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. Structured digital abstract GltSM and GltSM bind by molecular sieving ( View interaction) GltSM and GltSM bind by cross-linking study ( View interaction)

Cell-free methods to produce structurally intact mammalian membrane proteins

“…The cell-free methods are expected to become the standard procedures for crystallography-oriented membrane protein preparation, as they overcome a number of difficulties that unavoidably accompany the cell-based recombinant expression methods with respect to the preparation of mammalian membrane proteins. These expectations have been mentioned previously 38 39 , but have not been met successfully in the crystallography of mammalian membrane proteins at atomic resolution. In this study, we applied the P-MF and S-MF methods to the cell-free protein production and purification of mammalian α-helical membrane proteins.…”

A facile approach for the in vitro assembly of multimeric membrane transport proteins

“…To investigate whether our in vitro subunit reassembly procedure is applicable to other multimeric transporters, we initially tested the procedure on Glt Sm , a glutamate transporter homolog from Staphylothermus marinus that shares 58% sequence identity to Glt Ph ( Figure 5A )( Jaehme and Michel, 2013 ). We observed that SDS dissociated the Glt Sm transporter and that the dissociated subunits can be reassembled back to the trimeric state upon dilution of SDS coupled with protein incorporation into lipid vesicles ( Figure 5B ).…”

“…Glt Sm was amplified by PCR from a plasmid carrying the gene for a Glt Sm -GFP fusion protein ( Jaehme and Michel, 2013 ) and cloned into a pBAD-HisA vector (Fisher Scientific) with a N-terminal His 6 tag. The Glt Sm vector was transformed into TOP10 cells (Fisher Scientific) for protein expression.…”

A historical perspective on protein crystallization from 1840 to the present day

“…The alternate technology that eliminates most of these drawbacks is cell‐free in vitro protein synthesis. Presently, only a few crystallography groups have employed this technology to prepare soluble proteins and, recently, membrane proteins . In the case of RNAs as well, specific drawbacks have to be overcome for the preparation of homogeneous samples for crystallization.…”