CROSS-FLOW FILTRATION FOR THE SEPARATION OF INCLUSION-BODIES FROM SOLUBLE-PROTEINS IN RECOMBINANT ESCHERICHIA-COLI CELL LYSATE

Many proteins produced in large quantities in Escherichia coli form insoluble aggregates called inclusion bodies. The separation of inclusion bodies from soluble protein in lysed cell extracts by crossflow filtration was examined using a recombinant strain of E. coli containing a gene encoding a portion of gp41, the transmembrane protein of the AIDS (HTLV-III) virus. The effects of crossflow rate, transmembrane pressure, initial concentration, pore size and ionic environment on the removal of soluble proteins were examined on a laboratory scale crossflow filtration unit by comparing the protein concentration in the feed and the filtrate under various operating conditions. The retention of soluble protein increased dramatically with increasing flux or transmembrane pressure. Increasing the crossflow rate did not reduce the retention of soluble protein as expected. Examination of permeate samples by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE) revealed that particulate material at the membrane surface did not cause sieving of specific proteins. Careful control of flux and transmembrane pressure in a constant volume diafiltration experiment allowed the removal of 87% of the soluble protein from the inclusion body suspension after three volume exchanges of buffer. © 1990.