MembraneMax™ Protein Expression Kits | for expression of recombinant membrane proteins in a cell-free protein synthesis system

The MembraneMax™ Protein Expression Kits are designed for in vitro expression of soluble membrane proteins from template DNA in a single, scalable reaction that is amenable to high-throughput. This versatile system contains all the components for the production of recombinant membrane protein of interest from an expression construct in less than 4 hours with microgram to milligram yields. Once purified, the resulting recombinant membrane protein is suitable for use in other downstream applications including biochemical, biophysical, and structural characterization.

The MembraneMax™ Protein Expression Kits are offered in four configurations: MembraneMax™ Protein Expression Kit and MembraneMax™ HN Protein Expression Kit, each available in two sizes (20 reactions and scalable from 20-100 reactions).

Advantages of MembraneMax™ Protein Expression

• Production of soluble and monodispersed membrane protein population
• Microgram to milligram quantities of membrane protein in a homogeneous population achieved by cell-free expression
• Convenient purification/enrichment scheme for both native and tagged membrane proteins
• Expression of toxic membrane proteins that otherwise show poor expression in vivo and compromised yield in cell based systems
• Easy, scalable membrane protein synthesis reactions that are amenable to high-throughput for a wide range of expression needs
• Convenience of a complete kit with all the reagents supplied

Possible Applications

Recombinant membrane proteins that are expressed using the MembraneMax™ Protein Expression Kits can be used in a variety of downstream applications upon purification, such as structural and functional studies, ligand binding, antibody production, and X-Ray crystallography, and studies with mutant proteins.

Note: There are many factors that affect protein yield, solubility, and function. Therefore, your purified protein might not be suitable for all the downstream applications listed above.

Components of the System

The major components of the MembraneMax™ Protein Expression Kits include:

• MembraneMax™ Reagent containing native (MembraneMax™) or polyhistidine-tagged (MembraneMax™ HN  nanolipoprotein particles that are optimized for high-yield production, increased stability, and enhanced solubility of the membrane protein of interest
• An optimized E. coli extract (Zubay, 1973) for increased stability of DNA constructs during transcription and translation and increased production of soluble membrane protein
• An optimized reaction buffer composed of an ATP regenerating system (Kim et al., 1996; Lesley et al., 1991; Pratt, 1984) to provide an energy source for protein synthesis
• An optimized feed buffer containing salts and other substrates (Kim and Swartz, 1999) to replenish components depleted or degraded during protein synthesis, thus enhancing recombinant membrane protein yield
• Amino acids (–Met) required for protein synthesis to occur
• Methionine provided separately to allow production of labeled recombinant protein, if desired
• Proprietary T7 Enzyme Mix containing T7 RNA polymerase and other components optimized for T7-based expression from DNA templates (Studier et al., 1990)
• The pEXP5-CT/bR control plasmid (expressing bacteriorhodopsin) and all-trans retinal (cofactor for bacteriorhodopsin) for use as a positive control for membrane protein expression in a rapid colorimetric activity assay
• The pEXP5-NT/CALML3 control vector for use as a positive control for protein expression with Expressway™ Expression Modules

MembraneMax™ Reagent

The MembraneMax™ Protein Expression Kits deliver high yields of soluble membrane proteins using the proprietary MembraneMax™ Reagent, an optimized lipid-protein formulation with cellular membrane-like properties containing nanolipoprotein particles (NLPs) available in native (MembraneMax™) or polyhistidine-tagged (MembraneMax™ HN; HN: His-tagged NLP) formulations.

NLPs are discoidal particles of approximately 10 nm in diameter consisting of a proprietary formulation of a scaffold protein ring that encloses a planar DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) lipid bilayer. When combined with the highly efficient Expressway™ Cell-free E. coli Expression System from Invitrogen, the MembraneMax™ Reagent allows the production of microgram to milligram quantities of soluble membrane proteins (Katzen et al., 2008).

MembraneMax™ Reagent works conveniently without the need of any detergent or tedious reconstitution into membrane vesicles, because the membrane protein of interest is synthesized into an environment that mimics its cellular surroundings. Furthermore, with its high uniformity and consistent structure, MembraneMax™ Reagent minimizes the formation of membrane protein aggregates or clumping often caused by inconsistent size, shape and structure typically observed with microsomes or micelles. Upon completion of its synthesis, the membrane protein of interest is accessible from either side of the lipid bilayer.

How the System Works

The MembraneMax™ Protein Expression Kits combine the convenience of the Expressway™ Cell-free E. coli Expression System with the cellular membrane-like environment provided by the MembraneMax™ Reagent.

The addition of the DNA template into the reaction mixture initiates the MembraneMax™ Protein Synthesis reaction. While the MembraneMax™ Reagent ensures proper folding and stability of the membrane protein, the Expressway™ Cell-free E. coli Expression System (included in the kit) allows high levels of protein synthesis with a proprietary mix of an ATP regenerating reaction buffer, amino acids, and an optimized feed buffer that replenishes the salts, amino acids, and other substrates that are depleted during protein synthesis.

E. coli slyD–Extract

All Invitrogen Expressway™ Expression Modules supplied with MembraneMax™ and MembraneMax™ HN Protein Expression Kits contain an optimized E. coli slyD– extract. The slyD– extract promotes the high yield expression of fulllength, active protein from DNA constructs under the reaction conditions specified in this manual.
   
