Rapid One-Step Capturing of Native, Cell-Free Synthesized and Membrane-Embedded GLP-1R

Abstract

G protein-coupled receptors (GPCRs) are of outstanding pharmacological interest as they are abundant in cell membranes where they perform diverse functions that are closely related to the vitality of cells. The analysis of GPCRs in natural membranes is laborious, as established methods are almost exclusively cell culture-based and only a few methods for immobilization in a natural membrane outside the cell are known. Within this study, we present a one-step, fast and robust immobilization strategy of the GPCR glucagon-like peptide 1 receptor (GLP-1R). GLP-1R was synthesized in eukaryotic lysates harboring endogenous endoplasmic reticulum-derived microsomes enabling the embedment of GLP-1R in a natural membrane. Interestingly, we found that these microsomes spontaneously adsorbed to magnetic Neutravidin beads thus providing immobilized membrane protein preparations which required no additional manipulation of the target receptor or its supporting membrane. The accessibility of the extracellular domain of membrane-embedded and bead-immobilized GLP-1R was demonstrated by bead-based enzyme-linked immunosorbent assay (ELISA) using GLP-1R-specific monoclonal antibodies. In addition, ligand binding of immobilized GLP-1R was verified in a radioligand binding assay. In summary, we present an easy and straightforward synthesis and immobilization methodology of an active GPCR which can be beneficial for studying membrane proteins in general.

Keywords: CFPS; G protein-coupled receptors; GLP-1R; Sf21 cell lysate; cell-free expression; cell-free protein synthesis; immobilization to magnetic beads; membrane.

Figure 1

Analysis of cell-free synthesized GLP-1R in Sf21 and CHO lysate. Cell-free synthesized GLP-1R was analyzed in three lysate fractions (translation mixture (TM), soluble fraction (SN), microsomal fraction (MF)) and in TM of a cell-free synthesis reaction performed with microsome depleted lysate using the Sf21 (A) and the CHO cell-free system (B). Protein yields of 14C-leucine labeled GLP-1R were determined by precipitating aliquots of TM, SN and MF in hot TCA followed by liquid scintillation measurement using a scintillation counter (Hidex 600 SL). Standard deviations were calculated from triplicates. (C) Autoradiogram of GLP-1R synthesized using the cell-free Sf21 system. The qualitative analysis shows the expected molecular weight of the synthesized protein in its glycosylated and non-glycosylated form. Translation mixture (TM) of cell-free synthesized GLP-1R was separated by centrifugation into SN and MF. In order to prove the glycosylation, the TM and MF were treated with the enzymes PNGase F and Endo H. (D) Autoradiogram of GLP-1R synthesized in cell-free CHO system. No template control (NTC) was carried along as negative control.

Figure 2

Site-specific biotinylation of GLP-1R-L260amb using Biotin-Lys-tRNA_CUA. (A) For the determination of GLP-1R and GLP-1R-L260amb protein yields, 5 µL of TM was precipitated by hot TCA precipitation followed by liquid scintillation counting. Standard deviations were calculated from triplicates. (B) For qualitative characterization by autoradiography, a 5 µL aliquot of the translation reaction mixture (TM) was precipitated with acetone. The resulting pellets were resolved in sample buffer. Samples were then electrophoretically separated on a 10% SDS-PAGE gel followed by autoradiography. No template control (NTC) was carried along as negative control. Full-length suppression product and termination product are indicated by arrows. The structural model of the seven transmembrane domains of GLP-1R is indicated on the right. The red star represents the incorporated Biotin-label.

