The use of elements of the E-coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations

The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is s uitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammoni um as the sole nitrogen source in the culture medium is below 1 mM. This ni trogen regulatory system was optimized by introduction of expression casset tes consisting of additional elements of the ntr-system These artificial co nstructions result in enhanced recombinant gene expression in the productio n phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter prote in) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC similar to P via plasmidal co-e xpression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. I t is assumed that under nitrogen excess conditions sigma(70)-RNA polymerase binds at glnA pi and thereby prevents most of the binding of sigma(54)-RNA polymerase at glnAp2. The optimized expression systems were successfully a pplied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced t hrough external nitrogen limitation under FIA-controlled concentration of g lucose as carbon source. (C) 1999 Elsevier Science B.V. All rights reserved .