New England Biolabs, E5360L, NEBExpress® Cell-free E. coli Protein Synthesis System

The NEBExpress Related Categories NEBExpress, ®, Cell-free, E. coli, Protein Synthesis System,, Cell-Free Protein Expression,, Protein Expression Applications NEBExpress® Cell-free E. coli Protein Synthesis System,, High-throughput cloning and automation solutions,, Cell-Free Protein Expression, FAQ Q: Based on my application, should I use the NEBExpress® Cell-free E. coli Protein Synthesis System (NEB #E5360) or the PURExpress® In Vitro Protein Synthesis Kit (NEB #E6800)? A: The NEBExpress® Cell-free E. coli Protein Synthesis System and PURExpress In Vitro Protein Synthesis Kit are both suited for high yield protein synthesis of a variety of protein targets, under the control of T7 RNA Polymerase. Both products offer convenient reaction analysis by SDS-PAGE or by direct assay. Both systems are compatible with plasmid and linear DNA templates; mRNA can also be used as a template. PURExpress is a reconstituted system and therefore provides a more defined reaction environment with minimal nuclease and protease activity. Downstream applications such as directed protein evolution, translation studies and protein/protein interactions are particularly suited to PURExpress. PURExpress is also ideal for applications where components of the translational machinery are selectively left out (PURExpress Δ (aa, tRNA) Kit, PURExpress Δ RF123 Kit, PURExpress Δ Ribosome Kit), including non-natural amino acid incorporation, radiolabeling and ribosome studies. In PURExpress, all protein components (except ribosomes) are recombinant and His-tagged allowing for reverse purification. The NEBExpress® Cell-free E. coli Protein Synthesis System is preferable to isolate proteins that are His-tagged. In addition, it has a lower cost per reaction, making it more easily scalable to large volume applications. To compare PURExpress and the NEBExpress® Cell-free E. coli Protein Synthesis System by application, reference the application chart under the tools and resources tab. Q: What is the difference between NEBExpress® Cell-free E. coli Protein Synthesis System and the PURExpress In Vitro Protein Synthesis Kit? A: The PURExpress® In Vitro Protein Synthesis Kit is a reconstituted protein synthesis system where all necessary components needed for in vitro transcription and translation (including T7 RNA Polymerase) are purified from E. coli and mixed in a multicomponent solution. All protein components are His-tagged (except the ribosome). PURExpress® contains minimal nuclease and protease activities. The protein mix is combined with an optimized reaction buffer for coupled transcription and translation. The NEBExpress® Cell-free E. coli Expression System is a high activity lysate from E. coli, that in combination with an optimized reaction buffer, T7 RNA Polymerase, and RNase inhibitor, allow coupled transcription and translation. Both systems are compatible with plasmid and linear DNA templates; mRNA can also be used as a template. To compare PURExpress and the NEBExpress® Cell-free E. coli Protein Synthesis System by application, reference the application chart under the tools and resources tab. Q: Are the yields with NEBExpress® Cell-free E. coli Protein Synthesis System comparable with the PURExpress In Vitro Protein Synthesis Kit? A: Protein yield, using either the PURExpress In Vitro Protein Synthesis Kit or the NEBExpress Cell-free E. coli Protein Synthesis System, is dependent on the nature of the target protein and the reaction conditions. The NEBExpress Cell-free E. coli Protein Synthesis System routinely produces up to 0.5 mg/ml (up to 25 μg per 50 μl reaction). The PURExpress system routinely produces up to 0.25 mg/ml (up to 12.5 μg per 50 μl reaction). The conditions for a given target will require optimization of the incubation temperature and time, as well as the concentration of the template and supplements. Q: Can I express large proteins with NEBExpress Cell-free E. coli Protein Synthesis System? A: The NEBExpress Cell-free E. coli Protein Synthesis System can produce high molecular weight targets due to stabilization of the mRNA template in a low RNase environment. Proteins from 17 to 230 kDa have been successfully synthesized in an active form (unpublished results). For example, a functionally active 230 kDa protein, β-galactosidase from Streptococcus, was expressed using the NEBExpress Cell-free E. coli Protein Synthesis System. Q: Can I use the NEBExpress Cell-free E. coli Protein Synthesis System to incorporate unnatural amino acids or radiolabeled amino acids? A: For these applications, the PURExpress® In Vitro Protein Synthesis Kit (NEB #E6800) is the best option. Q: Can the reaction be scaled to smaller or larger volumes? A: Yes, the typical reaction volume is 50 µl, but can be scaled up or down linearly. All reaction components, including the DNA template, must be scaled proportionally. The system has been tested with reaction volumes as small as 1 µl and as large as 2 ml. Protein synthesis yields are comparable within this range. For small volumes, such as 1 µl, an acoustic droplet ejection liquid handler (or similar) should be used. For large volumes use a vessel with adequate headspace to allow for aeration and incubate with agitation. Q: What methods can be used to purify the synthesized protein? A: Any affinity purification method can be applied, as none of the endogenous proteins in the NEBExpress Cell-free E. coli Protein Synthesis System are tagged. If the synthesized protein contains a His-tag, NEBExpress Ni-NTA Magnetic Beads (NEB #S1423), NEBExpress Ni Spin Columns (NEB #S1427) or NEBExpress Ni Resin (NEB #S1428) can be used. If the synthesized protein contains a maltose-binding protein (MBP) tag, Amylose Resin (NEB #E8021), Amylose Resin High Flow (NEB #E8022) or Amylose Magnetic Beads (NEB #E8035) can be used. If the synthesized protein has a chitin binding domain (CBD) tag, Chitin resin (NEB #S6651) or Chitin Magnetic Beads (NEB #E8036) can be used. Q: Can I co-express different