If you have used other Invitrogen Expressway™ Cell-free E. coli Expression Systems, note that some of the components including the Expressway™ IVPS E. coli Extract and the Expressway™ 2.5X IVPS Reaction Buffer supplied with older Expressway™ kits contain different formulations and may not be compatible with the Expressway™ Expression Modules included in MembraneMax™ and MembraneMax™ HN Protein Expression Kits.

For optimal results, use the components supplied in this kit to perform the protein synthesis reaction.

Bacteriorhodopsin protein

The Halobacterium salinarum bacteriorhodopsin (bR) (GenBank acc # J02755) protein is light driven proton pump with 7 α-helical transmembrane domains. Although its mechanism does not involve the activation of G proteins, bR has been used as a structural model for rhodopsin and other GPCR family members When retinal is covalently bound to the Lys-216 residue in the cofactor binding pocket of bR via Schiff base formation, a purple color and characteristic absorption peaks at 558 and 568 nm (trimer) or at 546 and 553 nm (monomer) indicate a correctly folded, functional bR, thus rendering the utilization of the bRretinal complex an ideal positive control for the expression of membrane proteins using the MembraneMax™ Reagent.

Experimental Outline

The table below describes the major steps required to synthesize your recombinant membrane protein of interest using MembraneMax™ or MembraneMax™ HN Protein Expression Kits.



Choice of MembraneMax™ Reagents

Correct MembraneMax™ Reagent

The choice of the correct MembraneMax™ reagent is crucial for successful expression and purification of your membrane protein of interest. Follow the guidelines below to choose the correct MembraneMax™ reagent for your expression and purification needs.

• MembraneMax™ Protein Expression Kit: Use this kit if you wish to express your membrane protein as a fusion protein containing an N- or C-terminal polyhistidine tag (6×His) or any other tag.
• MembraneMax™ HN Protein Expression Kit: Use this kit if you wish to express your membrane protein in its native state (i.e., untagged) or if you wish to use a tag other than 6×His.

Note:   MembraneMax™ HN Reagent contains a 6×His tag, therefore it cannot be used to express fusion proteins tagged with 6×His.

You can utilize tandem purification methods to approach 100% membrane protein-NLP complex purity if:

• your fusion membrane protein of interest contains a tag other than 6×His, and
• you have used MembraneMax™ HN Protein Expression Kit to synthesize your protein.

MembraneMax™ Reagent Choice Scenarios

The table below provides several hypothetical scenarios to guide you in your choice of the MembraneMax™ Reagent. This table does not contain an exhaustive list of affinity tags or purification systems that can be used with the MembraneMax™ Protein Expression Kits. For more information on expression vectors and protein purifications systems available from Invitrogen, refer to www.invitrogen.com or contact Technical Support

Minimum Requirements

Many T7-based expression vectors are suitable for use as templates for the Expressway™ Expression Module in MembraneMax™ Kits. At a minimum, these vectors must contain the T7 promoter, RBS, and T7 terminator with the suitable spacing and sequence configuration for optimal expression of protein.

The following section includes important information on T7-based Gateway® and TOPO® Cloning vectors that are recommended for use with the Expressway™ Module in MembraneMax™ Kits.

Important

The pEXP1-DEST, pEXP3-DEST, pEXP4-DEST, pEXP5-NT/TOPO®, and pEXP5-CT/TOPO® vectors, as well as any other vector system that would allow the expression of polyhistidine-tagged proteins, must be used with the MembraneMax™ Protein Expression Kit, and not the MembraneMax™ HN Protein Expression Kit.  Any expression vectors containing a sequence for C-terminal polyhistidine tag, including pEXP4-DEST and pEXP5-CT/TOPO®, can be used with the MembraneMax™ HN Protein Expression Kit, only if:

• the polyhistidine tag is not used for the purification of the membrane protein,

or

• the gene for the membrane protein of interest and the sequence for the polyhistidine tag are separated by a stop codon in the expression construct.

We recommend using N-terminal 6×His tags for generating fusion membrane proteins because of higher expression levels in the MembraneMax™ protein synthesis reactions as compared to the C-terminally tagged fusion proteins.

We do not recommend the pEXP2-DEST vector for use with the MembraneMax™ Protein Expression Kits because of low expression levels observed.

Recommended Expression Vectors

The following expression vectors, available separately from Invitrogen, are ideally suited for use with the MembraneMax™ Protein Expression Kits. For detailed instructions on cloning your gene of interest into an expression vector, refer to the manual for the specific vector you are using. For more information on T7-based vectors, refer to our website at www.invitrogen.com or contact Technical Support.

Note: See above for important information on using vector systems that allow the expression of polyhistidine-tagged recombinant fusion membrane proteins with MembraneMax™ HN Protein Expression Kits.

pEXP5-NT/TOPO® and pEXP5-CT/TOPO® Vectors

The pEXP5-NT/TOPO® and pEXP5-CT/TOPO® vectors available from Invitrogen are ideally suited for use with MembraneMax™ Protein Expression Kits. Both vectors include the following features:

• T7 promoter, RBS, and T7 terminator with spacing and sequence configuration optimized to allow the high levels of protein expression in MembraneMax™ Protein Expression Kits

• Adapted with topoisomerase I to allow highly efficient, 5-minute, TOPO® Cloning of Taq polymerase-amplified PCR products for rapid generation of expression constructs

• An N-terminal peptide containing the 6×His tag and a Tobacco Etch Virus (TEV) recognition site to allow synthesis of a recombinant fusion membrane protein that may be easily detected and purified (pEXP5-NT/TOPO® only). The TEV recognition site allows the AcTEV™ Protease-mediated removal of the N-terminal tag to generate nearly native recombinant protein with only 2 amino acids added to N-terminus)

• A C-terminal peptide containing the 6×His tag to allow synthesis of a recombinant fusion membrane protein that may be easily detected and purified (pEXP5-CT/TOPO® only)

pEXP1-DEST Vector

The pEXP1-DEST vector allows T7-based, high-level expression of recombinant fusion proteins in the Expressway™ Cell-free E. coli Expression System and can be used with the MembraneMax™ Kits. The vector contains the following elements:

• Bacteriophage T7 promoter for high-level, inducible expression of the recombinant membrane protein of interest in the MembraneMax™ Protein Expression Kit
• N-terminal Xpress™ and 6×His fusion tags for detection and purification of recombinant fusion proteins
• Two recombination sites, attR1 and attR2 downstream of the T7 promoter for Gateway® cloning of the gene of interest from an entry clone
• Chloramphenicol resistance gene (CmR) located between the two attR sites for
• counterselection
• ccdB gene located between the attR sites for negative selection

Note: We do not recommend the pEXP2-DEST vector for use with the MembraneMax™ Protein Expression Kits because of the low membrane protein expression levels observed.

pEXP3-DEST and pEXP4 Vectors

The pEXP3-DEST and pEXP4-DEST vectors are designed to allow T7-based, highlevel expression of N- and C-terminal tagged recombinant fusion proteins using the Expressway™ Lumio™ Cell-free Expression and Detection System, and can be used with the MembraneMax™ Kits. The vectors contain the following elements:

• Bacteriophage T7 promoter for high-level, inducible expression of the recombinant membrane protein of interest in the MembraneMax™ Protein Expression Kit
• N- or C-terminal 6×His fusion and Lumio™ tags for detection and purification of recombinant fusion proteins (pEXP3-DEST and pEXP4-DEST, respectively)
• An N-terminal peptide containing TEV recognition site to allow the easy removal of the N-terminal tags from your recombinant fusion protein using the recombinant AcTEV™ Protease (pEXP3-DEST only)
• Two recombination sites, attR1 and attR2 downstream of the T7 promoter for Gateway® cloning of the gene of interest from an entry clone
• Chloramphenicol resistance gene (CmR) located between the two attR sites for counterselection
• ccdB gene located between the attR sites for negative selection

   
Important

Any vector system that would allow the expression of polyhistidine-tagged proteins must be used with the MembraneMax™ Protein Expression Kit, and not the MembraneMax™ HN Protein Expression Kit. Any expression vectors containing a sequence for C-terminal polyhistidine tag can be used with the MembraneMax™ HN Protein Expression Kit, only if:

• the polyhistidine tag is not used for the purification of the membrane protein,

or

• the gene for the membrane protein of interest and the sequence for the polyhistidine tag are separated by a stop codon in the expression construct.

Sequencing

Once you have generated your DNA template, we recommend that you sequence the expression construct to confirm the presence and orientation of the gene of interest. If you have generated a fusion construct verify that your gene of interest is in frame with the appropriate N- or C-terminal tag.

Purifying the DNA Template

After you have generated the DNA template, you must purify the DNA before proceeding to the protein synthesis reaction. You may use a variety of methods to purify your DNA template including commercial DNA purification kits (see below) or CsCl gradient centrifugation. When purifying your DNA template, keep the following in mind:

• Do not gel-purify your DNA template. Purified DNA solution obtained from agarose gels significantly inhibits the protein synthesis reaction.
• For rapid isolation of high quality purified plasmid DNA, we recommend using the PureLink™ HiPure Plasmid Midi- or Maxiprep Kits available from Invitrogen. Other commercial DNA purification kits are also suitable.
• For rapid isolation of high quality DNA from PCR reactions, we recommend using the PureLink™ PCR Purification Kit available from Invitrogen.
• Ammonium acetate is not recommended for use in DNA precipitation as any residual contamination may inhibit translation. Use sodium acetate.
• Make sure that the purified DNA is free of RNase contamination. Wear gloves and use RNase-free reagents when preparing DNA.
• Make sure that purified DNA is free of excess ethanol or salt as both can inhibit translation. Note: Carefully wash ethanol precipitated DNA with 70% ethanol to remove excess salt and dry.
• Resuspend purified DNA in 1X TE Buffer or water to a final concentration of at least 500 ng/μl.

Introduction

After you have obtained purified template DNA, you are ready to synthesize recombinant membrane protein using the MembraneMax™ or the MembraneMax™ HN Protein Expression Kit. This section provides guidelines and the standard protocol to synthesize your protein.

RNase contamination may affect protein yield. To reduce the chances of RNase contamination, wear gloves and use RNase-free reagents when performing the protein synthesis reaction. To eliminate RNase from surfaces, use RNase AWAY™ available separately from Invitrogen.

