SHuffle strains can be easily grown to high cell densities, OD600 25–50, in batch culture using rich growth medium. Although process optimization for production of one’s specific protein will be required there are some general principles to follow when beginning the optimization.
A general batch fermentation medium that can be used with SHuffle is: 2% soytone, 1% yeast extract, 2% glycerol, 37 mM KH2PO4, 120 mM Na2HPO4, 0.5 mM K2SO4, and 5 mM MgCl2. The pH of the medium should be adjusted to 7.0 with 30% NH4OH.
Glycerol is typically used as a carbon source when producing recombinant proteins in E. coli to achieve high cell densities. Typically, E. coli production strains grow well in media containing 5–10% glycerol.
However, our experience indicates that SHuffle does not grow well in high glycerol concentrations. SHuffle strains grow optimally when the glycerol concentration is below 2%. Accordingly, to achieve high cell densities multiple bolus doses of glycerol may be added so long as the final glycerol concentration in the fermentor is not greater than 2%. One bolus dose at the induction point is usually sufficient.
Other carbon sources such as glucose can be used but organic acid accumulation and inhibition of growth may occur when the cells are grown at low dissolved oxygen.
Other factors that affect recombinant protein yield in SHuffle include inducer concentration, induction temperature, and dissolved oxygen. A standard fermentation process involves pre-induction growth at 30°C followed by induction of protein expression at 16–20°C for 16–20 hours. Many of the recombinant proteins produced in SHuffle are more active when the dissolved oxygen is controlled between 5–10% of air saturation.
We also find it is sometimes beneficial to use a nonsaturating concentration of inducer to obtain correctly folded, soluble protein. For common promoters such a Ptac, Ptrc, Plac, and PT7, IPTG concentrations in the range of 20–100 µM may result in the greatest yield of soluble, active protein. This recommendation is especially relevant when expressing a target protein fused to maltose binding protein since the pMALc vectors employ a strong tac promoter.
A standard 10 L SHuffle fermentation scheme is depicted in Figure 1.
Preferably a single colony from a fresh transformation—a colony struck from a glycerol stock may also be used—is inoculated into 500 mL of LB broth. The culture is shaken at 30°C and 300 rpm for 12–16 hours until the OD600 reaches two. Then, the appropriate volume of seed culture is added to the fermentor to obtain an initial OD600 of 0.01.
The fermentor temperature is maintained at 30°C, pH is controlled at 7.0 by automatic addition of 30% NH4OH and 23 NH3PO4, and the dissolved oxygen level is maintained at 10% of air saturation.
Once the culture reaches 10 OD600 it is ready to induce (Figure 2). Typically the temperature is reduced to 16°C, IPTG is added to 0.1 mM, and the induced culture is allowed to grow for an additional 18 hours.
SHuffle strains yield proteins with complex arrays of disulfide bonds. These E. coli strains are ideally suited for rapid screening of targets that may eventually be produced in higher organisms such as yeast or CHO cells.
Commercial protein production using a high density fermentation process can also be achieved with SHuffle strains. For example, we have achieved purification yields of 3.2 grams of recombinant target protein per 562 grams of cells (wet weight) from a single 10 L fermentation run.
Other examples emerging from our studies and the work of collaborators highlight the potential of the SHuffle strains—full-length functional IgG molecules and single-chain polypeptides with up to 35 cysteines (17 possible dsbs) have been produced. In simpler cases, proteins with only one disulfide bond may also be produced at higher levels if cytoplasmic expression is employed.
Most proteins with multiple disulfide bonds require the activity of a disulfide bond isomerase in order to reach their native folded state. Therefore, the availability of DsbC within the SHuffle cytoplasm sets these strains apart from other first-generation trxB, gor protein-expression hosts.