Insertion of stabilizing loci in vectors of T7 RNA polymerase-mediated Escherichia coli expression systems: a case study on the plasmids involving foreign phospholipase D gene.
Plasmids carrying stabilizing loci were used in the expression of phospholipase D (PLD) gene fused with pelB signal sequence by a recombinant strain of Escherichia coli BL21 (DE3) using T7 RNA polymerase mediated expression system. By checking the living cell number and the percentage of the plasmid-bearing cells, it was found that the plasmids involving PLD gene were not stable under noninduced conditions and that, after the induction, the number of plasmid-bearing cells were rapidly decreased to almost zero. Then, a biologically stabilizing locus such as par B, ccd, or par was inserted into the plasmids. The newly constructed plasmids were maintained very stably in the recombinant cells until the cells were induced. However, after the induction, almost all the recombinant cells were rapidly killed due to highly toxic PLD. Using the best one of the stabilized PLD-expressing plasmids, PLD production was improved 2-fold.
Mishima
N
N
Laboratory of Molecular Biotechnology, Graduate School of Bio- and Agro-Sciences, Nagoya University, Japan.
Mizumoto
K
K
Iwasaki
Y
Y
Nakano
H
H
Yamane
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins; D014764:Viral Proteins
D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D010739:Phospholipase D