Membrane protein yields may vary depending on the nature of the protein expressed and the template utilized, as well as the reaction conditions used during synthesis. For highest membrane protein yields, the reaction conditions must be optimized. 

Reaction Volumes

The volume of the protein synthesis reaction may be scaled, based on your needs. For screening reactions, the standard volume is 100 μl (50 μl initial reaction + 50 μl Feed Buffer), but this can be decreased to 25 μl reaction volume and increased up to 10 ml reaction volume. For scaling the reaction, adjust the volumes of the reagents accordingly. You can also use 96-well plates (or any other plate configurations) for high-throughput membrane protein synthesis with the reaction volumes adjusted appropriately for the size of the wells.

Amount of DNA Template

For a 100 μl protein synthesis reaction, use 1 μg of template DNA (plasmid or linear DNA). For a 2 ml reaction, use 10–15 μg of template DNA. For optimal results, purify DNA template before use.

Reaction Vessel

Use a reaction vessel that contains a large enough surface area to allow moderate mixing to occur. We recommend performing the 100 μl membrane protein synthesis reaction in a sterile, RNase-free 1.5 ml microcentrifuge tube. If you require larger reaction volumes, you may use sterile, RNase-free 50 ml conical tubes. Other reaction vessels including 96-well, 6-well, or 12-well untreated culture plates are suitable.

Incubation Conditions

• To obtain optimal protein yield, it is critical to mix the reaction thoroughly throughout the incubation period. We recommend using a thermomixer set to 1,200 rpm or a shaking incubator set to 300 rpm. Do not use stationary incubators such as incubator ovens or water baths as protein yields may be reduced by up to 30–50%.
• Incubate the protein synthesis reaction at a temperature ranging from 30°C to 37°C. The optimal temperature should be determined empirically. Higher protein yields are generally obtained with incubation at higher temperatures (i.e., 37°C). The MembraneMax™ Reagent solubilizes the membrane protein at both 30°C and 37°C; however, lower temperatures decrease the rate of translation.
• You may obtain your protein of interest in 1.5 hours of incubation after feeding (2 hours total). Many reactions yield 75% of total protein within the first hour. Although longer incubation times up to more than 4 hours will increase total protein yield, the buildup of lipids in the reaction mix that are displaced from the NLP bilayer by membrane proteins during synthesis could trigger the formation of polydispersed micelles.

Amino Acid Concentration

Use an amino acid concentration ranging from 1 mM to 4 mM in the protein synthesis reaction. The recommended amino acid concentration is 1.25 mM each, but may be adjusted according to the protein being synthesized and your application (see Using Unnatural Amino Acids, below).

Feed Buffer

Add 1 volume of Feed Buffer (containing Expressway™ 2X IVPS Feed Buffer and amino acids) to the protein synthesis reaction after the initial 30-minute incubation. Higher protein yields may be obtained by adding one half-volume of Feed Buffer at 30 minutes and one half-volume of Feed Buffer again at 1 hour after initiating the protein synthesis reaction. If you choose longer incubation times, you may add the Feed Buffer at empirically determined intervals.

Using Unnatural Amino Acids

Methionine is supplied separately in the kit to allow you to incorporate unnatural amino acids into your recombinant protein and adjust the amino acid concentration in the protein synthesis reaction. Depending on your application, you may use the following unnatural amino acids:

• Radiolabeled methionine: Use [³⁵S]-Methionine to produce radiolabeled protein for use in expression and purification studies.
• Heavy metal-labeled methionine: Use selenomethionine (Budisa et al., 1995; Doublie, 1997; Hendrickson et al., 1990) to produce labeled protein for use in X-ray crystallographic studies.

Note: See Performing the MembraneMax™ Protein Synthesis Reaction, for the recommended amounts of labeled and unlabeled methionine. When using selenomethionine, do not use any unlabeled methionine in the protein synthesis reaction.

Handling Reagents

• Do not store the E. coli slyD– Extract, 2.5X IVPS Reaction Buffer (–amino acids), or 2X IVPS Feed Buffer at –20°C or room temperature as this may result in loss of activity.
• Freezing and thawing the E. coli slyD– Extract, 2.5X IVPS Reaction Buffer (–amino acids), and 2X IVPS Feed Buffer once or twice is acceptable. However, you should avoid multiple freeze/thaw cycles as this may result in loss of activity. You may also aliquot the reagents and freeze them to minimize multiple freeze/thaw cycles.
• Although multiple freeze/thaw cycles do not affect the performance of the MembraneMax™ Reagent, keep the freeze/thaw cycles to a minimum.

    
Important

All-trans retinal is light-sensitive. Although it is provided in an amber bottle, you should keep its exposure to light at a minimum and store it in the dark after use.

Positive Control Vectors

There are two positive control vectors included in the MembraneMax™ Protein Expression Kits.

• pEXP5-NT/CALML3 is for use with the Expressway™ Expression Module, and expresses the N-terminally-tagged human CALML3 protein.
• pEXP5-CT/bR is for use with the MembraneMax™ and MembraneMax™ HN Expression Modules in the rapid colorimetric control assay, and expresses bacteriorhodopsin.

