Abstract
The established Escherichia coli expression vectors ptrc99a, pKK223-3, pPLλ, pAsk75, pRA95, and pRA96, which differ in copy number, mode of induction, selection marker, and use of par sequences for stabilization, were investigated for the stable expression of recombinant L-leucine dehydrogenase from Bacillus cereus with a view to large-scale production. Best results were achieved with pIET98, a runaway-replication system derived from pRA96. Expression of L-leucine dehydrogenase was controlled by its constitutive B. cereus promoter and depended on host strain, cultivation temperature, induction time, and media composition. After cell cultivation at 30 °C and shifting to 41 °C to induce plasmid replication, E. coli BL21[pIET98] yielded 200 U LeuDH/mg protein, which corresponds to >50% of the soluble cell protein. Continuous cultivation in a semisynthetic high-cell-density medium verified structural and segregational stability over 100 generations in the absence of a selection pressure.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Author information
Authors and Affiliations
Additional information
Received: 19 July 1999 / Received revision: 4 November 1999 / Accepted: 5 November 1999
Rights and permissions
About this article
Cite this article
Ansorge, M., Kula, M. Investigating expression systems for the stable large-scale production of recombinant L-leucine-dehydrogenase from Bacillus cereus in Escherichia coli . Appl Microbiol Biotechnol 53, 668–673 (2000). https://doi.org/10.1007/s002539900290
Issue Date:
DOI: https://doi.org/10.1007/s002539900290