Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells

Abstract

Nanodiscs are emerging to serve as transfer vectors for the insertion of recombinant membrane proteins into membranes of living cells. In combination with cell-free expression technologies, this novel process opens new perspectives to analyze the effects of even problematic targets such as toxic, hard-to-express, or artificially modified membrane proteins in complex cellular environments of different cell lines. Furthermore, transferred cells must not be genetically engineered and primary cell lines or cancer cells could be implemented as well. We have systematically analyzed the basic parameters of the nanotransfer approach and compared the transfer efficiencies from nanodiscs with that from Salipro particles. The transfer of five membrane proteins was analyzed: the prokaryotic proton pump proteorhodopsin, the human class A family G-protein coupled receptors for endothelin type B, prostacyclin, free fatty acids type 2, and the orphan GPRC5B receptor as a class C family member. The membrane proteins were cell-free synthesized with a detergent-free strategy by their cotranslational insertion into preformed nanoparticles containing defined lipid environments. The purified membrane protein/nanoparticles were then incubated with mammalian cells. We demonstrate that nanodiscs disassemble and only lipids and membrane proteins, not the scaffold protein, are transferred into cell membranes. The process is detectable within minutes, independent of the nanoparticle lipid composition, and the transfer efficiency directly correlates with the membrane protein concentration in the transfer mixture and with the incubation time. Transferred membrane proteins insert in both orientations, N-terminus in and N-terminus out, in the cell membrane, and the ratio can be modulated by engineering. The viability of cells is not notably affected by the transfer procedure, and transferred membrane proteins stay detectable in the cell membrane for up to 3 days. Transferred G-protein coupled receptors retained their functionality in the cell environment as shown by ligand binding, induction of internalization, and specific protein interactions. In comparison to transfection, the cellular membrane protein concentration is better controllable and more uniformly distributed within the analyzed cell population. A further notable difference to transfection is the accumulation of transferred membrane proteins in clusters, presumably determined by microdomain structures in the cell membranes.

Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment

“…The functionality of CF synthesized H 2 R was analyzed using a recently developed technique for GPCR transfer from ND membranes into membranes of living cells 20 , 21 (Fig. 2a ).…”

“…In summary, the results show that our CF production pipeline can provide structural information of GPCRs with a good global resolution. We further demonstrate an alternative strategy for the functional transfer of membrane proteins from NDs to membranes of living cells 20 – 22 . Here, we used this approach to confirm the previously proposed homo- and heterooligomerization of the H 1 R and H 2 R using pull-downs and internalization assays.…”

“…The nanotransfer is a recently developed approach and several persistent limitations must still be considered 20 , 21 . Nanotransfer is currently much less efficient if compared with conventional transfection, reaching approx.…”

“…only 10% of a corresponding transfection efficiency. In addition, the transfer is not directed and, depending on the individual topology of the target, a significant fraction or even the majority of the transferred protein will become inserted in wrong orientation 20 . Flag-H 1 R and Flag-H 2 R were ordered from Addgene (#66400 & #66401) and de-tangonized using standard site-directed mutagenesis 67 .…”

“…The complex is formed with full-length H 2 R, G s and the G s stabilizing nanobody Nb35 in ND membranes composed of DOPG lipid. The biochemical characterization is complemented by analysis of the CF synthesized H 2 R after transfer into membranes of HEK293T cells using a recently developed nanotransfer technique 20 – 22 .…”