For details about the control vectors. To propagate and maintain the control plasmids:

1. Use 10 ng of plasmid to transform a recA, endA E. coli strain such as TOP10, DH5α™, or equivalent using your method of choice.


2. Select transformants on LB agar plates containing 100 μg/ml ampicillin.


3. Prepare a glycerol stock of a transformant containing plasmid for long-term storage.

Materials Needed

• Expression construct or other suitable DNA template (purified; resuspended in TE or water at a concentration greater than 500 ng/μl)
• For each reaction, sterile, RNase-free 1.5 ml microcentrifuge tubes or other suitable reaction vessels
• RNase-free pipette tips and microcentrifuge tubes
• Thermomixer (set to 30°C or 37°C and 1,200 rpm)
• Optional: Standard shaking incubator (set to 300 rpm) instead of thermomixer
• Optional: Labeled methionine (if producing labeled recombinant protein)

Supplied with the kit:

• MembraneMax™ or MembraneMax™ HN Reagent (thaw at room temperature)
• Expressway™ E. coli slyD– Extract (thaw on ice)
• Expressway™ 2.5X IVPS Reaction Buffer (–amino acids) (thaw on ice)
• Expressway™ 2X IVPS Feed Buffer (thaw on ice)
• T7 Enzyme Mix (keep on ice; store at –20°C after initial use)
• 50 mM Amino Acids (–Met)

Note: When thawing the 50 mM Amino Acids (–Met), the solution may have a brown or yellowish tint. This is normal and does not affect the activity of the amino acids.

• 75 mM Methionine
• DNase/RNase-free Water (supplied with the kit)
• Optional:   pEXP5-CT/bR control plasmid (0.5 μg/μl in TE Buffer, pH 8.0) and all-trans retinal substrate (10 mM in DMSO) (if performing the rapid colorimetric control reaction)

Scaling the MembraneMax™ Protein Synthesis Reaction

The MembraneMax™ Protein Expression Kits in their 100 reaction configuration are supplied with five tubes of the appropriate MembraneMax™ Reagent, as well as five tubes each of Expressway™ E. coli slyD– Extract (400 μl per tube), Expressway™ 2.5X IVPS Reaction Buffer (–amino acids) (400 μl per tube), and Expressway™ 2X IVPS Fee  Buffer (500 μl per tube).

It is possible to synthesize recombinant membrane protein in various reaction volumes, such as for screening experiments (i.e., 100 μl reaction) or to obtain larger amounts of protein (i.e., up to 10 ml per reaction). For small reaction volumes, we recommend the following:

1. Thaw on ice the MembraneMax™ or MembraneMax™ HN Reagent, E. coli slyD–Extract, 2.5X IVPS Reaction Buffer (–amino acids), and 2X IVPS Feed Buffer.


2. Remove the amount of MembraneMax™ or MembraneMax™ HN Reagent, E. coli slyD– Extract, 2.5X IVPS Reaction Buffer (–amino acids), and 2X Feed Buffer needed for the protein synthesis reaction and return tubes to a –80°C freezer.

Performing the MembraneMax™ Protein Synthesis Reaction

Use the protocol below to synthesize your recombinant membrane protein from the DNA template. The amounts given are for a standard 100 μl reaction; if you are scaling up the reaction, adjust the volume of reagents accordingly.

1. For each sample, add the following reagents to the appropriate reaction vessel
   
   

   Reagent	Protein Synthesis Rxn	Positive Control Rxn	Negative Control Rxn
   E. coli slyD– Extract	20 μl	20 μl	20 μl
   2.5X IVPS Reaction Buffer (–amino acids)	20 μl	20 μl	20 μl
   50 mM Amino Acids (–Met)	1.25 μl	1.25 μl	1.25 μl
   75 mM Methionine*	1 μl	1 μl	1 μl
   MembraneMax™ or MembraneMax™ HN Reagent	2 μl	2 μl	--
   T7 Enzyme Mix	1 μl	1 μl	1 μl
   DNA Template	1 μg	--	--
   pEXP5-CT/bR	--	2 μl	2 μl
   DNase/RNase-Free Water	To a final volume of 50 μl	To a final volume of 50 μl	To a final volume of 50 μl

   
   


2. Close the tube and incubate sample in the thermomixer (1,200 rpm) at 30–37°C for 30 minutes. During incubation, prepare the Feed Buffer by adding the following reagents to a sterile, RNase-free microcentrifuge tube for each sample. For multiple samples, you may scale up the volume of reagents used accordingly and prepare one master mix.
   
   

   Reagent	Protein Synthesis Rxn	Positive Control Rxn	Negative Control Rxn
   2X IVPS Feed Buffer	25 μl	25 μl	25 μl
   50 mM Amino Acids (–Met)	1.25 μl	1.25 μl	1.25 μl
   75 mM Methionine*	1 μl	1 μl	1 μl
   10 mM all-trans retinal	--	0.5 μl	0.5 μl
   DNase/RNase-Free Water	To a final volume of 50 μl	To a final volume of 50 μl	To a final volume of 50 μl

   
   
   *Note:  To generate radiolabeled membrane protein using [³⁵S]-methionine, add 1 μl each of [³⁵S]-methionine and unlabeled 75 mM methionine when initiating the reaction, then again in the feed buffer. To generate labeled protein using selenomethionine, use 2 μl of selenomethionine only, do not add unlabeled methionine.


