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5HQ3

Stable, high-expression variant of human acetylcholinesterase

Summary for 5HQ3
Entry DOI10.2210/pdb5hq3/pdb
DescriptorAcetylcholinesterase, O-ETHYLMETHYLPHOSPHONIC ACID ESTER GROUP, 2-(N-MORPHOLINO)-ETHANESULFONIC ACID, ... (5 entities in total)
Functional Keywordsdesign, de novo protein, hydrolase
Biological sourceHomo sapiens (Human)
More
Cellular locationCell junction, synapse . Isoform T: Nucleus. Isoform H: Cell membrane ; Lipid-anchor, GPI-anchor ; Extracellular side : P22303 P22303
Total number of polymer chains2
Total formula weight123155.34
Authors
Primary citationGoldenzweig, 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
Cited by
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
PDB entries with the same primary citation
Experimental method
X-RAY DIFFRACTION (2.6 Å)
Structure validation

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