TITLE: Functional Analysis of Membrane Proteins Produced by Cell-Free Translation
AUTHORS: Srujan Kumar Dondapati, Doreen A. Wüstenhagen, Stefan Kubick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20 μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from </span><span style = "font-style:italic;">Escherichia coli </span><span style = "font-style:italic;font-style:italic;">(</span><span style = "font-style:italic;">E. coli</span><span>) and </span><span style = "font-style:italic;">Spodoptera frugiperda </span><span style = "font-style:italic;font-style:italic;">(</span><span style = "font-style:italic;">Sf</span><span>21) lysates. In the case of the </span><span style = "font-style:italic;">E. coli </span><span>cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the </span><span style = "font-style:italic;">Sf</span><span>21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cell-free synthesis, these microsomes are directly used for the functional analysis of membrane proteins.</span></div></div>

[STEPS]
?. [3.1 Preparation of Nanodiscs (NDs)]
First prepare  () containing ,  with  and .
[MSP protein solution]
[Tris]
[NaCl]
[EDTA]
?. [3.1 Preparation of Nanodiscs (NDs)]
Dissolve DMPC, DOTAP and DOPG lipids in cholate buffer at a concentration of  (see Note 2).
?. [3.1 Preparation of Nanodiscs (NDs)]
Set up the reactions: mix the lipid solutions (see Notes 3 and 4) with MSP as indicated in Table 1.
?. [3.1 Preparation of Nanodiscs (NDs)]
Incubate the reactions at  for . Then, biobeads SM-2 are added almost to 80 vol % of the Lipid-MSP mixtures and left to react at  for  and then centrifuged at . Remove the upper supernatants.
25 °C
25 °C
Centrifuge: 1000 34, 1 min
?. [3.1 Preparation of Nanodiscs (NDs)]
For each reaction , add a second set of 80 vol % biobeads-SM2 and incubate for . After centrifugation at , recollect the top solution. The finally recovered solution is further centrifuged at  and just the top solution (leaving the lower  volume) is recollected and analyzed by zetasizer.
Centrifuge: 1000 34, 1 min
Centrifuge: 5000 34, 5 min
5 µl
?. [3.1 Preparation of Nanodiscs (NDs)]
Place  of the recovered solution in a zeta sizer cuvette and measure the size intensity of the particles present in the solution. Figure 1 shows an exemplary measurement: the presence of a strong peak with maximum intensity at around 11.5 nm indicates the presence of NDs.  ABCDEF1ReactionDMPC (15 mM) (uL)DOTAP (15 mM) (uL)DOPG (15 mM) (uL)MSP (200 uM) (uL)Total (uL)214010-50100322525-5010043-50-501005450--5010065--50501007640-10501008725-2550100
20 µl
ABCDEF1ReactionDMPC (15 mM) (uL)DOTAP (15 mM) (uL)DOPG (15 mM) (uL)MSP (200 uM) (uL)Total (uL)214010-50100322525-5010043-50-501005450--5010065--50501007640-10501008725-2550100
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
BR and MtlA proteins are synthesized in cell-free systems using E. coli lysates. A typical  standard reaction comprises: containing the complete amino acid mix ( each)14C-labeled leucine, final concentration: ; 4 DPM/pmol) (see Note 5), and 10% (v/v) of the prepared ND. Set up the following reactions and controls (without NDs) (Table 2).
50 µl
[E. coli lysate containing T7 RNA-polymerase]
[reaction buffer]
[respective plasmid (0.75 μg/mL)]
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
Perform the protein synthesis in a thermomixer with shaking at .
Centrifuge: 500 33, 90 min, 37 10
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
Once the reaction is completed, all the qualitative and quantitative measurements are done by SDS-PAGE combined with autoradiography and TCA precipitation using 14C-leucine. Initially 2 ×  of the reaction mixture (suspension) is collected for TCA precipitation and 1 ×  for SDS-PAGE analysis. Next, centrifuge the remaining reaction mixture at .
