Protein overproduction in Escherichia coli: RNA stabilization, cell disruption and recovery with a cross-flow microfiltration membrane.
After optimizing overproduction of a heterologous gene product (chloramphenicol acetyltransferase, CAT) using an RNA stabilization vector * in Escherichia coli (Chan et al., 1988), a single step cell disruption and recovery method * for obtaining a product stream essentially free of cell debris was developed. The behavior of an RNA stabilization plasmid (pKTN-CAT) containing stabilizing intron RNA was investigated in two different media both in batch and chemostat modes. CAT production of pKTN-CAT was consistently higher (3- to 7-fold) than that of the control lacking the stabilization sequences (pK-CAT). Highest CAT production was observed for cells grown in minimal medium in batch mode and induced for CAT expression early in growth. CAT production of cells grown in the chemostat mode exhibited an optimal dilution rate of about 0.1 h-1. Enhancement of protein production by pKTN-CAT as compared to pK-CAT tended to be higher when grown in rich medium rather than in minimal medium. Presence of the RNA stabilization plasmid did not significantly alter the growth rate of the cell. Using a combination of chemical treatment (1 mM EDTA) and shear stress resulting from cross-flow in a stainless steel microfiltration membrane *, CAT was released into the medium through disruption of the E. coli cells. The permeate flux increased from 2000 to 9000 kg m-2 h-1 with increasing axial Reynolds number from 10,000 to 60,000 or increasing mean shear stress from 12 to 47 Pa. The turbidity of the permeate was approximately 4% that of the retentate over this range of axial flow rates, indicating excellent removal of cell debris. Also, the concentration of CAT in the permeate was equal to that in the retentate over this range of axial flow rates, indicating complete passage of protein through the membrane. Thus, using a combination of chemical treatment and fluid-induced shear stress in a cross-flow membrane module, we were able to disrupt and recover the heterologous protein in a stream low in debris.
Chan
W K
WK
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany.
Belfort
M
M
Belfort
G
G
D001709:Biotechnology / Q000379:methods*; D002458:Cell Fractionation; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005374:Filtration / Q000379:methods; D008567:Membranes, Artificial; D010957:Plasmids; D012329:RNA, Bacterial / Q000378:metabolism*
D003470:Culture Media; D008567:Membranes, Artificial; D012329:RNA, Bacterial; D015500:Chloramphenicol O-Acetyltransferase