Increasing Inclusion Body Extractability And Recoverability By Altering Fermentation Conditions In High Cell Density Escherichia Coli Cultures
Volumetric protein production yields in Escherichia coli fermentations have improved with development of fed-batch high cell density cultures. The use of high cell densities has introduced need to obtain a better understanding of intercellular quorum sensing signals, glucose feeding strategies, and endotoxin removal during protein purification. The high purity of inclusion bodies (IBs) makes their extraction from host cell proteins in the cell lysate a standard procedure to simplify purification. The extraction is typically performed with centrifugation or tangential flow filtration, which are dependent on the IBs' size, density, and shape. The isolated IBs are then denatured, but the degree to which they are denatured can be dependent on their compactness. These qualities of IBs can all be influenced with fermentation growth conditions, but little research on this topic has been published. A synthetic version of melanoma antigen A (Syn-Melan-A) was produced in high cell density E. coli cultures in complex medium under various temperatures, inducer concentrations, and feeding strategies. IBs produced at 30 degrees C, 37 degrees C, and 42 degrees C were 316, 215, and 231 nm in size and had densities of 1.26, 1.30, and 1.31 g/cm3, respectively. No significant changes in IB size or density were detected in fermentations using inducer in the range of 0.05 to 0.50 mM. Changing from a pHstat feeding method that prevented glucose accumulation to an overfed culture could increase IB size to 340 nm and IB density to 1.31 g/cm3 at 37 degrees C. Similar results were obtained in a statistical design experiment in chemically defined medium. Scanning electron microscopy was used to show that Syn-Melan-A IBs were amorphous when produced at 30 degrees C, granulated at 37 degrees C, and smooth at 42 degrees C. Coomassie dye was used to show that these IBs had a 62%, 65%, and 68% accessibility, respectively, as determined by Bradford reaction. Different fermentation temperatures were used also to determine their influence on the IB characteristics of Melan-A, SSX-2, NY-ESO-1, and enhanced green fluorescent protein (EGFP). Different temperatures were only effective in altering IB characteristics of Syn-Melan-A. This susceptibility is associated with slower production rate of Syn-Melan-A compared to natural Melan-A.
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