5 µl
5 µl
Centrifuge: 16000 34, 10 min, 4 10
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
Collect the supernatant into a separate Eppendorf tube. 2 ×  of the supernatant is collected for TCA precipitation and 1 ×  for SDS-PAGE analysis.
5 µl
5 µl
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
For TCA precipitation, mix  aliquots (both suspension and supernatant) with  and incubate the mixture in a water bath at  for . Afterward, keep the reaction vessel  for .
5 µl
[trichloroacetic acid (TCA)]
80 °C
on ice
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
Filter the solution using the vacuum filtration system to retain the radiolabeled proteins on the surface of the filter paper. Wash proteins twice with TCA and twice with acetone, dry the filter papers for some minutes under the hood.
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
Transfer the dried protein-enriched filter papers to scintillation tubes (Zinsser Analytic), overlay with  and let it shake on the orbital shaker for at least . Measure the incorporation of 14C-leucine by liquid scintillation counting using the scintillation counter.
[scintillation cocktail]
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
For SDS-PAGE analysis, precipitate  aliquots (both suspension and supernatant) of the cell-free reaction mixtures by cold acetone precipitation: Add  of the water and  of ice cold acetone to the  aliquot and incubate  for . Next, centrifuge the mixture at . Discard the supernatant containing the acetone.
5 µl
45 µl
150 µl
5 µl
on ice
Centrifuge: 16000 34, 10 min, 4 10
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
Resuspend the pellets in  and load the samples on precast SDS-PAGE gels. Run the gel at 200 V for . After drying the gels for  at  using the Unigeldryer, visualize radioactively labeled proteins with the phosphorimager.
[NuPAGE® LDS Sample Buffer]
70 °C
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
BR in the presence of NDs is folded in a correct form and shows a purple color due to the conversion of all-trans retinal to 11-cis retinal (Fig. 2). Confirm this by analyzing the supernatant samples of BR (Step 10) by UV-Visible spectroscopy for measuring the BR-specific peak.  ABCDEFGH1ProteinE. coli lysate (uL)Reaction buffer (uL)14^C-Leucine (uL)Plasmid (5 nM) (uL)NDs (uL)Water (uL)Total (uL)2BR17.5201.252.553.75503MtlA17.5201.252.553.75504BR17.5201.252.503.75505MtlA17.5201.252.503.7550
ABCDEFGH1ProteinE. coli lysate (uL)Reaction buffer (uL)14^C-Leucine (uL)Plasmid (5 nM) (uL)NDs (uL)Water (uL)Total (uL)2BR17.5201.252.553.75503MtlA17.5201.252.553.75504BR17.5201.252.503.75505MtlA17.5201.252.503.7550
?. [3.2 Membrane Protein Synthesis in Prokaryotic Cell-Free Systems]
An absorbance peak at around 550 nm corresponds to the purple color of the BR protein. Supernatants from the control reaction (without NDs) should neither produce a purple color nor show an absorbance peak at 550 nm. This indicates that NDs help in correct folding of the BR. Figure 3 shows the synthesis of functional BR in the presence of NDs with saturated DMPC lipids doped with cationic (DOTAP) and anionic lipids (DOPG) (Reactions 1 and 6, Table 1). The intensity of the purple color, which indicates the presence of functional BR, varies with lipid composition with higher intensity in the presence of DOPG lipids. This clearly shows that doping of DOPG lipids along with DMPC helps in a better folding of the BR compared to DOTAP lipids. These results suggest that ND with different lipids can have influence on the functionality of the protein [14].
?. [3.3 Membrane Protein Synthesis in Eukaryotic Cell-Free Systems]
Cell-free protein synthesis is performed in the linked reaction mode by performing transcription and translation individually (see Notes 5 and 6). Details can be found in Refs. [4, 7]
?. [3.3 Membrane Protein Synthesis in Eukaryotic Cell-Free Systems]
In the first step, mRNA is generated by transcription from the plasmid DNA (plasmid encoding the KcsA protein, final concentration: 60 μg/mL) or E-PCR product (final concentration: 8 μg/mL) and is incubated for  at . The reaction is performed with , ,  ,, and 1 U/μL T7 RNA polymerase.
37 °C
[ HEPES–KOH buffer]
[MgCl2]
[NTPs]
[m7G(ppp)G-CAP analog]
?. [3.3 Membrane Protein Synthesis in Eukaryotic Cell-Free Systems]
Once the reaction is completed, purify the mRNA using DyeEx spin columns according to the manufacturer’s instructions.
?. [3.3 Membrane Protein Synthesis in Eukaryotic Cell-Free Systems]
Quantify the purified mRNA using the NanoDrop 2000c. After estimating the concentration, mRNA is used for protein synthesis.
?. [3.3 Membrane Protein Synthesis in Eukaryotic Cell-Free Systems]
In the second step, translation is performed by adding mRNA at a final concentration of . To monitor protein quality and quantity, add 14C-labeled leucine to the translation reaction mixture to yield a final concentration of . The translation mixture is incubated for  at .
27 °C
?. [3.3 Membrane Protein Synthesis in Eukaryotic Cell-Free Systems]
Quantification of the synthesized protein is done by hot trichloroacetic acid (TCA) precipitation followed by radioactivity measurements as described in Steps 10-13. To analyze homogeneity and molecular weight of in vitro translated proteins, take  aliquots of radiolabeled cell-free translation reaction mixtures for SDS-PAGE analysis using precast gels as explained in Step 14 and in Refs.[4, 5, 6, 6].
5 µl
?. [3.3 Membrane Protein Synthesis in Eukaryotic Cell-Free Systems]
KcsA forms a stable tetramer as seen in Fig. 4a. A prolonged time of translation leads to an increase in the intensity of the tetramer band in the vesicular fraction (VF). These observations correlate with the protein yields for different time periods (Fig. 4b).
?. [3.4 Preparation of Proteoliposomes]
Once the protein is synthesized, the vesicular fraction (VF) harboring the synthesized protein of interest is used for functional analysis. One can add either the translation reaction mixture harboring the microsomes and synthesized proteins directly onto the lipid bilayer or prepare proteoliposomes for a faster fusion, as described in the following steps:
?. [3.4 Preparation of Proteoliposomes]
of the cell-free translation mixture (TM) is centrifuged at . Supernatant (SN) is separated from the pellet (VF). VF contains the microsomes incorporating the MP of interest.
50 µl
Centrifuge: 16000 34, 10 min, 4 10
?. [3.4 Preparation of Proteoliposomes]
Resuspend the VF in  by pipetting up and down. Repeat the centrifugation step. After the second centrifugation, separate the SN from the VF. The VF contains the washed microsomes.
[PBS]
?. [3.4 Preparation of Proteoliposomes]
Dissolve the washed VF in . Resuspend vigorously.
[cholate buffer]
?. [3.4 Preparation of Proteoliposomes]
Mix  of the detergent resuspended microsomal fraction with  of the  lipid solution of choice (DOPG in our case) and keep the solution for rotation at  (see Note 3).
50 µl
50 µl
Centrifuge: 300 33, 60 min, 4 10
?. [3.4 Preparation of Proteoliposomes]
Add Biobeads-SM2 up to 80 vol% of the lipid mixture and incubate further  (ON) at .
4 °C
?. [3.4 Preparation of Proteoliposomes]
After ON incubation at , spin down all the Biobeads-SM2 by using a short centrifugation step for few seconds and collect the supernatant from the top. The proteoliposomes can be stored at  for few days when measured continuously (see Note 7).
4 °C
4 °C
?. [3.4 Preparation of Proteoliposomes]
For measuring the activity of the native translocon sec61 pore naturally present in the microsomes, add  to the vesicular fraction and incubate  for  (VF from step 1 in this section) to get a final concentration of  in . Puromycin combined with high salt concentration unplugs the ribosome from the microsomes and opens the sec61 pore [18].
[2.5 mM puromycin dissolved in 500 mM KCl]
on ice
[KCl]
?. [3.4 Preparation of Proteoliposomes]
Centrifuge the microsomal fraction once again at  and remove the SN. Prepare the proteoliposomes by repeating the steps 26–30 in this section.
