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Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis.

Samazan F, Rokbi B, Seguin D, Telles F, Gautier V, Richarme G, Chevret D, Varela PF, Velours C, Poquet I - Microb. Cell Fact. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Architecture of staphylococcal HtrA proteins and design of soluble proteins. Three HtrA family members are shown: from top to bottom, E. coli DegS, S. aureus HtrA1 and HtrA2 proteins (strain COL). They display the typical domain organisation of the family: transmembrane, catalytic and PDZ domains are shown as hatched, dark grey and light grey boxes respectively, with their boundaries (residue position) indicated below. The catalytic Serine residue (S, in bold) is shown. The recombinant DegS form whose structure has been solved after N-terminal transmembrane domain deletion [64] is named DegSΔTM. A similar deletion strategy was applied to HtrA1 and HtrA2 proteins leading to N-terminally truncated proteins named HtrA1ΔTM and HtrA2ΔTM. All truncated protein forms are shown as lines with the position of their first and last residues in the corresponding WT sequence indicated.
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Fig1: Architecture of staphylococcal HtrA proteins and design of soluble proteins. Three HtrA family members are shown: from top to bottom, E. coli DegS, S. aureus HtrA1 and HtrA2 proteins (strain COL). They display the typical domain organisation of the family: transmembrane, catalytic and PDZ domains are shown as hatched, dark grey and light grey boxes respectively, with their boundaries (residue position) indicated below. The catalytic Serine residue (S, in bold) is shown. The recombinant DegS form whose structure has been solved after N-terminal transmembrane domain deletion [64] is named DegSΔTM. A similar deletion strategy was applied to HtrA1 and HtrA2 proteins leading to N-terminally truncated proteins named HtrA1ΔTM and HtrA2ΔTM. All truncated protein forms are shown as lines with the position of their first and last residues in the corresponding WT sequence indicated.

Mentions: In S. aureus, there are two transmembrane members of the HtrA family, HtrA1 and HtrA2 [23, 57] (for example in strain COL: Q5HF46 and Q5HH63, and data not shown for other published genomes; here, we provide the sequence of htrA genes from the clinical strain Lowenstein: Genbank BankIt1643789 htrA1_LOW KF322112 and BankIt1643789 htrA2_LOW KF322111) which are highly conserved between strains (more than 95 or 61% identity respectively). Even though HtrA2 bears a large N-terminal domain of unknown function [23], both HtrA1 and HtrA2 proteins display the typical family architecture [25, 26] (Figure 1) with three regions: from their N- to C-terminus, (1) a transmembrane domain as the export signal, (2) a catalytic domain with a characteristic His Asp Ser triad (His144, Asp174 and Ser255 in the case of HtrA1, and His504, Asp534 and Ser619 in the case of HtrA2) and (3) one PDZ domain (a protein–protein interaction domain named for the three proteins (PSD95, DLG1, and ZO-1) where it was initially discovered [26]). In both staphylococcal HtrA proteins, like in other family members of Gram-positive species, the C-terminal region encompassing the catalytic and PDZ domains is predicted to be extra-cellular (predicted C-out topology by HMMTOP, http://www.enzim.hu/hmmtop/html/submit.html).Figure 1


Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis.

Samazan F, Rokbi B, Seguin D, Telles F, Gautier V, Richarme G, Chevret D, Varela PF, Velours C, Poquet I - Microb. Cell Fact. (2015)

Architecture of staphylococcal HtrA proteins and design of soluble proteins. Three HtrA family members are shown: from top to bottom, E. coli DegS, S. aureus HtrA1 and HtrA2 proteins (strain COL). They display the typical domain organisation of the family: transmembrane, catalytic and PDZ domains are shown as hatched, dark grey and light grey boxes respectively, with their boundaries (residue position) indicated below. The catalytic Serine residue (S, in bold) is shown. The recombinant DegS form whose structure has been solved after N-terminal transmembrane domain deletion [64] is named DegSΔTM. A similar deletion strategy was applied to HtrA1 and HtrA2 proteins leading to N-terminally truncated proteins named HtrA1ΔTM and HtrA2ΔTM. All truncated protein forms are shown as lines with the position of their first and last residues in the corresponding WT sequence indicated.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4502909&req=5

Fig1: Architecture of staphylococcal HtrA proteins and design of soluble proteins. Three HtrA family members are shown: from top to bottom, E. coli DegS, S. aureus HtrA1 and HtrA2 proteins (strain COL). They display the typical domain organisation of the family: transmembrane, catalytic and PDZ domains are shown as hatched, dark grey and light grey boxes respectively, with their boundaries (residue position) indicated below. The catalytic Serine residue (S, in bold) is shown. The recombinant DegS form whose structure has been solved after N-terminal transmembrane domain deletion [64] is named DegSΔTM. A similar deletion strategy was applied to HtrA1 and HtrA2 proteins leading to N-terminally truncated proteins named HtrA1ΔTM and HtrA2ΔTM. All truncated protein forms are shown as lines with the position of their first and last residues in the corresponding WT sequence indicated.
Mentions: In S. aureus, there are two transmembrane members of the HtrA family, HtrA1 and HtrA2 [23, 57] (for example in strain COL: Q5HF46 and Q5HH63, and data not shown for other published genomes; here, we provide the sequence of htrA genes from the clinical strain Lowenstein: Genbank BankIt1643789 htrA1_LOW KF322112 and BankIt1643789 htrA2_LOW KF322111) which are highly conserved between strains (more than 95 or 61% identity respectively). Even though HtrA2 bears a large N-terminal domain of unknown function [23], both HtrA1 and HtrA2 proteins display the typical family architecture [25, 26] (Figure 1) with three regions: from their N- to C-terminus, (1) a transmembrane domain as the export signal, (2) a catalytic domain with a characteristic His Asp Ser triad (His144, Asp174 and Ser255 in the case of HtrA1, and His504, Asp534 and Ser619 in the case of HtrA2) and (3) one PDZ domain (a protein–protein interaction domain named for the three proteins (PSD95, DLG1, and ZO-1) where it was initially discovered [26]). In both staphylococcal HtrA proteins, like in other family members of Gram-positive species, the C-terminal region encompassing the catalytic and PDZ domains is predicted to be extra-cellular (predicted C-out topology by HMMTOP, http://www.enzim.hu/hmmtop/html/submit.html).Figure 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus