+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases



Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases



Protein Expression and Purification 96: 32-38



TREM-2 (triggering receptor expressed on myeloid cells-2) is an innate immune receptor expressed on dendritic cells, macrophages, osteoclasts, and microglia. Recent genetic studies have reported the occurrence of point mutations in TREM-2 that correlate with a dramatically increased risk for the development of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. Structural and biophysical studies of wild-type and mutant TREM-2 ectodomains are required to understand the functional consequences of these mutations. In order to facilitate these studies, we undertook the production and crystallization of these proteins. Here we demonstrate that, unlike many single Ig domain proteins, TREM-2 could not be readily refolded from bacterially-expressed inclusion bodies. Instead, we developed a mammalian-cell based expression system for the successful production of wild-type and mutant TREM-2 proteins in milligram quantities and a single-chromatography-step purification scheme that produced diffraction-quality crystals. These crystals diffract to a resolution of 3.3 Å and produce data sufficient for structure determination. We describe herein the procedures to produce wild-type and mutant human TREM-2 Ig domains in sufficient quantities for structural and biophysical studies. Such studies are crucial to understand the functional consequences of TREM-2 point mutations linked to the development of neurodegenerative diseases and, ultimately, to develop patient-specific molecular therapies to treat them.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 055137645

Download citation: RISBibTeXText

PMID: 24508568

DOI: 10.1016/j.pep.2014.01.015


Related references

Crystallization and preliminary X-ray crystallographic analysis of a nonstructural protein 15 mutant from Human coronavirus 229E. Acta Crystallographica. Section F Structural Biology Communications 71(Pt 9): 1156-1160, 2015

Crystallization and preliminary X-ray crystallographic studies of wild-type human ornithine transcarbamylase and two naturally occurring mutants at position 277. Acta Crystallographica Section D Biological Crystallography 57(5): 719-721, 2001

Crystallization and preliminary X-ray analysis of wild-type and V103L mutant Myb R2 DNA-binding domain. Acta Crystallographica Section D Biological Crystallography 55(7): 1345-1347, 1999

Crystallization and preliminary X-ray crystallographic analysis of human peptidylarginine deiminase type III. Acta Crystallographica. Section F Structural Biology and Crystallization Communications 68(Pt 6): 668-670, 2012

Crystallization and preliminary crystallographic analysis of human common-type acylphosphatase. Acta Crystallographica. Section F Structural Biology and Crystallization Communications 62(Pt 1): 80-82, 2006

Crystallization and preliminary X-ray crystallographic analysis of human peptidylarginine deiminase type I. Acta Crystallographica. Section F Structural Biology and Crystallization Communications 69(Pt 12): 1357-1359, 2013

Crystallization and preliminary X-ray analysis of wild-type and K272A mutant glutamate 1-semialdehyde aminotransferase from Synechococcus. Journal of Molecular Biology 242(4): 591-594, 1994

Crystallization and preliminary X-ray crystallographic analysis of the human type 3 3alpha-hydroxysteroid dehydrogenase at 1.8 ANG resolution. Acta Crystallographica Section D Biological Crystallography 57(4): 589-591, 2001

Purification, crystallization and preliminary crystallographic analysis of the globular domain of the human type V myosin Myo5a. Acta Crystallographica. Section F Structural Biology and Crystallization Communications 69(Pt 11): 1220-1223, 2013

Crystallization and X-ray crystallographic studies of wild-type and mutant tryptophan synthase alpha-subunits from Escherichia coli. Molecules and Cells 19(2): 219-222, 2005

Expression, purification, crystallization and preliminary X-ray structure analysis of wild-type and L(M196)H-mutant Rhodobacter sphaeroides reaction centres. Acta Crystallographica. Section F Structural Biology and Crystallization Communications 69(Pt 5): 506-509, 2013

Crystallization and preliminary X-ray crystallographic analysis of the receptor-uncoupled mutant of Galphai1. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications 63(Pt 2): 139-141, 2007