Overproduction of recombinant proteins by escherichia coli — employing the combinations of two and three different types of multicopy plasmids — in a 60-litre working volume airlift tower loop reactor

Abstract

Bioprocess aspects of the overproduction of recombinant proteins were investigated in a 60-litre working volume airlift tower loop reactor by monitoring the cell mass concentration ( X), total cell count (TCC), number of colony-forming units (CFU), plasmid copy numbers, and the production of EcoRI and the fusion protein (EcoRI::SpA) activity produced by Escherichia coli JM 103 carrying the combination of the expression plasmids pRTF309 + or pMTC48, the repression plasmids pcI857 or pRK248cI, and the protection plasmid pEcoR4. The investigations were carried out in shake flasks, a 1·5-litre stirred-tank reactor and in a 60-litre airlift tower loop reactor (ATLR). In the absence of the protection plasmid, the cell mass concentration X, total cell count TCC, the number of colony-forming units CFU, and the product concentrations were very low due to the toxicity of EcoRI. The combination of expression, repression and protection plasmids yielded high enzyme activities in the shake flasks and in the small stirred-tank reactor with temperature shift induction of gene expression. By applying the expression plasmid pMTC48, it was possible to compare the enzyme activities with the indiction of the gene expression by the P R promotor (by thermal induction) as well as by the P LAcUV5 promotor (by chemical induction). The optimum thermal and chemical induction conditions were evaluated. In the 1·5-litre stirred tank, the optimal inducer (isopropylthiogalactoside, IPTG) concentration was much lower (0·5 m M) than that in the 60-litre airlift tower loop reactor (4 m M). In small reactors, the enzyme activity with thermal induction was five times higher than that with chemical induction and in agreement with the ratio of the corresponding promotor strengths. In the 60-litre reactor, however, the enzyme activities with chemical induction were higher than those with thermal induction. After chemical induction, the cell mass, total cell count, and number of CFU increased. After thermal induction, the cell concentration and total cell count stagnated and the number of CFU decreased to very low values, which could explain the lower performance in this reactor.