Cell-free translation experiments of integral membrane proteins in single-compartment liposomes

Summary

Membrane proteins provide the appropriate molecular mechanisms that allow the controlled entry of useful molecules into the cell and permit the export of intracellular products to the extracellular compartment. Enzymes in the cell membrane synthesize the molecules that make up the cell membrane (e.g., saturated and unsaturated fatty acids, mercurial lipids, glycerides, sphingolipids, sterols, polyisoprene). Enzyme complexes in the cell membrane are responsible for electron transfer, generating electrochemical gradients, which are utilized by the delicate cytosolic ATPase motor to produce ATP. Membrane proteins can also harvest light, provide mutant receptors to transduce information about the binding of external molecules to the cell membrane into cellular responses via signaling cascades; provide surface contacts necessary for the differentiation and assembly of embryonic stem cells into more complex tissues; and help to stimulate an antigenic response to pathogenic infections.

Author: Burgess et al., Translated by Wei Chen, this experiment is from "Protein Purification Guide".

Operation method

Cell-free translation experiments of integral membrane proteins in single-compartment liposomes

Move

gene cloning

This method requires two-step PCR (Blommel et al., 2006; Thao et al., 2004). Figure 37.2 provides a vector map of pEU-HSCB and an example of primer design for cloning His6-bacterial retinoids (Blommel andFox, 2007). Using this vector and primer design scheme, the T E V protease was able to cleave the MGHHHHHHHAIAENLYFQ sequence so that serine became the first amino acid of the mature protein. The tobacco mosaic virus omega sequence is a translational enhancer sequence (Sawasaki et al., 2000). The Flexi Vector cloning method (Blommel et al., 2006) can be used to transfer the cloned genes into any (or all) of the vectors mentioned in Table 37-1, as well as a number of other commercially available vectors (see http :// for other examples), by adding the Sg/ I and PmeI cleavage sites to the positions indicated in the corresponding primers. www.promega.com for other examples). The s a c & C A T cassettes shown in Fig. 37. 2 provide a toxicity screen when the corresponding transformants are grown on plates containing 5 % sucrose. In this way positive clones containing the target gene can be screened more efficiently, while sac& C A T also provides resistance against chloramphenicol. The p F I K homology region enhances the efficiency of transferring cloned genes between different Flexi vectors (Blommel et al., 2006).

In the example given in Figure 37. 2 B, the 5' forward primer used in the first step of the P C R contains a gene-specific nucleotide sequence and incorporates an invariant sequence at the end of the 5' segment that encodes part of the T EV cleavage site. The 3' reverse complementary primers shown in Figure 37. 2 C contain gene-specific nucleotide and PmeI sites. Other genes can be cloned by substituting gene-specific sequences when designing primers.

The second PCR step uses a universal forward primer (Fig. .37. 2B) containing sequences that complement the T E V digest site and the Sg/I site. Generic reverse PCR primers were used to replicate the PmeI site and spiked with additional nucleotides (Fig. 37.2 B) . In the second PCR step, a portion of the product from the first PCR step is added to a new PCR reaction system containing the universal forward and reverse primers to obtain a PCR product that is correct for Flexi Vector cloning.

PCR product purification

PCR products are purified before SG/I/PMeI digestion.

Materials and Reagents

PCR purification kit [Quickstep 2 PCR purification kit (EdgeBio, Gaithersburg, MD)] containing SOPE resin, secure sterile sealing tape, forma Ultra 96-well plates and V-Bottom 9 6-well plates.

Experimental protocols

(1) Add 4 uL of well-suspended SOPE resin to 20 jlxL of the PCR product (from the F L E X I - T E V PCR protocol described above).

(2) Seal the plate with a secure sterile sealing tape and vortex. The suspension is allowed to stand at room temperature. Simultaneously prepare; performaUltra 9 6-well plates.

(3) Remove the adhesive seal from the top and bottom of the Performa Ultra 9 6-well plate and add the lid.