Figure 3

Purification of cell-free synthesized, membrane-embedded GLP-1R and GLP-1R-L260amb comparing two different types of magnetic beads. Synthesis of GLP-1R was performed using the Sf21 cell-free system in batch mode. For the synthesis for GLP-1R-L260amb Biotin-Lys-tRNA_CUA was supplemented to enable the synthesis of full-length suppression product. (A,B) Autoradiograms showing the different purification fractions of microsomal membrane-embedded GLP-1R and GLP-1R-L260amb purified using either SpeedBead Sera-Mag Neutravidin beads or SpeedBead Sera-Mag Streptavidin-blocked magnetic particles: For acetone precipitation and further analysis by SDS-PAGE 4% from the microsomal fraction (MF) before purification (input) and 70% of binding supernatant (BSN), washing fraction 1 (W1) and elution fraction 1 (E1) were obtained. No template control (NTC) was carried along as negative control. (C,D) Pie chart showing the percentage distribution of the fractions collected during the purification of GLP-1R and GLP-1R-L260amb using either SpeedBead Sera-Mag Neutravidin beads (C) or SpeedBead Sera-Mag Streptavidin-blocked magnetic particles (D). Amount of cell-free synthesized protein which was added to the beads as input, was set to 100%.

Figure 4

Fluorescence analysis of GLP-1R-L260amb-eYFP fusion protein. GLP-1R-L260amb-eYFP was synthesized in the Sf21 cell-free system in batch mode either in presence of Ala-tRNA_CUA to obtain fluorescent suppression product or in absence of Ala-tRNA_CUA to obtain non-fluorescent termination product. For the detection of background fluorescence, no template control (NTC) was analyzed either. (A) Fluorescence image of prepared samples. After cell-free synthesis, an aliquot of 20 µL translation mixture (TM) was transferred to an IBIDI slide and analyzed by confocal laser scanning microscopy. Excitation wavelength: 488 nm (eYFP) and 633 nm (bright field), 60× objective with oil, 2.3× zoom. (B) Overlay of confocal fluorescence image and brightfield to visualize colocalization of microsomes and fluorescence signal. (C) Overlay of confocal fluorescence image and brightfield of magnetic beads incubated with microsomal membrane-embedded GLP-1R-L260amb-eYFP. In detail, the microsomal fraction (MF) of GLP-1R-L260amb-eYFP was incubated with 10 µg SpeedBead Sera-Mag Neutravidin beads for 1 h at 4 °C, washed twice and transferred to an IBIDI-slide for fluorescence analysis. As a control, untreated beads were analyzed in parallel. For better visibility, the contrast of all images was adjusted equally.

Figure 5

Probing the accessibility of bead-captured GLP-1R extracellular domain (ECD) by bead-based ELISA. GLP-1R was synthesized using the batch-formatted Sf21 cell-free synthesis system (1×) as well as repetitive cell-free protein synthesis (4×). Translation mixture (TM) was fractionated in supernatant (SN) and microsomal fraction (MF). (A) Scheme of the implementation of the bead-based ELISA with cell-free synthesized GLP-1R. (B,C) MF was immobilized using magnetic SpeedBead Sera-Mag Neutravidin beads without elution of target protein to obtain bead-captured GLP-1R for further analysis. Monoclonal anti-hGLP-1R antibody (B11, Santa Cruz Biotechnology) and human anti-GLP-1R recombinant antibody (clone 3F52 creative biolabs) were analyzed for the binding of microsomal membrane-embedded and bead-captured GLP-1R ECD. No template control (NTC) and a bead control were carried along as negative controls. Standard deviations were calculated from duplicates (antibody B11) or quadruplicates (antibody 3F52). For the statistical analysis, a t-test for two independent samples was performed with p ≤ 0.05 (*) and p ≤ 0.01 (**).

Figure 6

Ligand-binding assay of radioactive-labeled exendin-4 peptide ligand to bead-captured, microsome membrane-embedded GLP-1R. Microsomal fractions of cell-free synthesized GLP-1R were harvested from a standard Sf21 in vitro transcription/translation reaction and incubated with SpeedBead Sera-Mag Neutravidin beads. In addition, microsomal fractions of cell-free synthesized GIPR and a no template control (NTC) reaction were treated likewise and used as controls to monitor background binding activities. Bead-captured microsomal fractions were analyzed for the binding of radioactive-labeled exendin-4 peptide ligand. Percentage values show the fraction of radioactive-labeled peptide ligand bound to bead-captured microsomal fractions. GLP-1R and NTC values were calculated from quadruplicates, GIPR values were calculated from triplicates. For the statistical analysis, a t-test for two independent samples was performed with p ≤ 0.01 (**) and p ≤ 0.001 (***).