Anti-6X His tag monoclonal antibody, clone BE2.2.20

Employing immobilized metal-ion affinity chromatography and magnetic separation could ideally provide a useful analytical strategy for purifying His-tagged protein. In the current study, a facile route was designed to prepare CMPEI-Ni2+@SiO2@Fe3O4 (CMPEI=carboxymethylated polyethyleneimine) magnetic nanoparticles composed of a strong magnetic core of Fe3O4 and a Ni2+-immobilized carboxymethylated polyethyleneimine coated outside shell, which was formed by electrostatic interactions between polyanionic electrolyte of carboxymethylated polyethyleneimine and positively charged surface of 3-(trimethoxysilyl) propylamin modified SiO2@Fe3O4. The resulting CMPEI-Ni2+@SiO2@Fe3O4 composite nanoparticles displayed well-uniform structure and high magnetic responsiveness. Hexa His-tagged peptides and purified His-tagged recombinant retinoid X receptor alpha were chosen as the model samples to evaluate the adsorption, capacity, and reusability of the composite nanoparticles. The results demonstrated the CMPEI-Ni2+@SiO2@Fe3O4 nanoparticles possessed rapid adsorption, large capacity, and good recyclability. The obtained nanoparticles were further used to purify His-tagged protein in practical environment. It was found that the nanoparticles could selectively capture His-tagged recombinant retinoid X receptor protein from complex cell lysate. Owing to its easy synthesis, large binding capacity, and good reusability, the prepared CMPEI-Ni2+@SiO2@Fe3O4 magnetic nanoparticles have great potential for application in biotechnological fields.

A novel inducible artificial metalloenzyme obtained by covalent attachment of a manganese(III)-tetraphenylporphyrin (MnTPP) to the artificial bidomain repeat protein, (A3A3 ')Y26C, is reported. The protein is part of the alpha Rep family. The biohybrid was fully characterized by MALDI-ToF mass spectrometry, circular dichroism and UV/Vis spectroscopies. The peroxidase and monooxygenase activities were evaluated on the original and modified scaffolds including those that have a) an additional imidazole, b) a specific alpha Rep bA3-2 that is known to induce the opening of the (A3A3 ') interdomain region and c) a derivative of the alpha Rep bA3-2 inducer extended with a His(6)-Tag (His(6)-bA3-2). Catalytic profiles are highly dependent on the presence of co-catalysts with the best activity obtained with His(6)-bA3-2. The entire mechanism was rationalized by an integrative molecular modeling study that includes protein-ligand docking and large-scale molecular dynamics. This constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways.