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Bacterial Cell-Free Protein Expression
Bacterial in vitro expression systems use an efficient coupled transcription and translation reaction to produce high yields of full-length, functional protein from E. coli cell lysates. The time-consuming steps of cell-based protein production have been eliminated; in just a few hours, you can produce protein starting from plasmid or linear DNA.
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MembraneMax™ Protein Expression System
Membrane proteins play critical roles in cell-to-cell contact, surface recognition, cytoskeleton contact, signaling, enzymatic activity, and transport. Because of their varied cellular functions, they are ideal drug targets. However, membrane proteins are challenging because their expression can be toxic to cells or cause inclusion bodies, which limit protein yield and tedious optimization makes purification difficult and time consuming.
MembraneMax™ Protein Expression Kits overcome these challenges, allowing you to produce high yields of soluble (dispersed) membrane proteins using the MembraneMax™ reagent—a planar phospholipid membrane bilayer surrounded by a scaffold protein (also called a nanolipoprotein particle or NLP).
Key advantages
- High yields—nanogram to milligram
- Enhanced protein solubility—protein produced in NLP complexes
- Easy purification—His affinity tag
- Complete kit—simply add your gene of interest to get started
High yield for your downstream analysis
Expression of membrane protein is often very difficult due to insolubility in bacterial systems, in vivo toxicity, or simply low yield in mammalian systems. Using our Ultimate™ ORF collection as a source for membrane proteins, we tested membrane proteins representing differing sizes (8 kDa to 51k Da) and complexity (2 to 7 transmembrane domains). With the addition of the MembraneMax™ reagent, the majority of proteins were expressed >0.1mg/mL of protein (see table), which is sufficient and suitable for nearly every downstream application, including antibody production, crystallography, immunoprecipitation, ligand binding or functional assays, mass spectrometry, NMR, and protein array construction.
| Membrane protein (GenBank® accession no.) | Yield (mg/mL) | Membrane protein (GenBank® accession no.) | Yield (mg/mL) |
|---|---|---|---|
| Glycophorin (NM_002102.2) | 0.41 | Bacteriorhodopsin (J02755.1) | 0.33 |
| CRHR1 (NM_004382) | 0.30 | MGST2 (NM_002413.3) | 0.30 |
| Glycophorin E (NM_002102.2) | 0.30 | MGST2 (NM_004528.2) | 0.30 |
| Epiregulin (NM_001432.1) | 0.30 | b2 adrenergic receptor (NM_000024.3) | 0.30 |
| CKLF (NM_81640.1) | 0.30 | UNQ1887 (BC025781.1) | 0.30 |
| SPPL2B (BC001788.1) | 0.30 | CHRM1 (NM_000738.2) | 0.27 |
| PPAPDC1B (NM_032483.2) | 0.20 | CHRM2 (NM_000739.2) | 0.30 |
| The table above shows data for a small subset of proteins we have expressed using MembraneMax™ Protein Expression Kit. Over 64% of the proteins expressed at >0.1 mg/mL of reaction (n=32). | |||
Enhanced protein solubility
One of the most pervasive problems in protein expression is the insolubility of recombinant protein, often seen as inclusion bodies. This is especially acute when working with membrane proteins, which have both hydrophilic and hydrophobic domains as well as complex folding motifs. MembraneMax™ Protein Expression Kits use the novel MembraneMax™ reagent to enhance the solubility of overexpressed membrane proteins ranging in size and complexity.
Scalable and easy to use
Simply combine your T7 E. coli expression vector encoding your membrane protein with Expressway™ cell-free E. coli extract and MembraneMax™ reagent, incubate for 2 hours and purify. Within a day, you can generate milligrams of soluble membrane protein for downstream analysis, without detergents or tedious reconstitution protocols. MembraneMax™ Protein Expression Kits contain all the necessary reagents for successful expression of your membrane protein; the cell-free format allows high-throughput expression screening of multiple samples, or large-scale production of a single membrane protein.
Simple purification method
Purification challenges such as tedious and time-consuming testing of various detergents and extraction conditions to remove other endogenous membrane proteins while maintaining your protein's stability and function have notoriously impeded research on membrane proteins. In contrast, MembraneMax™ HN kits contain a His-tagged MembraneMax™ reagent, which simplifies purification without having to add a tag to your membrane protein. You also have the flexibility to design your DNA template with your favorite purification tag at the N- or C-terminus of your membrane protein, which when used with the MembraneMax™ HN module enables tandem purification for very clean membrane protein–NLP complexes. Whether you choose to work with a native membrane protein or a tagged version, our MembraneMax™ kits give you the flexibility you need.
