Protein Expression and Purification Selection Chart

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.

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.

Application Kit Advantages
High-Yield Expression NEBExpress MBP 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.
NEBExpress Cell-free E. coli Protein Synthesis System Uses a highly active cell extract and T7 RNA Polymerase promoter to routinely achieve yields of 0.5 mg/ml.
Cell-Free Expression NEBExpress Cell-free E. coli Protein Synthesis System Quickly generates analytical amounts of protein from DNA plasmids or linear templates; amenable to scale-up
PURExpress® In Vitro Protein Synthesis Kit Quickly generates analytical amounts of protein from DNA plasmids or linear templates
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 PURExpress® In Vitro Protein Synthesis Kit Fusion to MBP enhances solubility of proteins in E. coli.*
K. lactis Protein Expression Kit Utilizes K. lactis eukaryotic folding pathway.
Affinity Tag Chromatography IMPACT™ Kit Utilizes an intein-chitin binding domain (CBD) tag on either the N- or C- terminus, offering single-step purification.
NEBExpress MBP 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 resin.
NEBExpress Cell-free E. coli Protein Synthesis System Compatible with all types of affinity purification; target protein can be synthesized from any T7 promoter vector
Post-translational Modification K. lactis Protein Expression Kit Secretion of both N- and O- glycosylated proteins.
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).


* Kapust and Waugh (1999) Protein Science, 8, 1668–1674. PMID: 10452611