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- 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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| 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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