A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis.
Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of L. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only approximately 60% of total Nuc was present in a secreted form at approximately 5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to approximately 30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to approximately 80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to approximately 20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the alpha-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.
Le Loir
Y
Y
Laboratoire de Génétique Appliquée-URLEA, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy en Josas, France. leloir@biotec.jouy.inra.fr
Gruss
A
A
Ehrlich
S D
SD
Langella
P
P
D000595:Amino Acid Sequence; D013294:Lactococcus lactis / Q000378:metabolism*; D008836:Micrococcal Nuclease / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D011498:Protein Precursors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism*
D009842:Oligopeptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D008836:Micrococcal Nuclease