Cellular and Molecular Life Sciences

, Volume 71, Issue 24, pp 4895–4910 | Cite as

Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

  • Coilín Boland
  • Dianfan Li
  • Syed Tasadaque Ali Shah
  • Stefan Haberstock
  • Volker Dötsch
  • Frank Bernhard
  • Martin Caffrey
Research Article


Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.


Cell-free Crystallisation In meso Lipid cubic phase Macromolecular crystallography Membrane protein structure 



Adenosine diphosphate


Acetabularia acetabulum rhodopsin II


Adenosine triphosphate


n-octaethylene glycol monododecyl ether


n-octyl tetraethylene glycol monoether




Cytidine triphosphate


Column volume


Diacylglycerol kinase






Deoxyribonucleic acid




Ethylenediaminetetraacetic acid


Ethyleneglycoltetraacetic acid


Green fluorescent protein


Guanosine triphosphate


Hydrochloric acid


4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid


Horseradish peroxidase


Human voltage-dependent anion channel 1


Isopropyl β-D-1-thiogalactopyranoside




Lipid cubic phase


Lauryldimethylamine N-oxide






Molecular weight cut off


Nicotinamide adenine dinucleotide


Optical density


Polymerase chain reaction






Phosphenolpyruvic acid


Pyruvate kinase




Piperazine-1,4-bis(2-ethanesulfonic acid


Phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride


Root-mean-square deviation


Ribonucleic acid


Size-exclusion chromatography


tris (2-carboxyethyl) phosphine hydrochloride


Thin-layer chromatography


Transfer ribonucleic acid


Uridine triphosphate


Yeast phosphate tryptone glucose


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Copyright information

© Springer Basel 2014

Authors and Affiliations

  • Coilín Boland
    • 1
  • Dianfan Li
    • 1
  • Syed Tasadaque Ali Shah
    • 1
  • Stefan Haberstock
    • 2
  • Volker Dötsch
    • 2
  • Frank Bernhard
    • 2
  • Martin Caffrey
    • 1
  1. 1.Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and ImmunologyTrinity College DublinDublinIreland
  2. 2.Centre of Biomolecular Magnetic Resonance, Institute of Biophysical ChemistryJohann Wolfgang Goethe-University of FrankfurtFrankfurtGermany

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