This Isolation of proteins from cell lysate WORKS ® Optimization Procedure is intended to isolate proteins from lysed yeast or bacteria cultures. This optimization procedure uses a microfiltration (MF) or a high molecular weight cutoff ultrafiltration (UF) membrane that retains the cells and cell debris and allows the desired protein to pass freely through the membrane. The passage characteristics of the desired protein change with different buffers, temperatures, concentrations, and membranes. By examining the passage characteristics of the different MF membranes available in the appropriate process conditions, a well defined and executed process development study can identify the most efficient membrane and process conditions to achieve the required performance.
This Isolation of proteins from cell lysate protocol is intended for isolating recombinant proteins with a molecular weight of less than 30 kD from lysed cells. This process has been repeatedly implemented with consistent success in lysed E. coli and Pichia fermentations.
The concentration step passes the target protein from the fermentation broth by using a 150 kD regenerated cellulose (RC) membrane to pass the target protein freely into the permeate and retain the large molecular weight broth components, and the accumulated cell debris. The protocol calls for the broth to be concentrated to 5X prior to starting the diafiltration. The required diafiltration buffer is added in small semi-continuous aliquots to increase the efficiency of the diafiltration.
