title: Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis.
abstract: BACKGROUND
  Lactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development.
  
  
  RESULTS
  A recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development.
  
  
  CONCLUSIONS
  In this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.
authors.affiliation: INRA, UMR1319 Micalis (Microbiologie de l'Alimentation au service de la Santé), Domaine de Vilvert, 78352, Jouy-en-Josas Cedex, France. Frederic.Samazan@curie.fr.|Institut Curie/CNRS, UMR3244, 25 rue d'Ulm, 75248, Paris Cedex 05, France. Frederic.Samazan@curie.fr.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Samazan
authors.affiliation: Sanofi Pasteur, Campus Mérieux, 1541 avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Bachra.Rokbi@sanofipasteur.com.
authors.forename: Bachra
authors.initials: B
authors.lastname: Rokbi
authors.affiliation: Sanofi Pasteur, Campus Mérieux, 1541 avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Delphine.Seguin@sanofipasteur.com.
authors.forename: Delphine
authors.initials: D
authors.lastname: Seguin
authors.affiliation: Sanofi Pasteur, Campus Mérieux, 1541 avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Fabienne.Telles@sanofipasteur.com.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Telles
authors.affiliation: Stress molecules, Institut Jacques Monod, Université Paris 7, 15 rue Hélène Brion, 75013, Paris, France. valerie.gautier@univ-paris-diderot.fr.
authors.forename: Valérie
authors.initials: V
authors.lastname: Gautier
authors.affiliation: Stress molecules, Institut Jacques Monod, Université Paris 7, 15 rue Hélène Brion, 75013, Paris, France. richarme@paris7.jussieu.fr.
authors.forename: Gilbert
authors.initials: G
authors.lastname: Richarme
authors.affiliation: INRA, UMR1319 Micalis (Microbiologie de l'Alimentation au service de la Santé), Domaine de Vilvert, 78352, Jouy-en-Josas Cedex, France. didier.chevret@jouy.inra.fr.
authors.forename: Didier
authors.initials: D
authors.lastname: Chevret
authors.affiliation: CNRS, Avenue de la Terrasse, Bât. 34, 91190, Gif-Sur-Yvette, France. Paloma.Fernandez-Varela@lebs.cnrs-gif.fr.
authors.forename: Paloma Fernández
authors.initials: PF
authors.lastname: Varela
authors.affiliation: CNRS, Avenue de la Terrasse, Bât. 34, 91190, Gif-Sur-Yvette, France. Christophe.Velours@lebs.cnrs-gif.fr.
authors.forename: Christophe
authors.initials: C
authors.lastname: Velours
authors.affiliation: INRA, UMR1319 Micalis (Microbiologie de l'Alimentation au service de la Santé), Domaine de Vilvert, 78352, Jouy-en-Josas Cedex, France. isabelle.poquet@jouy.inra.fr.|LPBA, Institut Pasteur, Bât. Calmette, 75015, Paris, France. isabelle.poquet@jouy.inra.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Poquet
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D001428:Bacterial Vaccines / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013203:Staphylococcal Infections / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology* / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D010447:Peptide Hydrolases