recombinant antibodies and recombinant peptides) to CORMS for direct delivery to inflammatory sites, there was the need to produced human recombinant TNF-a protein.

Materials and methods: A synthetic recombinant tnf-a (rTNF-a), was cloned in the vector pNZY29V (NZYTECH), with the codon usage for expression in E.coli. This synthetic gene was then subcloned by PCR ligation in pHTP1 expression vector (NZYEasy Cloning & Expression System, NZYTECH). The protein rTNF-a was expressed in E.coli XJB (DES3) bacteria (Zymo Research, arabinose lysis dependent) by IPTG (1mM, 3h at 37ºC). The purification process occurred by affinity chromatography with immobilized nickel ions (IMAC, Fast-Flow GE Biosciences). The eluted samples were analyzed by reading the absorbance at 280 nm, ELISA, SDS-PAGE electrophoresis and western-blot, using commercial anti-TNF-a and anti-HIS Tag antibodies.

Results: The cloning and expression system used was a directional cloning of PCR-generated fragments or synthetic gene, a DNA ligase free, with this system we have achieve a high cloning efficiency (80-100%). The protein was ready expressed, and the lysis was very efficient after arabinose-induced expression of the bacteriophage λ endolysin protein, coupled to a single freeze-thaw cycle. After IMAC chromatography the purification was between 90 to 95% (gel quantification). The rTNF-a expression was ready detected after IPTG induction using ELISA and wester-blot assays.

Discussion and conclusions: We succeeded in producing a rTNF-a protein, capable of functioning as a antigen for the production of monoclonal and recombinant antiboides and recombinant peptide ligands.

Acknowledgements

The authors acknowledge funding from Fundação para a Ciência e a Tecnologia, PTDC/QEQ-MED/1902/2014 - Nanoagentes de monoxido de carbono COGSs para combater a Artrite Reumatoide Carbon monoxide guided shuttles (COGSs) to fight Rheumatoid Arthritis.