title: Evaluation of cell-free protein synthesis for the crystallization of membrane proteins--a case study on a member of the glutamate transporter family from Staphylothermus marinus.
abstract: Cell-free in vitro synthesis of proteins using coupled transcription/translation is considered to be a powerful alternative to the use of traditional cell-based expression systems. Recently, promising developments have been reported applying cell-free production to membrane proteins for structural biology and in particular for NMR spectroscopy. However, the general applicability of this system to produce large amounts of stable, functional and homogeneous membrane proteins as required for X-ray crystallography remains to be determined. Here, we present a systematic study comparing structural and functional properties of membrane proteins produced using Escherichia coli derived in vitro and in vivo expression systems. The function of the target membrane protein, a previously uncharacterized bacterial glutamate transporter homolog from Staphylothermus marinus, was analyzed using ligand binding and transport assays. In addition, the protein structure was investigated with respect to its overall fold and oligomeric state in different detergents. We found that the protein synthesized in vitro is highly stable and monodisperse. However, in contrast to the protein produced using an in vivo system, it was not able to assemble into the native trimeric state nor to bind substrate. We thus conclude that cell-free expression systems can compromise folding and function of such complex secondary active transporters. The expression product has to be carefully characterized prior to biophysical investigations like crystallography of membrane proteins.
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Jaehme
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000096:biosynthesis* / Q000737:chemistry; D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D003460:Crystallization; D019620:Desulfurococcaceae; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli; D005960:Glucosides / Q000737:chemistry; D005976:Glutaral / Q000737:chemistry; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
chemical_list: D027322:Amino Acid Transport System X-AG; D019843:Archaeal Proteins; D003432:Cross-Linking Reagents; D003902:Detergents; D005960:Glucosides; D010767:Phosphorylcholine; C028810:dodecylphosphocholine; C040358:dodecyl maltoside; D005976:Glutaral