Protein secretion controlled by a synthetic gene in Escherichia coli.
The inability of Escherichia coli to secrete proteins in growth medium is one of the major drawbacks in its use in genetic engineering. A synthetic gene, homologous to the one coding for the kil peptide of pColE1, was made and cloned under the control of the lac promoter, in order to obtain the inducible secretion of homologous or heterologous proteins by E. coli. The efficiency of this synthetic gene to promote secretion was assayed by analysing the production and secretion of two proteins, the R-TEM1 beta-lactamase, and the alpha-amylase from Bacillus licheniformis. This latter protein was expressed in E. coli from its gene either on the same plasmid as the kil gene or on a different plasmid. The primary effect of the induction of the kil gene is the overproduction of the secreted proteins. When expressed at a high level, the kil gene promotes the overproduction of all periplasmic proteins and the total secretion in the culture medium of both the beta-lactamase or the alpha-amylase. This secretion is semi-selective for most periplasmic proteins are not secreted. The kil peptide induces the secretion of homologous or heterologous proteins in two steps, first acting on the cytoplasmic membrane, then permeabilizing the outer membrane. This system, which is now being assayed at the fermentor scale, is the first example of using a synthetic gene to engineer a new property into a bacterial strain.
Blanchin-Roland
S
S
INSA, Département de Génie Biochimique et alimentaire, Centre de Transfert en Biotechnologie-Microbiologie, Toulouse, France.
Masson
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D010957:Plasmids; D011506:Proteins / Q000378:metabolism*; D000516:alpha-Amylases / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
D004274:DNA, Recombinant; D011506:Proteins; D000516:alpha-Amylases; D001618:beta-Lactamases