Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

Langella, Philippe; Loir, Yves Le

doi:10.1590/s0100-879x1999000200007

Cited by 42 publications

(34 citation statements)

References 23 publications

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“…We therefore considered an alternative system for native E7 production based on a food-grade lactic acid bacterium. The best-known lactic acid bacterium, Lactococcus lactis, has been extensively engineered for the production of heterologous proteins (5,6,10,18,20,21,28,29,35,37). Protein production in L. lactis offers advantages: L. lactis is a food-grade gram-positive bacterium that produces very low amounts of native exoproteins.…”

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Production of Human Papillomavirus Type 16 E7 Protein in Lactococcus lactis

Bermúdez‐Humarán

Langella

Miyoshi

et al. 2002

Appl Environ Microbiol

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108

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The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.Infection with human papillomavirus type 16 (HPV-16) is the main factor associated with development of cervical cancer (42). The HPV-16 E6 and E7 proteins are constitutively produced in cervical carcinomas, and E7 was shown to interact with several cell compounds, causing deregulation of the cell cycle and cell transformation (43). E7 is a 98-amino-acid nuclear phosphoprotein that is devoid of any known enzymatic activity (36). In eukaryotic cells, E7's half-life is short (30 to 40 min); its degradation is mediated by the ubiquitin-proteasome pathway (31). E7 protein is widely studied because of its implication in carcinoma onset. It is also considered to be a good antigen candidate for the development of new vaccines against cervical cancer.E7 production systems have been developed in both eukaryotes (1, 39) and prokaryotes (27,34). Since the 1990s, several workers have investigated the use of bacteria as E7 antigen delivery vehicles to elicit an immune response against 22). The gram-positive and generally regarded as safe (GRAS) commensal bacterium Streptococcus gordonii was used for this purpose to display E7 protein at the cell surface in fusion with export signals (30). These recombinant S. gordonii strains could elicit an immune response in mice and monkeys (23,26). Although encouraging, these results rely on a commensal, GRAS but non-food-grade bacterium. One risk of commensal, and hence persistent, microorganisms is the induction of immunotolerance. Thus, a transient presentation of the antigen to the immune system by a noncommensal bacterium may be needed to avoid this risk.None of the systems mentioned above seems to provide the combination of safety, sufficient yields, and simplified methods that would allow both purification and eventual oral immunization using E7. We therefore considered an alternative system for native E7 production based on a food-grade lactic acid bacterium. The best-known lactic acid bacterium, Lactococcus lactis, has been extensively engineered for the production of heterologous proteins (5,6,10,18,20,21,28,29,35,37). Protein production in L. lactis offers advantages: L. lactis is a food-grade gram-positive bacterium that produces very low amounts of native exoproteins. It is therefore a good candidate for heterologous protein secretion in different applications ranging from industrial production of high-added-value proteins to in vivo use as a live vaccine. As L. lactis is a noncommensal and transient bacterium in the digestive tract (12), t...

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Production of Human Papillomavirus Type 16 E7 Protein in Lactococcus lactis

Bermúdez‐Humarán

Langella

Miyoshi

et al. 2002

Appl Environ Microbiol

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108

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“…They are considered safe and even beneficial organisms. The potential of using LAB for new applications such as in production of heterologous proteins for biotechnology, in fermented food products, or in the digestive tract of humans or animals is currently under active study (3,11,13,17,19,22,30,49,54).…”

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confidence: 99%

“…We are focused on optimizing heterologous protein secretion and export in Lactococcus lactis (30,32), a well-characterized LAB for which genetic tools and the genome sequence are available (5,11). To date, heterologous proteins such as bovine plasmin (3), bovine beta-lactoglobulin (BLG [6a], bovine rotavirus nonstructural protein 4 (NSP4 [13a]), murine interleukin-2 (IL-2) and IL-6 (54), or Listeria monocytogenes bacteriophage lysin (17) have been fused to lactococcal signal peptides (SPs) to direct their secretion in the medium.…”

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confidence: 99%

“…Its enzymatic activity is readily detectable on petri plates as well as in zymograms (31,34). Translational fusions to the N terminus and/or the C terminus of the mature protein are enzymatically active (30,32,41). SP Nuc is atypical, as it is unusually long (60 residues) and contains two hydrophobic stretches that may form a hairpin in the cytoplasmic membrane during translocation (27).…”

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Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

Loir

Nouaille

Commissaire

et al. 2001

Appl Environ Microbiol

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115

104

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Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc [1895][1896][1897][1898][1899][1900][1901][1902][1903] 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP Usp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.

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“…Secretion of these proteins leads to the direct production of several proteins in fermented food products facilitating the interaction of the secreted proteins (enzymes or antigens) and their environment (the food products themselves or the digestive tract of animals that consume these engineered bacteria). 12 Due to this, it would be advantageous to use lactococci as chymosin producers because no foreign components of the host would be added to the cheese vat.…”

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confidence: 99%

RecombinantLactococcus lactisfails to secrete bovine chymosine

et al. 2014

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Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein.

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Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

Cited by 42 publications

References 23 publications

Production of Human Papillomavirus Type 16 E7 Protein in Lactococcus lactis

Production of Human Papillomavirus Type 16 E7 Protein in Lactococcus lactis

Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

RecombinantLactococcus lactisfails to secrete bovine chymosine

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