(4) Stack the Performa Ultra 96-well plate on top of a 9 6-well flat-bottomed microculture plate.

(5) Place the device in a lined plate carrier for centrifugation.

(6) Centrifuge at 850 g for 5 min.

(7) Centrifuge briefly to enrich the SOPE/PCR mixture to the bottom of the wells. Slowly transfer the S O P E / P C R reaction mixture directly to the Performs Ultra 96-well plate using a liquid pipette. Ensure that the liquid flows into the gel matrix. Add lid.

(8) Stack the Performa Ultra 96-well plate on top of the 96-well V - b o t t o m microplate.

(9) Place the device in a centrifuge plate tray and centrifuge at 850 g for 5 mIn. The eluate containing the purified PCR product is retained. The fraction can be stored at 20°C until ready for use.

Flexi vectors and PCR product digests

The following procedure involves digestion of pEU vector variants and purified PCR products with SG/I and PMEI prior to ligation. Additional descriptions of cloning using the FIexi Vector System can be found elsewhere in the literature (Blommer and Fox, 2007; Blommer and Fox, 2007; Blommer and Fox, 2007; Blommer and Fox, 2007).
et al., 2006). Successful Flexi V e c t o r cloning is important to avoid overdigestion of the P C R product and the Flexi vector. sgf1 has asterisk activity, and overdigestion removes the sticky ends and turns the digested vector into a flat end, which can result in the vector self-linking and producing a high background of clones without the inserted human fragment.

Reagents

5X Flexi Digestion Buffer (Promega); 10X Sgf1/PmeI Enzyme Mix (Promega); pEU Vector Variants (Table 37.1); purified PCR product [from step (9) of the PCR product purification protocol].

Ligation reaction

The digested and purified P C R product and p E U variant vector are ligated in this step. For the most efficient ligation reaction, it is important to use a high concentration of ligase.

Reagents

10X T4 DNA Ligase Buffer (Promega); high concentration T4 DNA Ligase (Promega); pEU Variants Carrier (from Flexi Carrier Digestion Reaction Step (2));

Purified PCR digest [from PCR product digestion reaction step (6)].

Experimental Program

(1) Set up a ligase master mix consisting of 225 ul of sterile deionized water, 100 ul of 10 X T4 ligase buffer, and 50 ul of highly concentrated T4 DNA ligase.

(2) Add 5.0 ul of purified PCR digest, 2.0 ul of digested pEU carrier, and 3.5 pL of Ligation Master Mix to each well of the new PCR plate. This is the ligation plate.

(3) Incubate the ligation plate at 25°C for 3 h in a thermocycler.

(4) Transform immediately or store the connection plate overnight at 4°C.

Conversion reaction

The following steps are for the transformation of receptor cells with the products of the linkage reaction. Receptor cells from both Invitrogen and P r o m e g a have been used successfully according to the manufacturer's experimental protocols.

Materials and Reagents

10 G chemoreceptor cells for transformation (LUCig en, Middleton, W I ); 10 G cell recovery solution (Lucigen); Junction plate [from step (4) of the Junction reaction]; Fishherb nmd sterile disposable Petri dishes (60 m m X 15 m m ) (Fisher Scientific, Pittsburgh, P A ); Y T agar plates containing 0.5% (W/V) glucose, 50 ug/m L kanamycin, and 5% sucrose. ,P A ); Y T agar plates containing 0.5% (W /V) glucose, 50 ug/m L kanamycin, and 5% sucrose for screening of p E U -H S C B carriers (Fig. 37. 2); ColiR oiler plate beads (N o v a g e n , Gibbstow n , N J ) .

The experimental program

(1) 10 G chemoreceptor cells were removed from an 80°C refrigerator and melted on ice.

(2) Dispense 10 ul of cells into pre-cooled PCR plates or strip tubes.

(3) Add I.0 ligand and mix well with the tip of the pipette gun.

(4) Incubate on ice for at least 5 m i n . Lock the thermocycler setting to 34°C.