3. After 30 minutes of incubation (from Step 2 above), add 50 μl of the Feed Buffer to the sample (total volume = 100 μl).


4. Cap the tube and return the sample to thermomixer (1,200 rpm). Incubate for 1.5–2 hours at 30–37°C as appropriate.


5. Place the reaction on ice and proceed to Analyzing Samples, or store the sample at –20°C for future processing or analysis.

Introduction

Once you have performed the membrane protein synthesis reaction, you may use any method of choice to analyze your sample. Generally, the amount of membrane protein produced in a MembraneMax™ Protein Synthesis reaction is sufficient to allow detection on a Coomassie-stained protein gel or by western blot analysis. If you hav  trace-labeled your membrane protein with [ ³⁵S]-methionine during synthesis, you can analyze your protein by autoradiogram. Alternatively, you can use enzymatic activity assays or affinity purification for more robust biochemical analysis.

• The rapid colorimetric control assay performed in parallel to the membrane protein synthesis reaction (see below) indicates whether the components of the MembraneMax™ Protein Expression Kit are functioning as intended.

• If you plan to analyze your sample using polyacrylamide gel electrophoresis, first precipitate the proteins with acetone to remove background smearing. A protocol to perform the acetone precipitation and other general guidelines for gel electrophoresis are provided on below.

• If you have performed trace labeling using [³⁵S]-methionine, you may use TCA precipitation to determine the amount of radiolabeled methionine incorporated and to calculate the yield of protein (see Determining Protein Yield).

Interpreting the MembraneMax™ Colorimetric Control Assay

Successful expression of the correctly-folded, functional bacteriorhodopsin (bR) in the presence of its cofactor all-trans retinal causes the reaction mixture to change from a pale yellow to a characteristic pink color in as little as 15 minutes. Longer incubation times (up to the recommended 2 hours or more) results in higher bR yields and stronger color change. See above for more information on the bacteriorhodopsin protein.

If the MembraneMax™ Protein Synthesis reaction proceeds correctly, you will observe the emergence of the characteristic pink color in your positive control reaction mixture, while the negative control mixture remains pale yellow. The control sample may also be analyzed by any one of the methods described below.

The expression levels of membrane proteins may vary from protein to protein depending on the nature of the protein, the configuration of the DNA template, and the conditions of the MembraneMax™ Protein Synthesis reaction. If you have used small reaction volumes for preliminary experiments or for screening purposes, you may not be able to visualize your membrane protein on a Coomassie-stained protein gel. In such cases, we recommend silver staining the protein gel, or using western blot analysis or autoradiography to visualize your membrane protein.

SDS-PAGE Analysis

To facilitate separation and visualization of your recombinant membrane protein by SDS polyacrylamide gel electrophoresis, Invitrogen offers a wide range of precast NuPAGE® and Novex® Tris-Glycine polyacrylamide gels and electrophoresis apparatus, as well as a large selection of staining kits and molecular weight protein standards, including Novex® Sharp™, BenchMark™, and HiMark™. For ordering information. For more information on the various electrophoresis products available from Invitrogen, visit www.invitrogen.com or
contact Technical Support.

Before SDS-PAGE analysis, you must first precipitate the protein with acetone to remove background smearing. A protocol to perform acetone precipitation and other general guidelines for gel electrophoresis are provided below.

Materials Needed for SDS-PAGE Analysis

• Acetone (room temperature)
• 1X NuPAGE® LDS or Novex® Tris-Glycine SDS Sample Buffer (or any other compatible sample buffer appropriate to the gel used)
• Appropriate polyacrylamide gel to resolve your protein of interest
• Appropriate protein standard and stain (e.g., Novex® Sharp™ Protein Standard, SimplyBlue™ SafeStain)

Note: You may also use standard SDS-PAGE gels and sample buffers.

Acetone Precipitation

Use the following protocol to precipitate your proteins prior to loading on the polyacrylamide gel for analysis. Before starting this procedure, make sure that you have an appropriate gel for your protein size, or use one of the pre-cast polyacrylamide gels available from Invitrogen.

1. Add 5 μl of the protein reaction product from Step 5, to 20 μl of acetone. Mix well.


2. Centrifuge for 5 minutes at room temperature in a microcentrifuge at 10,000 × g.


3. Carefully remove the supernatant, taking care not to disturb the protein pellet.


4. Resuspend pellet in 20 μl of 1X NuPAGE® LDS Sample Buffer.


5. Heat at 70–80°C for 10–15 minutes and centrifuge briefly.

Proceed to Polyacrylamide Gel Electrophoresis

Note: Alternatively, samples may be stored at –20°C until needed.

Polyacrylamide Gel Electrophoresis

1. Load 5–10 μl of the sample from Step 5, above, on an SDS-PAGE gel and electrophorese following manufacturer’s recommendations. You may also save your sample by storing at –20°C, if desired.


2. Depending on your assay of choice, perform the following:

Note: For radiolabeled proteins, the signal may be enhanced by placing the gel in a commercially available reagent that enhances the signal. Dry the gel and expose to x-ray film for 1–4 hours.

