| 1. |
- Ollila, Hanna M., et al.
(författare)
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Narcolepsy risk loci outline role of T cell autoimmunity and infectious triggers in narcolepsy
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Narcolepsy type 1 (NT1) is caused by a loss of hypocretin/orexin transmission. Risk factors include pandemic 2009 H1N1 influenza A infection and immunization with Pandemrix (R). Here, we dissect disease mechanisms and interactions with environmental triggers in a multi-ethnic sample of 6,073 cases and 84,856 controls. We fine-mapped GWAS signals within HLA (DQ0602, DQB1*03:01 and DPB1*04:02) and discovered seven novel associations (CD207, NAB1, IKZF4-ERBB3, CTSC, DENND1B, SIRPG, PRF1). Significant signals at TRA and DQB1*06:02 loci were found in 245 vaccination-related cases, who also shared polygenic risk. T cell receptor associations in NT1 modulated TRAJ*24, TRAJ*28 and TRBV*4-2 chain-usage. Partitioned heritability and immune cell enrichment analyses found genetic signals to be driven by dendritic and helper T cells. Lastly comorbidity analysis using data from FinnGen, suggests shared effects between NT1 and other autoimmune diseases. NT1 genetic variants shape autoimmunity and response to environmental triggers, including influenza A infection and immunization with Pandemrix (R).
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| 2. |
- Eriksson, Hanna M., 1978-, et al.
(författare)
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High-yield expression and purification of a monotopic membrane glycosyltransferase
- 2009
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Ingår i: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 66:2, s. 143-148
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins are essential to many cellular processes. However, the systematic study of membrane protein structure has been hindered by the difficulty in obtaining large quantities of these proteins. Protein overexpression using Escherichia coli is commonly used to produce large quantities of protein, but usually yields very little membrane protein. Furthermore, optimization of the expressing conditions, as well as the choice of detergent and other buffer components, is thought to be crucial for increasing the yield of stable and homogeneous protein. Herein we report high-yield expression and purification of a membrane-associated monotopic protein, the glycosyltransferase monoglucosyldiacylglycerol synthase (alMGS), in E. coli. Systematic optimization of protein expression was achieved through controlling a few basic expression parameters, including temperature and growth media, and the purifications were monitored using a fast and efficient size-exclusion chromatography (SEC) screening method. The latter method was shown to be a powerful tool for fast screening and for finding the optimal protein-stabilizing conditions. For alMGS it was found that the concentration of detergent was just as important as the type of detergent, and a low concentration of n-Dodecyl-β-D-maltoside (DDM) (~1× critical micelle concentration) was the best for keeping the protein stable and homogeneous. By using these simply methods to optimize the conditions for alMGS expression and purification, the final expression level increase by two orders of magnitude, reaching 170 mg of pure protein per litre culture.
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| 3. |
- Eriksson, Hanna M., 1978-, et al.
(författare)
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Increased amounts of overexpressed membrane proteins in Escherichia coli by co-expression with a foreign vesicle-inducing protein
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Escherichia coli has a limited capacity to overexpress integral membrane proteins to amounts needed for structural studies. This is usually attributed to the limited capacity of the Sec transport machinery, shortage of accessory chaperons, sub-optimal codon usage, potentially “wrong” lipids, and lack of membrane space for the new proteins. A foreign, monotopic lipid glycosyltransferase was recently shown to induce the formation of extensive amounts of intracellular vesicles in E. coli. We show here that such vesicles can improve the expressed levels up to 3-4 times for a substantial fraction of integral membrane proteins tested. These had 2 to 12 transmembrane helices, and all had a C-terminally fused GFP reporter. Strongly overexpressed proteins yielded intensely green vesicles, of slightly lower buoyant density than the inner membranes. Most proteins could be detected in the vesicles. Multivariate sequence analyses indicated a correlation between sequence property features and expression levels, and factors analyzed involved protein mass, transmembrane segments, inside/outside loops, etc. It is concluded that this vesicular system can yield substantial improvements in expression levels, by creation of extra membranes and lateral space in E. coli.
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| 4. |
- Wikström, Malin, et al.
(författare)
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Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function.
- 2009
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:2, s. 954-65
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.
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