| 1. |
- Kanje, Sara, 1986-, et al.
(författare)
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Protein engineering allows for mild affinity-based elution of therapeutic antibodies
- 2018
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 430:18, s. 3427-3438
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Tidskriftsartikel (refereegranskat)abstract
- Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification
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| 2. |
- Midtgaard, Søren Roi, et al.
(författare)
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Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering
- 2018
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 285:2, s. 357-371
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Tidskriftsartikel (refereegranskat)abstract
- A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D2 O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca2+ ATPase (SERCA). In all, the synthesis of isotope-substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to non-specialists. This article is protected by copyright. All rights reserved.
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| 3. |
- Venskutonytė, Raminta, et al.
(författare)
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Expression and purification of rat glucose transporter 1 in Pichia pastoris
- 2018
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745. ; 1713
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Bokkapitel (refereegranskat)abstract
- Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein. The purification protocol describes all steps needed to obtain a pure and homogenous GLUT1 protein solution, including cell growth, membrane isolation, and chromatographic purification methods.
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