(5) Heat shock at 34°C for 30 s (follow manufacturer's instructions for small reaction volumes).

(6) Return the transformed reaction to ice and incubate for 2 m i n .

(7) Remove from ice and add 80 fxL of recovery medium at room temperature.

(8) Incubate at 37°C for I h. Do not shake. Do not shake.

(9) While incubating the transformants, mark the bottom of % Y T plates containing the appropriate antibiotic and A1-H12, and add 5 to 10 sterile ColiRoller glass beads to each plate.

(10) Add all of the transformation reagents to each corresponding labeled plate. Shake the plate in a circular motion to disperse the liquid. Carefully remove the ColiRoller beads by turning the plates into a suitable waste container or place 1 000 % ethanol in the plate for next use.

(11) Incubate plates at 37°C overnight.

Purification of plasmid D N A

All reagents used for in vitro transcription and translation must be free of RNAase. Therefore, all glassware used to prepare cell-free reactions must be baked at 1800 °C for 3 h to remove the RNA enzyme. In addition, wear gloves and avoid talking and sneezing while handling reagents to prevent RNAase contamination of hands and saliva. Unless otherwise noted, all buffers must be sterilized by filtration with a 0.2 um filter and stored at 20 °C.

In addition, all reagents are prepared in 18 MΩ water (Milli-Q water, Millipore, Billerica, M A ) unless otherwise noted. Diethyl pyrocarbonate (DEPC)-treated water should not be used in this protocol because the degradation products of DEPC inhibit in vitro transcription and translation reactions.

Reagents

The 10X buffer for proteinase K contains 10 mmol/L Tris-HCl (pH 8.0), 50 mmol/L
EDTA and l % ( m/ V ) S D S

Protease K was purchased from S i g m a/Aldrich (St. Louis, M O ). An IO X protease K solution was prepared by adding 0.5 m g of protease K to I.0 m L of IO X protease K buffer. Dispense 10 ul of this protease K solution and store it at 80°C.

mRNA Preparation

In cell-free translation, m R N A is a necessary reactant for protein synthesis reactions. In order to achieve the highest transcriptional efficiency, a sufficient amount of m RNA must be added to saturate the ribosomes in the translation reaction.

Reagents

Contains Mg Transcription Buffer (5X ): 400 m m o l / L HEPE-KOH (p H 7.8), 100 m m o l / L magnesium acetate, 10 m m m o l / L spermidine hydrochloride, and 50 m m m o l / L DTT. Store at 20° C.

NTP solution: Contain 25 m m o l /L of ATP, GTP, CTP and UTP, prepared by sterile filtration of 100 m m o l /L NTP (Milli-Q water soluble) through a 0.2 um filter. The NTP solution was stored at 180°C. The NTP solution was also preserved in the refrigerator.

SP6 RNA polymerase and RNA enzyme inhibitor (RNI) were purchased from Promega.

Preparation of liposomes

Single-compartment liposomes are added to a cell-free translation reaction to capture newly translated membrane proteins. This step provides an alternative approach to solubilizing membrane proteins with detergents. The presence of a decontaminating agent would result in an inability to directly measure the function of the membrane protein.

Materials and Reagents

Soybean complete extract (200 % lecithin) was purchased from Avanti Polar Lipids (Alabaster, A L ).

Lipid rehydration buffer: 25 m m o l / L HEPES (p H 7.5), IOO m m o l / L NACL.

0.4 um and 0.I um track-etch polycarbonate membranes (Track-etch polycarbonate membrane) were purchased from Nucleopore (Pleasanton, CA).

A mini-extruder for the preparation of single-chamber liposomes was purchased from Avanti Polar Lipids.

Experimental Program

(1) Dissolve I g of soybean complete extract (200 % lecithin) in 3 m L of chloroform.

(2) Wash the lipid solution with a stream of N2 to remove most of the organic solvents. Dry the residual lipids in vacuum for 30 mIn.

