Expression, purification, and characterization of soluble recombinant TNFR1

Abstract

Tumor Necrosis Factor Alpha (TNF-α) is a trimeric cytokine secreted by macrophages and monocytes. It belongs to type II transmembrane protein family and is involved in both innate and adaptive immune system. TNF-α exists in two forms: transmembrane TNF (tmTNF), which is synthesized as a precursor form, and solubilized TNF (sTNF), which is created after further processing. TNF-α functions in a variety of biological processes by interacting with specific receptors namely as TNFR1 and TNFR2. TNFR1 consists of intracellular, transmembrane, and extracellular domains. The extracellular part consists of four cysteine-rich parts. When interacting to TNFR1, TNF-α can activate various signaling pathways for instance inflammation and apoptosis. TNF-α level is undetectable in normal individuals, yet under inflammation conditions the protein concentration in serum increases proportionally to the inflammation level in the body, thus making the protein detectable. Controlled production of TNF-α is crucial for tissue healing and fight against infection, but a constantly high level of TNF-α can cause various diseases including rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis/psoriatic arthritis, and Chron's disease. TNF-α inhibitors are designed and used in treatment of diseases caused by overexpression of TNF-α. An alternative pathway includes employment of TNFR1 extracellular domain as a biotherapeutic tool to inhibit the effects of overexpressed TNF-α. The latter approach seems to be way more preferred than other therapeutics due to its targetselectivity and high affinity. Production of proteins like TNFR1 are characterized by cysteine-rich domains by using bacterial systems is quite advantageous in terms of cost, yield, and time. However, lack of an effective translation system in Escherichia coli (E. coli) may hinder production of such disulfide bond containing proteins mainly in terms of generating mismatched cysteine residues or failing in acquiring a sulfide bridge formation thus leading to protein aggregation and inclusion body formation. Recovering proteins from inclusion bodies is time-consuming and expensive while considering the protein loss and incorrectly folded proteins due to the wrongly formed disulfide bonds. Dsb protein family including DsbA, DsbB, DsbC, DsbD, and DsbG, is responsible for the formation of disulfide bonds in bacteria. The DsbA-DsbB complex plays role in the oxidative pathways, while DsbC-DsbD complex functions in the isomerization pathway. Among all Dsb proteins, DsbA and DsbC are the most extensively studied and widely used in the field of biotechnology. Our previous studies showed that the extracellular portion of TNFR1 forms inclusion bodies in E. coli, and in vitro refolding steps are needed to obtain the correct xxvi conformation. Protein aggregations during the refolding process also caused a decrease in yield. The purpose of this study is to obtain the extracellular part of TNFR1 as a soluble protein in E. coli. Initially, a SHuffle T7 strain with a cytoplasmic DsbC copy was selected as the host cell and expression experiments were carried out accordingly. However, the target protein formed an inclusion body. To assess the effect of DsbC in the correctly folded-soluble TNFR1 production, a expression vector containing fusion protein DsbC-TNFR1 was constructed. Formation of DsbC-TNFR1 fusion protein was held on the basis of plasmid design conferring the order of a 6x histidine tag, DsbC, TEV cleavage site, and TNFR1 from N to C terminus respectively. It was assured that periplasm target sequence was removed from DsbC gene, allowing its production within the bacterial cytoplasm. DsbC-TNFR1 production was verified by SDS-PAGE and immunoblotting analyses, which revealed that some conditions led to the production of the fusion protein in soluble form. In order to detect the optimum conditions to produce DsbC-TNFR1, four different E. coli strains as host cells were employed, namely as Rosetta (DE3), Rosetta-gami 2, SHuffle T7, and BL21 (DE3). However, since production of the Rosetta (DE3) strain was higher than others, the research was conducted on that particular strain. Induction conditions including IPTG induction and autoinduction medium were also assessed to get higher yield from the selected cells. As a result of induction trials, the target protein was obtained in soluble form when autoinduction was utilized. Following sonication and centrifugation of the cells, the affinity chromatography was performed for separation of the recombinant protein from other host cell proteins. Anion exchange chromatography (AEX) was used as a second purification step to remove remaining impurities. The efficiency of purification was evaluated by using SDS-PAGE and immunoblotting analyses. Then, protein characterization studies were performed. The isoelectric point of the pure protein was calculated by isoelectric focusing via capillary electrophoresis device, and the purity of the protein was checked using the purity determination method. The secondary structure of the fusion protein was analyzed by circular dichroism spectroscopy and it was determined to be in the α-helix structure. Intact mass analysis through LC/MS was also performed to calculate the molecular weight of the protein. In addition, the peptide mapping was used to identify the amino acid sequence. The native structure of the pure protein was investigated by using blue native polyacrylamide gel electrophoresis (BN-PAGE), which revealed that the protein exists in both dimers and different oligomer structures. The functionality of the fusion protein was assessed by performing a pull-down assay, which showed that DsbC-TNFR1 is functional as it was capable of binding to TNFR1 ligand TNF-α. As a consequence of the research, it was revealed that the implementation of such a fusion protein is a successful tool for expressing TNFR1 in its soluble form in bacterial cells. After analysis of the pure protein generated as the outcome of downstream steps, it was shown that the TNFR1 produced by using the implemented fusion method is functional.