See a detailed protocol for MembraneMax™ protein expression
See the MembraneMax™ Protein Expression Kits user guide
| Product | Features | 20 rxn | 100 rxn |
|---|---|---|---|
| MembraneMax™ Protein Expression Kit |
| A10632 | A10633 |
| MembraneMax™ HN Protein Expression Kit |
| A10634 | A10635 |
Expressway™ Expression System
Expressway™ Mini and Maxi Cell-Free Expression Systems
The Expressway™ Cell-Free E. coli Expression Systems are designed for in vitro transcription and translation of target DNA to protein in a single reaction. These flexible systems allow production of recombinant protein of interest from an expression construct in as little as 3 hours. Once purified, the resulting recombinant protein is suitable for use in other downstream applications including biochemical, physical, and structural characterization. The system is available in two formats: Expressway™ Maxi (scalable from 20–100 reactions) and Expressway™ Mini (20 reactions).
The Expressway™ Cell-Free E. coli Expression Systems provide a means to produce high levels of recombinant protein that may be easily detected and purified. These systems offer:
- High yields—milligram-level protein production within 4–6 hours through the use of a uniquely formulated Feed Buffer
- High-throughput compatibility—reactions can be performed in 96-well plates and once positive expression is detected, scale-up production can be performed
- Optimized TOPO® TA vectors—designed to minimize additional amino acid sequences that may interfere with native protein folding and functionality
- Producing proteins that are toxic to cells
Expressway™ Lumio™ Expression and Detection System
The Expressway™ Lumio™ Expression and Detection System takes advantage of the Lumio™ recognition sequence, a small, six amino acid sequence (Cys-Cys-Pro-Gly-Cys-Cys). The Lumio™ detection reagent binds the recognition sequence with high specificity and affinity, resulting in a bright fluorescent signal for real-time protein production analysis and immediate in-gel protein detection. In addition, Expressway’s specialized E. coli lysate, derived from a slyD mutant, eliminates nonspecific binding of the Lumio™ Green Detection Reagent to the endogenous SlyD protein and provides optimal background for detection of recombinant proteins. The Lumio™ Green Detection Kit is included in the system for real-time detection of protein synthesis as well as easy in-gel protein detection.
Vectors for bacterial cell-free expression
Find vectors compatible with Expressway™ Cell-Free Expression System and MembraneMax™ Protein Expression Kits.
| Vector | Cloning system | Fusion tag | Recommended cell-free expression systems |
|---|---|---|---|
| pEXP1-DEST | Gateway® | N-terminal Xpress™, 6xHis | E, M |
| pEXP2-DEST | Gateway® | C-terminal V5, 6xHis | E |
| pEXP3-DEST | Gateway® | N-terminal 6xHis, Lumio™ | E, M |
| pEXP4-DEST | Gateway® | C-terminal 6xHis, Lumio™ | E, M |
| pEXP5-NT/TOPO® | TOPO® | N-terminal 6xHis | E, M |
| pEXP5-CT/TOPO® | TOPO® | C-terminal 6xHis | E, M |
| E= Expressway™ Cell-Free Expression System. M = MembraneMax™ Protein Expression Kits. | |||
Antibodies for fusion tagged-proteins
Choose antibodies for your expressed protein based on the vector for the cell-free protein expression system you're using.
| For use with | Product | Epitope |
|---|---|---|
| pEXP1-DEST | Anti-Xpress™ Antibody | Detects the 8 amino acid Xpress™ epitope DLYDDDDK |
| pEXP1-DEST, pEXP3-DEST, pEXP5-NT/TOPO® | Anti-HisG Antibody, Anti-HisG-HRP Antibody, Anti-HisG-AP Antibody | Detects the N-terminal polyhistidine (6xHis) tag followed by glycine: HHHHHHG |
| pEXP2-DEST, pEXP4-DEST, pEXP5-CT/TOPO® | Anti-His (C-term) Antibody, Anti-His (C-term)-HRP Antibody, Anti-His (C-term)-AP Antibody | Detects the C-terminal polyhistidine (6xHis) tag: HHHHHH-COOH (requires the free carboxyl group for detection (Linder et al., 1997) |
| pEXP1-DEST, pEXP2-DEST, pEXP3-DEST, pEXP4-DEST, pEXP5-NT/TOPO®, pEXP5-CT/TOPO® | Penta-His Mouse IgG1 Monoclonal Antibody | Detects both N- and C-terminal polyhistidine (6xHis) tag |
| pEXP2-DEST | Anti-V5 Antibody, Anti-V5-HRP Antibody, Anti-V5-AP Antibody | Detects 14 amino acid epitope derived from the P and V proteins of the paramyxovirus; SV5 GKPIPNPLLGLDST (Southern et al., 1991) |
Ni-NTA and cobalt IMAC purification resins
Find immobilized metal affinity chromatography (IMAC) media for the purification of poly histidine-tagged proteins.
For Research Use Only. Not for use in diagnostic procedures.