protein targets from different plasmids? A: Yes, more than one target can be expressed in a single reaction, the yields are similar to what is observed when each protein is expressed independently. Q: Do I need to purify my plasmid or linear DNA template prior to synthesis? A: Template purity is important for successful in vitro transcription/translation; we recommend the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110) or Monarch® Spin PCR & DNA Cleanup Kit (5 μg) (NEB #T1130) to isolate plasmid or linear DNA. In addition, we recommend using a DNA template concentration ≥100 ng/µl. Q: Are genes under bacterial promoter control expressed with the NEBExpress® Cell-free E. coli Protein Synthesis System? A: Although endogenous E. coli RNA polymerase is present in the cell lysate, there will be minimal expression of genes under bacterial promoters. Protein yields will be very low in comparison to the high protein synthesis driven by T7 RNA Polymerase. Q: What systems does NEB offer for protein expression and purification? A: The NEBExpress® MBP Fusion and Purification System (NEB# E8201) takes advantage of the strong Ptac promoter and the translation initiation signals of maltose binding protein (MBP) to enhance solubility and expression levels of a desired protein in E. coli. The resulting product is an MBP fusion protein, which is then purified and isolated in two easy steps: amylose elution followed by TEV Protease cleavage and Ni resin isolation results in a highly pure tag-free target protein The IMPACT™ System (NEB# E6901) utilizes engineered protein splicing elements (inteins) to purify recombinant proteins by a single chitin affinity column. This system distinguishes itself from other protein fusion systems by its ability to separate a recombinant protein from the affinity tag without the use of a protease. In addition, native recombinant proteins possessing a C-terminal thioester can be isolated for applications such as protein semisynthesis and site specific labeling. The K.lactis Protein Expression Kit (NEB# E1000) provides an easy method for expressing a gene of interest in the yeast Kluyveromyces lactis. Proteins may be produced intracellularly or be secreted using the supplied integrative expression vector pKLAC1. The PURExpress® In Vitro Protein Synthesis Kit (NEB# E6800) is an E.coli cell-free transcription/translation system reconstituted from purified components. The E.coli transcription/translation factors (except for ribosomes) are His-tagged. Target protein may be expressed from either linear of plasmid DNA templates containing a T7 promoter. The NEBExpress™ Cell-Free E.coli Protein Synthesis System (NEB #E5360) is an extract-based system derived from E. coli cells that have been engineered for high-level in vitro production of recombinant protein from genes cloned downstream of a T7 promoter in either linear or plasmid DNA templates. Efficient isolation of the recombinant target protein may be accomplished by using NEBExpress Ni-NTA Magnetic Beads (NEB #S1423), spin columns (NEB #S1427) or resin (NEB #S1428). NEBExpress™ Ni-NTA Magnetic Beads (NEB #S1423), NEBExpress™ Ni Spin Columns (NEB #S1427) and NEBExpress™ Ni Resin (NEB #S1428) – a portfolio of immobilized Nickel products for the isolation of poly-histidine tagged (His-tagged) proteins from cell lysates or cell-free protein synthesis reactions. Q: Will the target protein remain stable during the synthesis incubation at 37°C? A: Although this is a cell-extract based system, the protease level is minimal. The protein product remains stable during synthesis until the reaction is complete and ready for downstream analysis or purification. Reactions can also be stored at -20°C for further analysis. Q: Following the NEBExpress cell-free protein synthesis reaction, how should I analyze the protein? Is it possible to use native SDS-PAGE or other functional assays to determine the activity of the expressed protein? A: In vitro protein synthesis reactions produced by the NEBExpress Cell-free E coli Protein Synthesis System can be directly loaded onto either native or denaturing SDS-PAGE gels without the need for acetone or TCA precipitation. Reactions can also be directly tested, without protein purification, in functional assays (enzymatic, colorimetric, fluorescent, gel based, etc.), as long as the protein synthesis components (nucleotides, amino acids, buffer) do not interfere with the specific assay measurements. Q: Can I add an RNA polymerase (RNAP) other than T7 RNAP to my NEBExpress Cell-free E. coli Protein Synthesis reactions? A: No. The NEBExpress Cell-free E. coli Protein Synthesis System was designed to work specifically with the T7 RNAP provided. If your gene of interest is under control of a promoter other than T7, it is best to replace it using our Q5 site-directed mutagenesis kit (NEB #E0554S). Q: Can I use a different concentration or formulation of T7 RNA Polymerase in my NEBExpress Cell-free E. coli Protein Synthesis reactions? A: No. The NEBExpress Cell-free E. coli Protein Synthesis System was designed to work specifically with the T7 RNA Polymerase provided. Q: The protocol recommends shaking the reactions vigorously. Is this necessary? A: It is not necessary to shake reactions vigorously if there is sufficient headspace in the tubes containing the reactions. Shaking may improve yield, but it is not required. If you choose to shake your reactions, we recommend shaking at 800-1000 rpm in a temperature-controlled mixer (e.g. Eppendorf ThermoMixer or equivalent). Q: What size is the NEBExpress® Control DHFR-His Plasmid? A: The NEBExpress® Control DHFR-His Plasmid is 2775 bp Q: The protocol recommends shaking the reactions vigorously. Is this required? A: If there is sufficient headspace in the tubes containing the reactions, it is not necessary to shake reactions vigorously. Shaking may help to improve yield but is not required. If you choose to shake your reactions, we recommend shaking at 800-1000 rpm in a temperature-controlled mixer (e.g. Eppendorf ThermoMixer or equivalent).