ABCA2 p.Gln2135Ser
| Predicted by SNAP2: | A: D (59%), D: D (75%), E: D (71%), F: D (63%), G: D (75%), H: D (63%), I: D (53%), K: D (71%), L: D (63%), M: D (63%), N: D (66%), P: D (75%), Q: D (71%), R: D (71%), S: D (66%), T: D (63%), V: N (53%), W: D (80%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Expression, purification and structural properties... Protein Expr Purif. 2014 May;97:50-60. doi: 10.1016/j.pep.2014.02.010. Epub 2014 Feb 28. Tsybovsky Y, Palczewski K
Expression, purification and structural properties of ABC transporter ABCA4 and its individual domains.
Protein Expr Purif. 2014 May;97:50-60. doi: 10.1016/j.pep.2014.02.010. Epub 2014 Feb 28., [PMID:24583180]
Abstract [show]
ABCA4 is a member of the A subfamily of ATP-binding cassette transporters that consists of large integral membrane proteins implicated in inherited human diseases. ABCA4 assists in the clearance of N-retinylidene-phosphatidylethanolamine, a potentially toxic by-product of the visual cycle formed in photoreceptor cells during light perception. Structural and functional studies of this protein have been hindered by its large size, membrane association, and domain complexity. Although mammalian, insect and bacterial systems have been used for expression of ABCA4 and its individual domains, the structural relevance of resulting proteins to the native transporter has yet to be established. We produced soluble domains of ABCA4 in Escherichia coli and Saccharomyces cerevisiae and the full-length transporter in HEK293 cells. Electron microscopy and size exclusion chromatography were used to assess the conformational homogeneity and structure of these proteins. We found that isolated ABCA4 domains formed large, heterogeneous oligomers cross-linked with non-specific disulphide bonds. Incomplete folding of cytoplasmic domain 2 was proposed based on fluorescence spectroscopy results. In contrast, full-length human ABCA4 produced in mammalian cells was found structurally equivalent to the native protein obtained from bovine photoreceptors. These findings offer recombinantly expressed full-length ABCA4 as an appropriate object for future detailed structural and functional characterization.
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No. Sentence Comment
201 The resulting mutants containing from six down to two Cys showed unaltered expression levels and were refolded after purification from IB (Fig. 6b, left) except for the C2135S mutation construct that led to protein precipitation.
X
ABCA2 p.Gln2135Ser 24583180:201:169
status: NEW