We designed an expression and export system that enabled the targeting of the smurfp to specific locations in Lactococcus lactis cells, cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (∼95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated.
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Usp45 is a signal peptide used for M6 anchoring protein.
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===reference===
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[1] Dieye Y, et al. Design of a protein-targeting system for lactic acid bacteria. J Bacteriol. 2001.
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Latest revision as of 09:56, 13 October 2017
usp45
Usage and Biology
Usp45 is a signal peptide used for M6 anchoring protein.
