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J Bacteriol, April 1998, p. 1895-1903, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in Lactococcus lactis

Y. Le Loir,1,* A. Gruss,1 S. D. Ehrlich,2 and P. Langella1

Laboratoire de Génétique Appliquée-URLEA,1 and Laboratoire de Génétique Microbienne,2 Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France

Received 1 December 1997/Accepted 29 January 1998

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 ~60% of total Nuc was present in a secreted form at ~5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to ~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 ~80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to ~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.

* Corresponding author. Mailing address: Laboratoire de Génétique Appliquée-URLGA, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France. Phone: 33 01 34 65 20 83. Fax: 33 01 34 65 20 65. E-mail: leloir{at}biotec.jouy.inra.fr.




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