Protein expression and purification can be very challenging because there is no single approach that is appropriate for every protein or every downstream application. Successful expression can be affected by each individual protein's intrinsic ability to fold, its solubility, its toxicity or its need for post-translational modifications. Additionally, the application for which the protein is ultimately to be used may dictate how the protein needs to be expressed or purified. It is therefore important to have a flexible set of tools and methods that can be applied to each unique project.
Each NEB expression technology offers different advantages which enable you to choose the strategy that best suits your protein expression and purification needs. Many of the expression vectors share a compatible poly-linker, enabling the gene of interest to be easily shuffled between systems. Various applications along with the recommended NEB kit are highlighted below.
|High-Yield Expression||pMAL™ Protein Fusion and Purification System||Substantial yields (up to 100 mg/L) in more than 75% cases tested; uses the strong Ptac promoter.|
|K. lactis Protein Expression Kit||Uses the strong LAC4 promoter; multiple integrations of plasmid results in higher yield.|
|IMPACT™ Kit||Uses the T7 promoter for high level regulated expression.|
|Cell-Free Expression||PURExpress® In Vitro Protein Synthesis Kit||Quickly generates analytical amounts of protein.|
|Co-Expression of Multiple Proteins||K. lactis Protein Expression Kit||Easily co-express 2–4 proteins.|
|PURExpress® In Vitro Protein Synthesis Kit||Bicistronic constructs or multiple plasmids with appropriate transcription and translation regulatory elements can be used.|
|Enhanced Solubility||pMAL™ Protein Fusion and Purification System||Fusion to MBP enhances solubility of proteins in E. coli.*|
|K. lactis Protein Expression Kit||Utilizes K. lactis eukaryotic folding pathway.
|IMPACT™ Kit||Utilizes an intein-chitin binding domain (CBD) tag on either the N- or C- terminus, offering single-step purification.|
|pMAL™ Protein Fusion and Purification System
||Fusion to MBP allows for purification on amylose resin.|
|K. lactis Protein Expression Kit||Vectors are sold separately that generate fusions to MBP allowing for purification on amylose restin.|
|Post-translational Modification||K. lactis Protein Expression Kit||Secretion of both N- and O- glycosylated proteins.|
|Periplasmic Expression||pMAL™ Protein Fusion and Purification System||Periplasmic expression enhances folding of proteins with disulfide bonds.|
|Secreted Expression||K. lactis Protein Expression Kit||Eliminates cell lysis step, simplifying purification.|
|Toxic Proteins||K. lactis Protein Expression Kit||Secretion of protein from the cell.|
|IMPACT™ Kit||Can express the toxic gene in two pieces and ligate proteins together using intein mediated protein ligation (IPL).|
|pMAL™ Protein Fusion and Purification System||Can export toxic proteins into periplasmic space.|
|PURExpress® In Vitro Protein Synthesis Kit||Cell-free environment not affected by "toxicity" of expressed protein.|
|Protein Modification||IMPACT™ Kit||
Generates proteins with reactive ends (N-terminal cysteine and/or C-terminal thioester) allowing for labeling or ligation of proteins or peptides.
|PURExpress® In Vitro Protein Synthesis Kit||Allows introduction of modified, unnatural or labeled amino acids.|
|No Additional Amino Acid Residues||IMPACT™ Kit||Start of native protein is fused adjacent to site of intein cleavage.|
|pMAL™ Protein Fusion and Purification System||Start of protein is fused adjacent to protease site.|
* Kapust and Waugh (1999) Protein Science, 8, 1668–1674. PMID: 10452611