Investigating expression systems for the stable large-scale production of recombinant L-leucine-dehydrogenase from Bacillus cereus in Escherichia coli.
The established Escherichia coli expression vectors ptrc99a, pKK223-3, pPLlambda, 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 degrees C and shifting to 41 degrees 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.
Institut für Enzymtechnologie der Heinrich-Heine-Universität Düsseldorf, Jülich, Germany.
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D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D050803:Leucine Dehydrogenase; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase