Functional expression of a two-transmembrane HtrII protein using cell-free synthesis.

An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of the halobacterial transducer protein, HtrII, was translated with various large soluble tags added ( protein and maltose binding protein). In this system, all fusion HtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-d-maltoside was added for enhancing the solubilization of the hydrophobic region of HtrII. The activity of the expressed HtrII, having various tags, was checked using a pull-down assay, using the fact that HtrII forms a signaling complex with phoborhodopsin (pR) in the membrane, as also in the presence of a detergent. All tagged HtrII showed a binding activity with pR. Interestingly, the binding activity with pR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.