کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1987005 1540257 2014 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression of human AChR extracellular domain mutants with improved characteristics
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Expression of human AChR extracellular domain mutants with improved characteristics
چکیده انگلیسی

The muscle nicotinic acetylcholine receptor (AChR) has a central role in neuromuscular transmission, and is the major target in the autoimmune disease myasthenia gravis (MG). We created mutants of the extracellular domains (ECDs) of the human α1, β1, δ and ɛ AChR subunits, whereby their Cys-loop was exchanged for that of the acetylcholine binding protein. The mutants were expressed in Pichia pastoris and had improved solubility resulting in 2- to 43-fold higher expression yields compared to the wild type. An additional mutant was created for the α1 ECD restoring its glycosylation site within the Cys-loop and its α-bungarotoxin binding ability. Furthermore, we constructed dimeric and pentameric concatamers of the mutant ECDs. All concatamers were successfully expressed as soluble secreted proteins, although the pentamers had about 10-fold lower expression than the dimers and were more susceptible to fragmentation. Initial crystallizations with the mutant ECDs were promising, and we reproducibly obtained crystals of the β1 ECD, diffracting at ∼12 Å. Further optimization is underway to obtain crystals suitable for high resolution crystallography. The proteins described herein are useful tools in structural studies of the human muscle AChR and can be used in applications requiring high yields such as therapeutic adsorbents for MG autoantibodies.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: International Journal of Biological Macromolecules - Volume 63, February 2014, Pages 210–217
نویسندگان
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