Poly-Histidine-Tagged Protein Purification Using Immobilized Metal Affinity Chromatography (IMAC)

Loughran, Sinéad T.; Bree, Ronan T.; Walls, Dermot

doi:10.1007/978-1-0716-3362-5_11

Cited by 3 publications

(1 citation statement)

References 60 publications

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“…The resulting cells were tested for expression of target proteins by Western blotting using mouse anti-His tag (1:5,000) and goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000), with β-actin as the loading control detected by anti-β-actin antibody (1:1,000) as described (77).…”

Section: Expression Of Murine Proteins In Cho Cellsmentioning

confidence: 99%

Liver macrophages and sinusoidal endothelial cells execute vaccine-elicited capture of invasive bacteria

Wang,

An,

Ding

et al. 2023

Sci. Transl. Med.

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Vaccination has substantially reduced the morbidity and mortality of bacterial diseases, but mechanisms of vaccine-elicited pathogen clearance remain largely undefined. We report that vaccine-elicited immunity against invasive bacteria mainly operates in the liver. In contrast to the current paradigm that migrating phagocytes execute vaccine-elicited immunity against blood-borne pathogens, we found that invasive bacteria are captured and killed in the liver of vaccinated host via various immune mechanisms that depend on the protective potency of the vaccine. Vaccines with relatively lower degrees of protection only activated liver-resident macrophage Kupffer cells (KCs) by inducing pathogen-binding immunoglobulin M (IgM) or low amounts of IgG. IgG-coated pathogens were directly captured by KCs via multiple IgG receptors FcγRs, whereas IgM-opsonized bacteria were indirectly bound to KCs via complement receptors of immunoglobulin superfamily (CRIg) and complement receptor 3 (CR3) after complement C3 activation at the bacterial surface. Conversely, the more potent vaccines engaged both KCs and liver sinusoidal endothelial cells by inducing higher titers of functional IgG antibodies. Endothelial cells (ECs) captured densely IgG-opsonized pathogens by the low-affinity IgG receptor FcγRIIB in a “zipper-like” manner and achieved bacterial killing predominantly in the extracellular milieu via an undefined mechanism. KC- and endothelial cell–based capture of antibody-opsonized bacteria also occurred in FcγR-humanized mice. These vaccine protection mechanisms in the liver not only provide a comprehensive explanation for vaccine-/antibody-boosted immunity against invasive bacteria but also may serve as in vivo functional readouts of vaccine efficacy.

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Section: Expression Of Murine Proteins In Cho Cellsmentioning

confidence: 99%

Liver macrophages and sinusoidal endothelial cells execute vaccine-elicited capture of invasive bacteria

Wang,

An,

Ding

et al. 2023

Sci. Transl. Med.

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Facile Synthesis of Aptamer‐Functionalized Polydopamine‐Coated Magnetic Graphene Oxide Nanocomposites for Highly Efficient Purification of His‐Tagged Proteins

Qin,

Liu,

Wang

et al. 2024

J of Separation Science

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Recombinant proteins hold significant importance in numerous disciplines. As the demand for expressing and purifying these proteins grows, the scientific community is in dire need of a simple yet versatile methodology that can efficiently purify these proteins. Aptamers as synthetic nucleic acid–based ligands with high affinity have shown promise in this regard, as they can capture targets through molecular recognition. In this study, novel aptamer‐functionalized polydopamine‐coated magnetic graphene oxide nanocomposites were facilely prepared, achieving an impressive average aptamer coverage density (45 nmol/mg). These nanocomposites exhibited a uniform structure and robust magnetic responsiveness. The findings indicated that they possess several advantages, such as rapid adsorption, substantial capacity (171.4 mg/g), and excellent reusability. Notably, due to the inherent properties of nucleic acids, the immobilized aptamer‐magnetic beads can be utilized repeatedly with high purification efficiency. Finally, the nanocomposites were further employed to purify His‐tagged proteins from actual samples. Remarkably, they were able to selectively and efficiently isolate His‐tagged retinoid X receptor alpha protein from complex Escherichia coli lysate. The purified His‐tagged retinoid X receptor alpha protein was analyzed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. This confirmed the efficacy of developed nanocomposites, reinforcing their vast potential for purification of His‐tagged recombinant proteins.

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Displaying alphavirus physicochemical consensus antigens on immunogenic liposomes enhances antibody elicitation in mice

Huang,

Baker,

Lovell

et al. 2024

Virology

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Poly-Histidine-Tagged Protein Purification Using Immobilized Metal Affinity Chromatography (IMAC)

Cited by 3 publications

References 60 publications

Liver macrophages and sinusoidal endothelial cells execute vaccine-elicited capture of invasive bacteria

Liver macrophages and sinusoidal endothelial cells execute vaccine-elicited capture of invasive bacteria

Facile Synthesis of Aptamer‐Functionalized Polydopamine‐Coated Magnetic Graphene Oxide Nanocomposites for Highly Efficient Purification of His‐Tagged Proteins

Displaying alphavirus physicochemical consensus antigens on immunogenic liposomes enhances antibody elicitation in mice

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