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| 2. |
- Martinez Molina, Daniel, 1979-, et al.
(författare)
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Engineering membrane protein overproduction in Escherichia coli
- 2008
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 17:4, s. 673-680
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent-solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent-solubilized membrane protein that can be overexpressed and purified from recombinant Escherichia coli. Libraries of random mutants for nine membrane proteins were screened for expression using a novel implementation of the colony filtration blot. In only one cycle of directed evolution were significant improvements of membrane protein yield obtained for five out of nine proteins. In one case, the yield of detergent-solubilized membrane protein was increased 40-fold.
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| 3. |
- Sabirsh, A, et al.
(författare)
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Residues from transmembrane helices 3 and 5 participate in leukotriene B-4 binding to BLT1
- 2006
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 45:18, s. 5733-5744
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Tidskriftsartikel (refereegranskat)abstract
- Leukotrienes are inflammatory mediators that bind to seven transmembrane, G-protein-coupled receptors (GPCRs). Here we examine residues from transmembrane helices 3 and 5 of the leukotriene B-4 (LTB4) receptor BLT1 to elucidate how these residues are involved in ligand binding. We have selected these residues on the basis of (1) amino acid sequence analysis, (2) receptor binding and activation studies with a variety of leukotriene-like ligands and recombinant BLT1 receptors, (3) previously published recombinant BLT1 mutants, and (4) a computed model of the active structure of the BLT1 receptor. We propose that LTB4 binds with the polar carboxylate group of LTB4 near the extracellular surface of BLT1 and with the hydrophobic LTB4 tail pointing into the transmembrane regions of the receptor protein. The carboxylate group and the two hydroxyls of LTB4 interact with Arg178 and Glu185 in transmembrane helix 5. Residues from transmembrane helix 3, Val 105 and Ile 108, also line the pocket deeper inside the receptor. LTB4 is becoming increasingly important as an immunomodulator during a number of pathologies, including atherosclerosis. Detailed information about the LTB4 binding mechanism, and the receptor residues involved, will hopefully aid in the design of new immunomodulatory drugs.
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| 4. |
- Wetterholm, Anders, et al.
(författare)
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High-level expression, purification, and crystallization of recombinant rat leukotriene C4 synthase from the yeast Pichia pastoris
- 2008
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 60:1, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- Leukotriene C(4) synthase (LTC4S) is a member of the MAPEG family of integral membrane proteins and catalyzes the conjugation of leukotriene A(4) with glutathione to form leukotriene C(4), a powerful mediator of allergic inflammation and anaphylaxis. Structural information on this class of proteins would be highly useful for rational drug design. Here, we report the expression, purification, and crystallization of recombinant LTC4S from rat. The enzyme was expressed as an N-terminal hexa-histidine-tagged fusion protein in Pichia pastoris and purified with two steps of affinity chromatography on Ni-Sepharose and S-hexyl-glutathione agarose, followed by gel filtration. From 1l culture, we obtained 0.5-1 mg of apparently homogeneous protein with a specific LTC4S activity ranging between 36 and 49 micromol/mg/min. A small-scale screen identified dodecyl maltoside as a useful detergent for protein extraction and yielded a highly active protein. When tested separately in crystallization trials of the purified LTC4S, six out of seven detergents from all the maltoside family yielded diffracting crystals with the highest resolution at approximately 6 A. Hence, our approach holds promise for solving the structure of rat LTC4S and other members of the MAPEG family of integral membrane proteins.
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