Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers

Krishnarjuna, Bankala; Sharma, Gaurav; Ravula, Thirupathi; Ramamoorthy, Ayyalusamy

doi:10.1016/j.bbamem.2023.184240

Cited by 5 publications

(5 citation statements)

References 40 publications

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“…The result also indicates the feasibility of pentyl-inulin for isolating challenging membrane-binding proteins like APP. Further optimization of solu-bilizing factors such as pH, metal ions, salt and polymer con-centration could be helpful to increase the yield of solubilized APP-C99 (53).…”

Section: Resultsmentioning

confidence: 99%

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Nanodisc reconstitution and characterization of amyloid-β precursor protein C99

Krishnarjuna,

Sharma,

Hiiuk

et al. 2024

Preprint

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Amyloid precursor protein (APP) plays a pivotal role in the pathology of Alzheimers disease. Since the fragmentation of the membrane-bound APP that results in the production of amyloid-beta peptides is the starting point for amyloid toxicity in AD, it is important to investigate the structure and dynamics of APP in a near-native lipid-bilayer environment. However, the reconstitution of APP into a stable/suitable membrane-mimicking lipid environment is a challenging task. In this study, the 99-residue C-terminal domain of APP is successfully reconstituted into polymer nanodiscs and characterized using size-exclusion chromatography, mass spectrometry, solution NMR, and magic-angle spinning solid-state NMR. In addition, the feasibility of using lipid-solubilizing polymers for isolating and characterizing APP in native E. coli membrane environment is demonstrated.

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Section: Resultsmentioning

confidence: 99%

“…Membrane solubilization using pentyl-inulin. Cell membranes were weighed using an analytical balance and resuspended (25 mg/mL) in 10 mM Tris buffer (pH 7.4) containing 100 mM NaCl and mixed with polymer at a 1:1 w/w membrane-to-polymer ratio (53). The solubilization was carried out at 4, 25 and 37 °C.…”

Section: Introductionmentioning

confidence: 99%

Nanodisc reconstitution and characterization of amyloid-β precursor protein C99

Krishnarjuna,

Sharma,

Hiiuk

et al. 2024

Preprint

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“…The result also indicates the feasibility of pentyl-inulin for isolating challenging membrane-binding proteins like APP. Further optimization of solubilizing factors such as pH, metal ions, salt, and polymer concentration could be helpful to increase the yield of solubilized APP-C99 …”

Section: Resultsmentioning

confidence: 99%

“…Further optimization of solubilizing factors such as pH, metal ions, salt, and polymer concentration could be helpful to increase the yield of solubilized APP-C99. 57 Detergent-Solubilization and Purification of APP-C99 Using Ni-NTA Affinity Chromatography. The E. coli membranes were solubilized overnight using 20 mM Tris pH 7.8, 150 mM NaCl, 8 M urea, and 0.2% SDS.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Cell membranes were weighed using an analytical balance and resuspended (25 mg/mL) in 10 mM Tris buffer (pH 7.4) containing 100 mM NaCl and mixed with polymer at a 1:1 w/ w membrane-to-polymer ratio. 57 The solubilization was carried out at 4, 25, and 37 °C. Then, the insoluble components were removed by centrifugation at 10,000 rpm, 4 °C for 45 min.…”

Section: ■ Introductionmentioning

confidence: 99%

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Nanodisc Reconstitution and Characterization of Amyloid-β Precursor Protein C99

Krishnarjuna,

Sharma,

Hiiuk

et al. 2024

Anal. Chem.

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Amyloid precursor protein (APP) plays a pivotal role in the pathology of Alzheimer’s disease (AD). Since the fragmentation of the membrane-bound APP that results in the production of amyloid-β peptides is the starting point for amyloid toxicity in AD, it is important to investigate the structure and dynamics of APP in a near-native lipid-bilayer environment. However, the reconstitution of APP into a stable and suitable membrane-mimicking lipid environment is a challenging task. In this study, the 99-residue C-terminal domain of APP is successfully reconstituted into polymer nanodiscs and characterized using size-exclusion chromatography, mass spectrometry, solution NMR, and magic-angle spinning solid-state NMR. In addition, the feasibility of using lipid-solubilizing polymers for isolating and characterizing APP in the native Escherichia. coli membrane environment is demonstrated.

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Discovery of Therapeutic Antibodies Targeting Complex Multi-Spanning Membrane Proteins

Stephens,

Wilkinson

2024

BioDrugs

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Complex integral membrane proteins, which are embedded in the cell surface lipid bilayer by multiple transmembrane spanning polypeptides, encompass families of proteins that are important target classes for drug discovery. These protein families include G protein-coupled receptors, ion channels, transporters, enzymes, and adhesion molecules. The high specificity of monoclonal antibodies and the ability to engineer their properties offers a significant opportunity to selectively bind these target proteins, allowing direct modulation of pharmacology or enabling other mechanisms of action such as cell killing. Isolation of antibodies that bind these types of membrane proteins and exhibit the desired pharmacological function has, however, remained challenging due to technical issues in preparing membrane protein antigens suitable for enabling and driving antibody drug discovery strategies. In this article, we review progress and emerging themes in defining discovery strategies for a generation of antibodies that target these complex membrane protein antigens. We also comment on how this field may develop with the emerging implementation of computational techniques, artificial intelligence, and machine learning.

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Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers

Cited by 5 publications

References 40 publications

Nanodisc reconstitution and characterization of amyloid-β precursor protein C99

Nanodisc reconstitution and characterization of amyloid-β precursor protein C99

Nanodisc Reconstitution and Characterization of Amyloid-β Precursor Protein C99

Discovery of Therapeutic Antibodies Targeting Complex Multi-Spanning Membrane Proteins

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