Detecting Fusion Proteins

If you have expressed your recombinant membrane protein with a fusion tag from pEXP1-DEST, pEXP3-DEST, pEXP4-DEST, pEXP5-NT/TOPO®, or pEXP5-CT/TOPO®, you may perform western blot analysis and detect protein expression utilizing an antibody appropriate to the epitope tag. For more information on antibodies, refer to www.invitrogen.com, or contact Technical Support

Detection of Lumio™-tagged Proteins

If you are using pEXP3-DEST or pEXP4-DEST expression vectors, you can choose to express your recombinant membrane protein fused to the Lumio™-tag, a tetracysteine sequence tag that is specifically recognized and bound by the Lumio™ Detection Reagent, thus allowing both real-time and in-gel protein detection with high sensitivity. For more information on the Lumio™ Technology, see the Lumio™ Green Detection Kit manual or the Expressway™ Lumio™ Cell-free Expression and Detection System manual, both available at www.invitrogen.com, or by contacting Technical Support


Detecting CALML3 Control Protein

If you use pEXP5-NT/CALML3 as a positive control for protein expression with the Expressway™ Expression Module, you may be able to detect the CALML3 fusion protein on a Coomassie-stained gel using 5 μl of the reaction. A typical 100 μl protein synthesis reaction should yield at least 75 μg of the 19.5 kDa fusion protein.

The CALML3 fusion protein expressed from pEXP5-NT/CALML3 is not a membrane protein. It is provided as a positive expression control for the Expressway™ Expression Module. If the rapid colorimetric control assay provides a positive result (i.e., the reaction mixture turns pink), you do not need to use the pEXP5-NT/CALML3 plasmid. However, if the positive control reaction mixture remains a pale yellow, you may try expressing CALML3 using the Expressway™ Expression Module in order to pinpoint the source of the glitch in your reaction.

A negative colorimetric assay followed by positive expression of the CALML3 fusion protein would implicate a degraded MembraneMax™ Reagent, while the lack of CALML3 protein expression would point to the degradation of the reagents in the Expressway™ Expression Module as a potential source.

Note: All MembraneMax™ Protein Expression Kit reagents are guaranteed for six months if stored properly.


What to Do Next

Once you have verified expression, you may use the recombinant membrane protein in any downstream application of your choice. If you plan to use the recombinant protein for structural analyses including x-ray crystallography, note that you must purify the recombinant protein before use. Use any method of choice to purify your recombinant protein.

• If you have cloned your gene in frame with the N-terminal or C-terminal polyhistidine tag in your expression vector, you may purify your recombinant fusion protein using a metal-chelating resin such as Ni-NTA or ProBond™.
  
• Similarly, affinity purification with a metal-chelating resin can be employed to purify a native recombinant membrane protein or a recombinant protein containing a tag other than polyhistidine if it is synthesized using the MembraneMax™ HN Kit.
• A recombinant membrane protein containing an affinity tag other than a polyhistidine tag (e.g., GST or MBP) can be purified using the appropriate affinity chromatography method in tandem with a metal-chelating resin (such as Ni-NTA or ProBond™) provided that the membrane protein is expressed using the MembraneMax™ HN Kit.

Note: Other metal-chelating resins are also suitable.

Introduction

If you have included radiolabeled methionine in the membrane protein synthesis reaction, you may use TCA precipitation to determine the amount of radiolabeled methionine incorporated and to calculate the yield of protein.

Determining Total Counts

1. Mix and spot 5 μl of each radiolabeled reaction from Step 5, on a glass microfiber filter (Type GF/C; Whatman, Cat. no. 1822-021).


2. Set aside and let dry. Do not wash or TCA precipitate these filters.

Performing TCA Precipitation

Two protocols are provided below for performing TCA precipitation; one to perform standard TCA precipitation and one to perform TCA precipitation using a vacuum filtration device (e.g. Millipore 1225 Sampling Manifold). Choose the protocol that best fits your needs.

Performing Standard TCA Precipitation

1. Mix and spot 5 μl of each radiolabeled reaction from Step 5, on a separate set of individual glass fiber (GF/C) filters and allow to air dry for approximately 5–10 seconds.


2. Place filter in a beaker and wash once with cold 10% TCA for 10 minutes at room temperature while shaking gently (use approximately 10–20 ml per filter).


3. Wash with 5% TCA for 5 minutes at room temperature while shaking gently. Repeat wash.


4. Rinse filters with methanol to facilitate drying.


5. Allow filters to dry, place in scintillation vials, and add scintillation fluid. Count samples in a scintillation counter.


6. Proceed to Calculating Protein Yield.

Performing TCA Precipitation Using a Vacuum Filtration Device

1. Aliquot 5 μl of each radiolabeled reaction from Step 5 - Experimental Overview, into separate glass tubes.


2. Add 100 μl of 1 N NaOH to each reaction and incubate for 5 minutes at room temperature.


3. Add 3 ml of 10% TCA to each glass tube and incubate tubes at +4ºC for 20 minutes.


4. Wet individual glass fiber (GF/C) filters with 10% TCA and place onto the vacuum filtration device.


5. Turn the vacuum on and pour the TCA solution from each glass tube into a sample well.


6. Wash filters twice with 5% TCA.


7. Wash filters once with 100% ethanol. Leave the vacuum on for 1 minute to allow the filters to dry.


8. Turn the vacuum off and remove the filters. Place the filters in scintillation vials, and add scintillation fluid. Count samples in a scintillation counter.


