The expression of the fusion protein may be affected by a variety of factors such as the (a) E. coli strain, (b) cell growth conditions (e.g. temperature, aeration, cell density, IPTG concentration, etc.), (c) toxicity of the target protein, (d) codon usage and (e) structure and stability of mRNA. E. coli ER2566 cells are supplied in the kit (not competent) as a host for fusion protein expression from an IMPACT vector. High efficiency competent cells of this strain, T7 Express (NEB #C2566 ) are available seperately. For expression of toxic proteins T7 Express Iq Competent E. coli (High Efficiency) (NEB #C3016 ), T7 Express IysY/Iq Competent E. coli (High Efficiency) (NEB #C3013 ) and T7 Express IysY Competent E. coli (High Efficiency) (NEB #C3010 ) are also available. For some proteins soluble expression is achieved with the SHuffle T7 Express E. coli (NEB #C3029). Expression of a toxic protein may require lowering the culture temperature.
Induction of protein expression at 12-15°C can often help the folding and the solubility of the fusion protein and increase the cleavage efficiency of the intein.
For all protein samples remove a 40 μl aliquot and mix with 20 μl 3X SDS Sample Buffer. The samples are then boiled for five minutes before loading onto a gel. If necessary, the samples can be stored at -20°C for a few days.
The following protocol is provided as a general guideline (see Figure 5 on main product page).
- Inoculate 1 liter of LB medium, containing 100g/ml ampicillin, with a freshly grown colony. Using cells stored at 4°C for an overnight culture may lower the protein yield.
- Incubate the culture in an orbital shaker at 37°C until the OD600 reaches 0.5.
- IPTG is added to a final concentration of 0.4 mM for induction of protein expression. Before the addition of IPTG, an aliquot of cell culture should be removed and incubated separately as an uninduced control (sample 1, uninduced). Initially induction at 37°C for 2-4 hours can be tested for expression and solubility.
- Remove a sample (40 μl) and mix with 20 μl 3X SDS Sample Buffer for the total cell extract or induced protein sample (sample 2, crude cell extract or induced). The cells from the IPTG-induced culture are spun down at 5000 x g for 15 minutes at 4°C and the supernatant is discarded. The cell pellet can be stored at -80°C.
- The cell pellet from a one-liter culture is resuspended in 100 ml of the appropriate ice-cold Column Buffer.
The inclusion of nonionic detergents in the buffer can reduce nonspecific protein binding to the chitin resin during the affinity column step. Oxidation-sensitive proteins can be stabilized during purification by using the reducing agents TCEP [tris-(2-carboxyethyl)phosphine] or TCCP [tris-(2-cyanoethyl)phosphine] (0.1 mM) in the Column buffer. Egg white lysozyme is not recommended for cell lysis because it is known to bind and degrade chitin. Cell lysis reagents, such as B-PER® (Thermo Scientific, Rockford, IL), can be used.
- Lyse the cells by sonication on ice.
Sonicate in short pulses, keep the cell culture cold during sonication and do not allow the build up of bubbles/foam. The release of protein can be monitored by using Bradford Reagent (BioRad, Hercules, CA) or OD280. Continue sonication until the level of the released protein level reaches a maximum.
- Centrifuge the cells at 15,000 x g for 30 minutes at 4°C; the supernatant is the clarified extract to be loaded on the chitin column. Remove 40 μl from the supernatant for the clarified protein sample (sample 3, clarified lysate). Save the pellet at –20°C for future analysis.
Optimization of Expression
If the target fusion protein is present as an induced band in the crude cell extract but absent from the clarified cell extract, this may indicate a solubility problem. Also if in vivo cleavage is detected (the induced sample contains the intein-CBD and target protein) different induction conditions should be tested. Usually induction at lower temperatures and/or with lower IPTG concentrations results in increased solubility and improved folding and subsequent thiol induced cleavage. In order to optimize expression conditions we recommend splitting a one liter culture into several samples (100-200 ml each and testing for optimal expression conditions. The optimal incubation temperature and time for induction will vary depending on the target protein. We recommend varying induction temperature and time to optimize expression (37°C for 2-4 hours, 30°C for 4-6 hours, 22-25°C for 6-16 hours and 12-15°C overnight using 0.4 mM IPTG). One sample with no IPTG should be incubated as a control for uninduced cells. Varying IPTG concentrations (up to 1 mM) can also be tested. Lowering the IPTG concentration (0.01-0.1 mM) may also reduce the fusion protein expression in inclusion bodies. For low temperature induction (e.g. 12-15°C) the culture can be incubated at 37°C until the OD600 reaches 0.6-0.7.
Different E. coli T7 expression strains, such as T7 Express (NEB #C2566) and SHuffle T7 Express (NEB #C3029), can be tested for optimal soluble expression. The SHuffle strain has been shown to help soluble expression of some proteins with disulfide bonds.
Fusion protein expression can be examined by SDS-PAGE, followed by Coomassie staining or western blot analysis, or by an activity assay. Analyze samples from both the total cell extract (soluble and insoluble proteins) and clarified extract (soluble). If the fusion protein is not detected by Coomassie staining, a Western blot with the Mouse Anti-CBD Monoclonal Antibody may be performed.
If the cell pellet needs to be tested, dissolve the pellet from a 100-200 ml culture in 10-20 ml column buffer and mix 40 μl of the pellet suspension with 20 μl of the 3X SDS Sample Buffer. The pellet can also be dissolved in a buffer containing urea. This sample can be saved for analysis (by loading 5-10 μl on a SDS-PAGE) if the fusion protein is not detected in the clarified (soluble) extract.