Detection and localization of peptidases inLactococcus lactiswith monoclonal antibodies

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Abstract
Summary: Monoclonal antibodies against peptidases ofLactococcus lactiswere isolated and characterized: PEPN1–4 against a lysyl aminopeptidase PepN, PEPT1–5 against a tripeptidase PepT and PEPD1–3 against a dipeptidase PepD. These monoclonal antibodies reacted specifically with their respective antigens in crude cell extracts ofLc. lactissubspp.cremorisandlactis. A number of monoclonal antibodies cross reacted with proteins of other (lactic acid) bacteria. PEPT1, 2, 4 and 5 cross reacted weakly with a 35 kDa protein inLactobacillus delbrueckii, while PEPT1 and PEPT2 reacted with proteins in the cell-free extract ofStreptococcus thermophilusandClostridium fervidus. Of the four isolated monoclonal antibodies against PepN, only PEPN3 cross reacted weakly with a 90 kDa protein inEscherichia colicell-free extract, and the other three antibody species against PepN cross reacted with 80 kDa proteins ofLb. casei, Lb. delbrueckii, andStr. bovis, but not ofEsch. coli. Of the three monoclonal antibodies against PepD, only PEPD1 and PEPD2 cross reacted with 40 kDa proteins ofLb. casei, Lb. delbrueckiiandStr. bovis. All PEPN, PEPD and PEPT antibodies reacted with components in cell-free extracts of eleven differentLc. lactisstrains, indicating that the peptidases of these strains were very similar to those ofLc. lactissubsp.cremorisWG2. However,Lc. lactissubsp.hordniaeappeared to differ from the otherLc. lactissubspecies since only PEPT1, 2 and 5 reacted with a protein in the cell-free extract. Immunogold labelling ofLc. lactisWG2 with the isolated monoclonal antibodies revealed that PepN, PepD and PepT were located intracellularly. The intracellular location of these peptidases is discussed in relation to the supply of essential amino acids and peptides.