Lactobacillus bulgaricus proteinase expressed in Lactococcus lactis is a powerful carrier for cell wall-associated and secreted bovine beta-lactoglobulin fusion proteins.
Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine beta-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB partial differential, PrtB partial differential-BLG, and PrtB partial differential-T6) or 807 (PrtBdelta) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB partial differential-BLG yielded up to 170 microg per 10(9) CFU in the culture supernatant and 9 microg per 10(9) CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.
Bernasconi
Eric
E
R & D Laboratory of the Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, CH-1000 Lausanne 11, Switzerland.
Germond
Jacques-Edouard
JE
Delley
Michèle
M
Fritsché
Rodolphe
R
Corthésy
Blaise
B
D001426:Bacterial Proteins; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism*; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology*; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D007782:Lactoglobulins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007782:Lactoglobulins; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C099750:PrtB protein, bacteria