scholarly journals New insights on the interaction mechanism of rhTNFα with its antagonists Adalimumab and Etanercept

2020 ◽  
Vol 477 (17) ◽  
pp. 3299-3311
Author(s):  
María Angélica Contreras ◽  
Luis Macaya ◽  
Pedro Neira ◽  
Frank Camacho ◽  
Alaín González ◽  
...  
Keyword(s):  
High Performance ◽  
Interaction Mechanism ◽  
Protein Detection ◽  
Size Exclusion ◽  
Reducing Conditions ◽  
Kd Values ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
New Association ◽  
Linear Epitopes

TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS–PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10−9 M and >10−8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.

scholarly journals New insights on the interaction mechanism of rhTNFα with its antagonists Adalimumab and Etanercept

2020 ◽  
Author(s):  
María Angélica Contreras ◽  
Luis Macaya ◽  
Pedro Neira ◽  
Frank Camacho ◽  
Alaín González ◽  
...  
Keyword(s):  
Interaction Mechanism ◽  
Protein Detection ◽  
E Coli ◽  
Reducing Conditions ◽  
High K ◽  
Sds Page ◽  
New Association ◽  
Molecular Sizes ◽  
Two Phases ◽  
Binding Curves

AbstractTNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed E. coli. Protein detection was assessed by SDS-PAGE and western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis. Molecular sizes of the complexes were characterized by SEC-HPLC. Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to lineal epitopes and to one protomer of rhTNFα. Binding curves of two phases with low and high KD values (<10−9 M and >10−8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. This pioneer investigation revealed interactions of rhTNFα with Adalimumab and Etanercept never described before, which constitute valuable data for future approaches into the study of their interaction mechanism.

scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

scholarly journals Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miriam F. Suzuki ◽  
Larissa A. Almeida ◽  
Stephanie A. Pomin ◽  
Felipe D. Silva ◽  
Renan P. Freire ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Osmotic Shock ◽  
Size Exclusion ◽  
Specific Expression ◽  
E Coli ◽  
Human Prolactin ◽  
Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

scholarly journals Human Plasma and Recombinant Hemopexins: Heme Binding Revisited

2021 ◽  
Vol 22 (3) ◽  
pp. 1199
Author(s):  
Elena Karnaukhova ◽  
Catherine Owczarek ◽  
Peter Schmidt ◽  
Dominik J. Schaer ◽  
Paul W. Buehler
Keyword(s):  
Protein S ◽  
Soret Band ◽  
Cell System ◽  
Size Exclusion ◽  
Heme Binding ◽  
Systematic Analysis ◽  
Molar Ratios ◽  
Recombinant Production ◽  
Wide Range ◽  
Exclusion Chromatography

Plasma hemopexin (HPX) is the key antioxidant protein of the endogenous clearance pathway that limits the deleterious effects of heme released from hemoglobin and myoglobin (the term “heme” is used in this article to denote both the ferrous and ferric forms). During intra-vascular hemolysis, heme partitioning to protein and lipid increases as the plasma concentration of HPX declines. Therefore, the development of HPX as a replacement therapy during high heme stress could be a relevant intervention for hemolytic disorders. A logical approach to enhance HPX yield involves recombinant production strategies from human cell lines. The present study focuses on a biophysical assessment of heme binding to recombinant human HPX (rhHPX) produced in the Expi293FTM (HEK293) cell system. In this report, we examine rhHPX in comparison with plasma HPX using a systematic analysis of protein structural and functional characteristics related to heme binding. Analysis of rhHPX by UV/Vis absorption spectroscopy, circular dichroism (CD), size-exclusion chromatography (SEC)-HPLC, and catalase-like activity demonstrated a similarity to HPX fractionated from plasma. In particular, the titration of HPX apo-protein(s) with heme was performed for the first time using a wide range of heme concentrations to model HPX–heme interactions to approximate physiological conditions (from extremely low to more than two-fold heme molar excess over the protein). The CD titration data showed an induced bisignate CD Soret band pattern typical for plasma and rhHPX versions at low heme-to-protein molar ratios and demonstrated that further titration is dependent on the amount of protein-bound heme to the extent that the arising opposite CD couplet results in a complete inversion of the observed CD pattern. The data generated in this study suggest more than one binding site in both plasma and rhHPX. Furthermore, our study provides a useful analytical platform for the detailed characterization of HPX–heme interactions and potentially novel HPX fusion constructs.

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