Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli.
BACKGROUND
Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process.


RESULTS
In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.l-1 of purified active LCI.


CONCLUSIONS
These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli.
Puertas
Juan-Miguel
JM
Unité de Biochimie Structurale, Institut Pasteur, URA-CNRS 2185, 75724 Paris Cedex 15, France. jmbetton@pasteur.fr.
Betton
Jean-Michel
JM
D000595:Amino Acid Sequence; D003062:Codon / Q000378:metabolism; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D003062:Codon; D029968:Escherichia coli Proteins; C116332:LCI protein, Hirudo medicinalis; D021382:Protein Sorting Signals; D011506:Proteins; D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli