High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins

Abstract

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.

Keywords::

• High-throughput
• inner transmembrane protein
• detergents
• crystallization

Acknowledgements

This work was funded by the Membrane Protein Structure Initiative (MPSi) from the Biotechnology and Biology Sciences Research Council (BBSRC) for IB, NWI and KMcL, a BBSRC grant (BB/G011389/1) for MG and AJR, and the University of Glasgow and Westchem for a studentship for FK. The funding sources had no influence on the work done. We thank Diamond Light Source for access to beamline I02 and I03 (Proposal number MX1229) that contributed to the results presented here. We acknowledge Dr Dan Daley, University of Stockholm, Sweden, for his generous gift of all constructs, Mr Ryan Ritchie for assistance with the IVIS, and Dr Aleks Roszak for useful discussions.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.