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Research

Recombinant Protein Expression (E. coli system)

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Construction of Recombinant Protein in E. coli

We provide free consultation for all projects and can assist with the design, construction, and expression testing of vectors featuring various affinity tags (i.e., His, GST, FLAG) and/or fusion partners (i.e., TRX, MBP, EGFP) in order to maximize the expression level of the target protein in E. coli.

Choice of E. coli Host Cells

Depending on the nature of the heterologous target protein, we provide and utilize a number of different E. coli host strains to improve expression levels and to maximize the odds of proper folding. For example, Rosetta™ cells are used for protein constructs containing a high number of rare E. coli codons, and Origami™ cells are used to encourage proper disulfide bond formation.

Choice of Tag-cleaving Proteases

We maintain and utilize common proteases, such as Thrombin, PreScission and TEV. If there are concerns about affinity tags or fusion partners interfering with the structure and function of the target protein, we can provide reliable methods for removing affinity tags.

Improving Protein Solubility

In some cases, especially during overexpression in E. coli, the expressed protein accumulates as insoluble inclusion bodies, several strategies can be used to improve the solubility of the expressed protein. Our core is equipped with Thermo MaxQ high performance shakers for optimization of expression temperature to maximize soluble expression.

Insoluble Expression (of Protein and Peptide) and Refolding Screen

For proteins that express insolubly, we can provide purification under denaturing condition (e.g. in high concentrations of urea). We are also quite experienced in using DSF guiding refolding to determine suitable conditions to refolding by which to produce properly refolded protein from insoluble inclusion body preparations. We also routinely produce small peptides via insoluble expression in E. coli.