Formation of recombinant proteins in Escherichia coli under control of a nitrogen regulated promoter at low and high cell densities.
The use of a modified Escherichia coli glnAP2 promoter results in the formation of both homologous and heterologous, cytoplasmic and periplasmic recombinant proteins in a nitrogen concentration dependent manner. As in the E. coli nitrogen regulatory system, glnAP2 controlled gene expression is induced when ammonium concentration in the growth medium is below 1 mM (nitrogen limitation), otherwise only extremely low expression of recombinant genes is observed. Both high cell density cultivations (HCDC) and low cell density cultivations (LCDC) gave similar results for inducibility and formation of the following recombinant proteins: chloramphenicol-acetyltransferase, phosphorylcholine binding mini-antibodies (scFv-dhlx of McPC603) and K1-streptokinase. Recombinant proteins were formed in quantities of about 2-3% of total cellular protein. At low cell densities, slightly higher quantities resulted under partial nitrogen limitations than under total nitrogen limitation. In contrast, high cell density cultivations resulted in lower product concentrations at partial nitrogen limitation compared with total nitrogen limitation. These lowered product concentrations were probably due to the very high amounts of K+ or Na+ ions which accumulated during pH-regulation, thereby disturbing growth. HCDC under partial nitrogen limitation decreased proteolysis of recombinant proteins, therefore reduced amounts of proteases are considered to be responsible.
Schroeckh
V
V
Hans Knöll Institute for Natural Products Research, Dept. Bioregulation, Jena, FRG.
Kujau
M
M
Knüpfer
U
U
Wenderoth
R
R
Mörbe
J
J
Riesenberg
D
D
D002455:Cell Division / Q000235:genetics; D002478:Cells, Cultured; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods; D009584:Nitrogen / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D009584:Nitrogen