Increased efficiency of alkaline phosphatase production levels in Escherichia coli using a degenerate PelB signal sequence.
To obtain an expression vector that will optimize secretion of proteins with disulfide bridges in Escherichia coli, we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence encoding the pectate lyase B signal sequence (PelBSS). We used an extensively degenerate pelBSS with silent mutations to study their effects on the production level and activity of PhoA. 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production levels. The efficiency of translocation seems to be the result of an equilibrium between production and secretion levels that favours the secretion of active PhoA according to the competence of the fusion protein being translocated. Free energy calculations and the predicted mRNA secondary structures of the translation initiation regions showed that the high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa. A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels.
Le Calvez
H
H
Laboratoire d'Ingénierie et Dynamique des Systèmes Membranaires du C.N.R.S., Institut de Biologie Structurale et de Microbiologie, Marseille, France.
Green
J M
JM
Baty
D
D
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport; D003553:Cystine / Q000378:metabolism*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000378:metabolism*; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011133:Polysaccharide-Lyases / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D003553:Cystine; D000469:Alkaline Phosphatase; D011133:Polysaccharide-Lyases; C074124:pectin lyase B