5HQ3

Stable, high-expression variant of human acetylcholinesterase

5HQ3 の概要

• エントリーDOI: 10.2210/pdb5hq3/pdb
• 分子名称: Acetylcholinesterase, O-ETHYLMETHYLPHOSPHONIC ACID ESTER GROUP, 2-(N-MORPHOLINO)-ETHANESULFONIC ACID, ... (5 entities in total)
• 機能のキーワード: design, de novo protein, hydrolase
• 由来する生物種: Homo sapiens (Human); 詳細
• 細胞内の位置: Cell junction, synapse . Isoform T: Nucleus. Isoform H: Cell membrane ; Lipid-anchor, GPI-anchor ; Extracellular side : P22303 P22303
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 123155.34

構造登録者

Goldenzweig, A.,Goldsmith, M.,Hill, S.E.,Gertman, O.,Laurino, P.,Ashani, Y.,Dym, O.,Albeck, S.,Unger, T.,Prilusky, J.,Lieberman, R.L.,Aharoni, A.,Silman, I.,Sussman, J.L.,Tawfik, D.S.,Fleishman, S.J. (登録日: 2016-01-21, 公開日: 2016-07-27, 最終更新日: 2024-10-23)

主引用文献

Goldenzweig, A.,Goldsmith, M.,Hill, S.E.,Gertman, O.,Laurino, P.,Ashani, Y.,Dym, O.,Unger, T.,Albeck, S.,Prilusky, J.,Lieberman, R.L.,Aharoni, A.,Silman, I.,Sussman, J.L.,Tawfik, D.S.,Fleishman, S.J.
Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.
Mol.Cell, 63:337-346, 2016

PubMed Abstract: Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.

PubMed: 27425410
DOI: 10.1016/j.molcel.2016.06.012

実験手法

X-RAY DIFFRACTION (2.6 Å)