Large-scale production of genetically engineered CYP3A4 in E. coli: application of a jarfermenter.
To produce a large amount of CYP3A4, we applied a jarfermenter (ABLE, BMJ-PI or BMS-PI) to culture the genetically engineered E. coli cells harboring CYP3A4 along with NADPH-cytochrome P450 reductase (OR). The jarfermenter is a stirred bacterial culture vessel in which the pH, the dissolved oxygen (DO) and the temperature of a culture medium can be controlled. The expression of CYP3A4 in E. coli cells in the 500 mL of culture medium contained in the BMJ-PI (1 L vessel) (JFM-1) was examined by altering the parameters mentioned above. The highest expression of CYP3A4 in E. coli cells was attained when cultured at pH 6.0, at 30 degrees C under the DO of 0.1 ppm. The incubation was performed 18 hr after the addition of 1.5 mM isopropyl beta-D(-)-thiogalactopyranoside. The expression levels of CYP3A4 and the OR in the membrane fraction of E. coli cells were 267 nmol/L culture and 552 units/L culture, respectively. The CYP3A4 level was about three times higher than that obtained by incubation in a 500 mL flask (100 mL of medium) (84 nmol/L culture). The testosterone 6beta-hydroxylase activity of CYP3A4 expressed in the membrane fraction of E. coli obtained with the JFM-1 was examined. The apparent K(m) and V(max) values were 66.4 microM and 57.8 nmol/min/nmol CYP, respectively. Expecting the mass production of the CYP3A4 by a culture of E. coli, the possibility of a scale up of the culture with the BMS-PI (10 L vessel) (JFM-10) was examined. The optimal culture condition to achieve the highest expression of CYP3A4 with JFM-1 was employed. The expression levels of CYP3A4 and the OR obtained with JFM-1 and JFM-10 were almost equal. The total level of CYP3A4 obtained by using JFM-10 (5 L of medium) was calculated to be about 1.4 mumol. Based on these results, we confirm that the jarfermenter is a useful tool to produce large amounts of CYP3A4.
Kanamori
Yoichi
Y
Laboratory of Drug Metabolism, Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan.
Fujita
Ken-Ichi
K
Nakayama
Kazuo
K
Kawai
Hideki
H
Kamataki
Tetsuya
T