Centrifuge: 16000 34, 10 min, 4 10
?. [3.5 Formation of Lipid Bilayers]
Lipids of interest are dissolved in octane at a concentration of 2 mg/mL. All the stocks of lipids are stored at .
-20 °C
?. [3.5 Formation of Lipid Bilayers]
Lipid bilayers are formed on MECA array chips mounted on the Orbit 16 System [16].
?. [3.5 Formation of Lipid Bilayers]
Lipid bilayers are formed as described in [16, 17]: Briefly,  is added to the measurement chamber containing the MECA chip. Once the buffer is added, all the electrodes will be in open (seal resistance of few MΩ).
[electrolyte solution]
?. [3.5 Formation of Lipid Bilayers]
For the automated bilayer formation on the 16 cavities in parallel, a small amount of approx. is pipetted on to the chip surface and painted with the help of a magnetic bar lying on the MECA chip [16]. Following pipetting of lipids, the bar is moved across the apertures in a circuitious fashion by performing one slow (45–180°/s) rotation of a counter magnet positioned below the chip with the help of the electromotor.
[DPhPC at 2 mg/mL in octane]
?. [3.5 Formation of Lipid Bilayers]
Lipid bilayer formation will be indicated by the change in resistance from MΩ to GΩ (for confirmation of lipid bilayer formation, see Notes 8 and 9).
?. [3.6 Functional Assessment of MPs]
Once the lipid bilayer is formed, add  prepared from the puromycin treated native microsomes (no protein synthesized) directly into the buffer chamber containing the lipid bilayers and wait for fusion.
[proteoliposomes]
?. [3.6 Functional Assessment of MPs]
After incubating the lipid bilayer with the proteoliposomes, measure activity from the voltage-clamped lipid bilayers of the sec61 pore by a single channel amplifier single channel amplifier (EPC-10) and the data acquisition software Patchmaster connected to the multiplexer electronics port of the Orbit16 system [14, 15, 16]. Recordings are done at a sampling rate of 50 kHz with a 10 kHz Bessel filter (see "Instrumentation and Software" section in Materials). Unitary currents are recorded from the voltage-clamped lipid bilayers.
?. [3.6 Functional Assessment of MPs]
Analyze data: Parameters like voltage ramps for monitoring currents are measured by using Patchmaster software and analyzed by the Clampfit software version 10.7.
?. [3.7 Case Study: Exemplary Data for sec61 and KcsA Proteins]
Figure 5 shows the functional analysis of native endogenous translocon sec61 protein activity from the microsomal membranes. The diagram shows the typical voltage-gating behavior of native sec61 translocon pore typically present in the microsomes obtained after fusion to the planar lipid bilayer. At lower voltages of −20 and −40 mV, the channel is open without any subconductance states. When the potential is increased to −60, −80, and −100 mV, one can notice the subconductance states. The probability of the channel to be either closed or in one of its open states was constantly reduced at larger potentials. From these activity studies, we observed that the rate of fusion of the protein to the lipid bilayer is increased with modified microsomes (proteoliposomes) when compared to the unmodified. This also shows us the efficiency of the fusion of microsomal proteoliposomes to the lipid bilayer. From Fig. 5a, we can see that with increase in the holding potential, the probability of the channel to be open continuously is reduced [18].The next step was to prove that this observation is applicable to the newly expressed proteins into the microsomes. The results of the activity measurements of KcsA synthesized in the eukaryotic cell-free system are presented in Fig. 6. After the synthesis reaction, steps 1and 2 in the Subheading 3.4 were repeated and the microsomes were extracted and used for the functionality measurements. Electrophysiology measurements were recorded and analyzed as shown in Subheading 3.6, steps 2 and 3. KcsA present in the microsomes showed the functional activity after fusion with the lipid bilayer. Parameters like single-channel conductance events (in the case of KcsA), all point histograms (see Note 10) were measured and analyzed. The protein exhibits a typical single-channel gating characteristic of KcsA at a transmembrane voltage of +60 mV, with a varying mean open time of few ms followed by the closure of the channel (inactivation) [13] (see Note 11).