9. Proceed to Calculating Protein Yield

Calculating Protein Yield

Use the equations below to calculate the yield of protein obtained. You need to determine the pmoles of methionine present in your specific reaction. Remember to account for both radiolabeled and unlabeled methionine. You also need to determine the total counts incorporated using TCA precipitation (see above).


Background counts=             Precipitable counts obtained in a mock reaction in the absence of DNA

Total counts=                                         total cpm per 5 μl spotted ×   total reaction volume
                                                                                                                               5                          
                                                                                             
                                                                                                    total counts
Specific activity=                                                                pmoles of methionine



pmoles methionine                                  [ ( TCA precipitable counts-background counts)  X  total reaction volume
incorporated=                                                                                                                                             5
                                                                        ______________________________________________________
                                                                                                                     special activity



pmoles of protein=                                             pmoles of methionine incorporated into protein
                                                                                        number of methionines in protein

Yield of protein (in μg)=                                        moles of protein × molecular weight of protein
                                                                                                                   10 ⁶

Introduction

The presence of the N-terminal or C-terminal 6xHis tag in the pEXP1-DEST, pEXP3-DEST, pEXP4-DEST, pEXP5-NT/TOPO®, and pEXP5-CT/TOPO® vectors allows purification of your recombinant protein with a metal-chelating resin such as ProBond™ and Ni-NTA. This section provides guidelines for purification.

pEXP3-DEST and pEXP5-NT/TOPO® vectors contain a Tobacco Etch Virus (TEV) recognition site that allows the removal of the N-terminal tag from your recombinant fusion protein using the AcTEV™ Protease.

ProBond™ and Ni- NTA resins

ProBond™ and Ni-NTA are nickel-charged agarose resins that can be used for affinity purification of:

• Fusion membrane proteins containing the 6xHis tag
• Native membrane proteins synthesized using the MembraneMax™ HN
• Membrane proteins containing an affinity tag other than polyhistidine if they are expressed using MembraneMax™ HN (tandem affinity purification)

To purify your fusion protein using another metal-chelating resin,  refer to the manufacturer’s instructions.

Guidelines for Purification

Use these guidelines for purifying your polyhistidine-tagged membrane protein (expressed using the MembraneMax™ Protein Expression Kit) or if you have expressed your protein using the MembraneMax™ HN Protein Expression Kits (either native or as a fusion containing a tag other than polyhistidine). Remember to use criteria appropriate for purification under native conditions. For details, refer to the ProBond™ or the Ni-NTA manual, as appropriate.

1. Prepare the purification column containing ProBond™ or Ni-NTA agarose resin. After applying the resin to the column, wash with 4 volumes of water followed by 8 volumes of Binding Buffer (supplied with the kit;= 50 mM NaPO4, pH 8.0, 500 mM NaCl) to equilibrate.


2. Centrifuge the protein synthesis reaction at 15,000 × g for 10 minutes at room temperature to remove insoluble material.


3. Load the supernatant containing soluble protein onto the equilibrated resin and incubate (i.e., batch binding) for 30 minutes at the desired temperature.


4. Wash the column twice with 2 volumes of Binding Buffer each time.


5. Wash the column twice with 2 volumes of Binding Buffer containing 20 mM imidazole.


6. Elute the protein using an Elution buffer containing an appropriate amount of imidazole (e.g., 250 mM imidazole).


7. Analyze the fractions using SDS-PAGE.


8. Pool the desired fractions and dialyze, if necessary.

Introduction

This section provides an example of a typical protein synthesis experiment performed using the MembraneMax™ Protein Expression Kit and results obtained.

Experimental Conditions

In this experiment, Halobacterium salinarum bacteriorhodopsin gene (bR; GenBank accession no. J02755) was PCR amplified and TOPO® cloned into pEXP5-CT/TOPO® to generate the 3435 bp pEXP5-CT/bR expression vector.

Upon purification, 1 μg of pEXP5-CT/bR was used in a 100 μl protein synthesis reaction with the components supplied in the MembraneMax™ HN Protein Expression Kit (containing polyhistidine-tagged MembraneMax™ HN Reagent) according to the Experimental Overview above. The reaction mixtures contained 0.2 μCi/μl [35S]-methionine for trace labeling and 0.5 μl 10mM all-trans retinal for the colorimetric control assay. As a negative control, pEXP5-CT/bR plasmid from the same preparation was used to express bR in an in vitro protein synthesis reaction that lacked the MembraneMax™ HN Reagent.

5 μl aliquot from each reaction was electrophoresed on a NuPAGE® Novex® 4–12% Bis-Tris Gel and stained (B, Coomassie Stained) or exposed to x-ray film (C, Autoradiogram), while the remainder of the synthesis reactions was purified using the Ni-NTA Purification System, separated by SDS electrophoresis and Coomassie-stained (D). Panel A depicts the results of the colorimetric assay in the absence (–) or the presence (+) of the MembraneMax™ HN Reagent.

Introduction

Review the information in this section to troubleshoot your cell-free membrane protein expression experiment.