(3) Resuspend dried lipids with 67 mL of lipid rehydration buffer. Vortex the lipid solution until homogenization. 3 to 5 freeze/towa cycles will assist in the hydration of the lipids.

(4) Formation of single chamber liposomes by means of a miniature extruder. The liposome solution is passed 11 times through a 0.4 m Tracketch polycarbonate membrane and 11 times through a 0.im Track-etch polycarbonate membrane.

(5) Dispensing and rapid freezing of liposomes. Liposomes prepared in this way can be stored at 1 80°C.

Wheat Germ Translation Reaction

Figure 37.3 compares the construction of a bilayer reaction with that of a dialysis reaction. The bilayer reaction utilizes the difference in density between the extract and the upper buffer to isolate the extract and mRNA from the other reactants. In this case, diffusion of substrate and product occurs throughout the buffer interface. Due to the simplicity of the setup, the double-layer cell-free translation reaction can be operated automatically (Sawasaki et al. 2002a; Tyler et al. 2005; Vinarov et al. 2004; 2006). According to our results, the bilayer reaction can produce about 0.2 m g / m L of different membrane proteins. However, the yield is limited due to the diffuse dilution of the reaction.

The method can be scaled up from 50 uL to 10 mL or larger volumes without an overall change in volumetric productivity. This approach allows for simple screening of a small number of proteins in smaller volumes, while larger volumes can be scaled up for proteins that have been characterized by small-scale protocols and found to be amenable to further study. Experimental protocols for cell-free translation using bilayers and automated batching of analytical reactions have been published (E n d o andSawasaki, 2006; Sawasaki et al.) For the translation of intact membrane proteins, we have found that the addition of unilamellar liposomes effectively modifies the translation reaction (Gorene and Fox, 2008; Nozawaet al., 2007).

(2) Add 5 porphyrin of the mRNA preparation to a single well of a %-well U-bottom plate.

(3) Add an additional 20uL of the translation mixture to the mRNA sample in each well and mix well.

(4) Carefully add 125 fxL I X Cover Buffer to form a bilayer. Be careful not to disrupt the bilayer structure as this will dilute the extract and reduce protein yield.

(5) Incubate the reaction at 26°C for 20 h without disrupting the bilayer structure.

(6) Protein translation levels can be determined by denaturing SDS-PAGE, with creatine kinase as an internal reference.

Experimental protocol for dialysis reaction

(1) Dissolve the purified mRNA starch with 50 uL of the translation mixture described in step (1) of the two-layer reaction protocol.

(2) Place the translation mixture into a 12 k D a M W C ◦ dialysis cup.

(3) Prepare a stock dialysis buffer by mixing 6.5 m L Milli-Q water, 2.0 m L 5 X reaction buffer, and 1.5 m L of 2 m m o l / L unlabeled amino acids. Ultrasonically mix the solution for 5 m i n and filter through a 0.2 um filter. Add 2.5 mL of Reserve Dialysis Buffer to each well of a 2 4-well plate.

(4) Suspend the dialysis cup into the Reserve Dialysis Buffer. Be careful not to get bubbles under the dialysis cups, as this may interfere with the replenishment of the additives.

(5) Cover the 24-well plate with Saran wrap to prevent evaporation of the Reserve Dialysis Buffer. Incubate the translation reaction for 16 h at 26°C.

(6) Protein translation levels can be determined by denaturing SDS-PAGE with creatine kinase as an internal reference.

Density gradient ultracentrifugation purification

Figure 37. 4 shows a schematic diagram of the purification of liposomes containing membrane proteins (produced by the cell-free translation technique in wheat germ). After the density gradient is assembled, one-step centrifugation separates the liposomes from the cell-free extracted proteins. In most cases, liposomes are greatly concentrated at the interface between the 3 0 % A c c u d e n z solution and the upper buffer.

The partitioning surface of the mass rehydration buffer.
(6) Analyze the protein content of each fraction by denaturing SDS-PAGE.

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