High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins.
An important factor in the crystallization, and subsequent structural determination, of integral membrane proteins is the ability to produce a stable and monodisperse solution of the protein. Obtaining the correct purification detergent to achieve this can be laborious and is often serendipitous. In this study, high-throughput methods are used to analyze the suitability of eight different detergents on the stability of 12 inner transmembrane proteins from Escherichia coli. The best results obtained from the small-scale experiments were scaled up, the aggregation state of the proteins assessed, and all monodisperse protein solutions entered into crystallization trials. This resulted in preliminary crystallization hits for three inner membrane proteins: XylH, PgpB and YjdL and this study reports the methods, purification procedures and crystallization conditions used to achieve this.
Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
Mads
M
Gabrielsen
Frank
F
Kroner
Isobel
I
Black
Neil W
NW
Isaacs
Andrew J
AJ
Roe
Karen
K
McLuskey
D003460:Crystallization / Q000379:methods; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D057166:High-Throughput Screening Assays; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification*
D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C544083:YjdL protein, E coli; C043426:inner membrane protein, E coli