title: Physiological effects of TGF(alpha)-PE40 expression in recombinant Escherichia coli JM109.
abstract: Physiological effects of isopropyl-thiogalactopyranoside (IPTG) induction were examined in Escherichia coli strain JM109 expressing a fusion protein composed of transforming growth factor alpha and a 40-kD portion of Pseudomonas aeruginosa exotoxin A (TGF(alpha)-PE40) under control of the tac promoter. Fermentations at the 15-L scale in complex medium compared growth and metabolite profiles of the untransformed JM109 host strain, the strain transformed with the vector lacking the TGF(alpha)-PE40 open reading frame (JM109[pKK2.7]), and the strain with the complete plasmid for TGF(alpha)-PE40 expression (JM109[pTAC-TGF57-PE40]). Metabolite and growth profiles of JM109 (pTAC-TGF57-PE40) cultures changed significantly in IPTG-induced versus uninduced cultures. Prior to induction, glucose was metabolized to acetate or completely oxidized to CO(2). Following induction, pyruvate was also excreted in addition to acetate. In the absence of inducer, pyruvate was excreted by JM109 (pTAC-TGF57-PE40) only when dissolved oxygen levels fell to less than 10% of saturation (microaerobic rather than anaerobic conditions). The untransformed JM109 host strain or JM109 (pKK2.7) did not excrete pyruvate in the presence or absence of inducer, although JM109 (pKK2.7) exhibited a pattern of growth following addition of IPTG that closely resembled JM109 (pTAC-TFG57-PE40). Fermentations of JM109 (pTAC-TFG57-PE40) in a synthetic medium supported lower expression levels, but resulted in similar alterations in metabolite profiles. Induction in synthetic medium resulted in pyruvate excretion without further acetate accumulation. Taken together, these data suggest that one consequence of TGF(alpha)-PE40 expression in JM109 is altered patterns of pyruvate oxidation.
authors.affiliation: Merck Sharp & Dohme Research Laboratories, Department of Biochemical Process R & D, Rahway, New Jersey, USA.
authors.forename: H A
authors.initials: HA
authors.lastname: George
authors.forename: A L
authors.initials: AL
authors.lastname: Powell
authors.forename: M E
authors.initials: ME
authors.lastname: Dahlgren
authors.forename: W K
authors.initials: WK
authors.lastname: Herber
authors.forename: R Z
authors.initials: RZ
authors.lastname: Maigetter
authors.forename: B W
authors.initials: BW
authors.lastname: Burgess
authors.forename: S M
authors.initials: SM
authors.lastname: Stirdivant
authors.forename: R L
authors.initials: RL
authors.lastname: Greasham