Construction and characterization of a Lactococcus lactis strain deficient in intracellular ClpP and extracellular HtrA proteases

Cortes-Perez, Naima G.; Poquet, Isabelle; Oliveira, Maricê Nogueira de; Gratadoux, Jean-Jacques; Madsen, Søren; Miyoshi, Anderson; Corthier, Gérard; Azevedo, Vasco; Langella, Philippe; Bermúdez‐Humarán, Luis G.

doi:10.1099/mic.0.28698-0

Cited by 45 publications

(42 citation statements)

References 22 publications

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“…Precipitation of protein by TCA method has been very well documented in case of L. lactis expression system [1,8]. However, we failed to see any band in cell pellet and even in supernatant when TCA was employed for precipitation; hence TCA was replaced with methanol.…”

Section: Protein Precipitating Agentmentioning

confidence: 75%

“…There has been recent efforts in developing L. lactis as a host for production of heterologous proteins of medical and technological interest [1]. Several inducible expression systems have been developed for expression of heterologous proteins.…”

Section: Introductionmentioning

confidence: 99%

“…A unique protease, Hightemperature requirement A (HtrA), found in the extracellular matrix of L. lactis, is known to degrade the secreted protein [1]. Hence, we evaluated its impact on protein expression by using L. lactis VEL1153 (NZ9000 DhtrA).…”

Section: Introductionmentioning

confidence: 99%

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Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of Shigella dysenteriae Type-1 in Lactococcus lactis Using Nisin Inducible Controlled Expression (NICE)

et al. 2015

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Potential use of Lactococcus lactis (L. lactis) as a heterologous protein expression host as well as for delivery of multiple therapeutic proteins has been investigated extensively using Nisin Inducible Controlled Expression (NICE) system. Optimum inducible expression of heterologous protein by NICE system in L. lactis depends on multiple factors. To study the unexplored role of factors affecting heterologous protein expression in L. lactis using NICE, the present study outlines the optimization of various key parameters such as inducer concentration, host's proteases and precipitating agent using Outer membrane protein A (OmpA). For efficient expression and secretion of OmpA, pSEC:OmpA vector was successfully constructed. To circumvent the troubles encountered during detection of expressed OmpA, the precipitating agent was switched from TCA to methanol.Nevertheless, detection was achieved accompanied by degraded protein products. Speculating the accountability of observed degradation at higher inducer concentration, different nisin concentrations were evaluated. Lower nisin concentrations were found desirable for optimum expression of OmpA. Consistently observed degradation was eliminated by incorporation of protease inhibitor cocktail which inhibits intracellular proteases and expression in VEL1153 (NZ9000 DhtrA) strain which inhibits extracellular protease leading to optimum expression of OmpA. Versatility and complexity of NICE system in L. lactis requires fine-tuning of target protein specific parameters for optimum expression.

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Section: Protein Precipitating Agentmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 99%

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Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of Shigella dysenteriae Type-1 in Lactococcus lactis Using Nisin Inducible Controlled Expression (NICE)

et al. 2015

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“…These results suggest that the secretion of recombinant proteins is impaired in L. lactis NZ9000 htrA, irrespective of the protein or expression system used. Some reports in the literature have concluded that the secretion of recombinant proteins (and not secretion efficiency) is better in L. lactis htrA mutants (3,12,13). These conclusions were based on estimations made from Western blot analyses of the extracellular fraction only.…”

mentioning

confidence: 99%

HtrA Is Essential for Efficient Secretion of Recombinant Proteins by Lactococcus lactis

Sriraman

Jayaraman

2008

Appl Environ Microbiol

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HtrA is a unique protease on the extracellular surface of Lactococcus lactis. It is known to take part in the proteolysis of many secreted recombinant proteins, and the mutation of htrA can lead to the complete stabilization of recombinant proteins. In this work, we have shown that htrA mutation also leads to significant reduction of the efficiency of recombinant-protein secretion. We also show that the level of HtrA can be lowered by the suppression of the acid tolerance response (ATR) in L. lactis. Instead of using an L. lactis htrA mutant, the reduction of the HtrA level in wild-type recombinant cultures of L. lactis by ATR suppression may serve as a better strategy for the production of secreted recombinant proteins.Lactococcus lactis is a potential host for the production of therapeutic recombinant proteins. L. lactis strains used for recombinant protein production are known to express very few membrane-bound or secreted proteases. In such strains, HtrA is the only protease that has been characterized on the extracellular surface (15). HtrA is a key factor in the L. lactis response to specific stress conditions (5) and is known to be involved in the maturation of native proteins, processing of propeptides (15), and proteolysis of secreted recombinant proteins (12). Hence, L. lactis htrA mutants have been considered as potential hosts for the production of stable recombinant proteins (13, 15). The objectives of this work were to compare the efficacy of an L. lactis htrA mutant with that of wild-type L. lactis for the production of secreted recombinant proteins and to investigate the role of HtrA in the processing of recombinant proteins.We analyzed the secretion efficiencies of two recombinant proteins, streptokinase (a therapeutic protein) and staphylococcal nuclease (a reporter protein), in wild-type L. lactis and an L. lactis htrA mutant. The efficiency of secretion of both proteins was found to be severely affected in the L. lactis htrA mutant compared to their secretion in wild-type L. lactis. In an earlier report, we showed that the suppression of the acid tolerance response (ATR) in L. lactis leads to a decrease in the degradation of recombinant streptokinase (18). In this paper, we correlate our earlier observations with the observed downregulation of htrA expression during ATR suppression. We also demonstrate an enhancement in the accumulation of recombinant staphylococcal nuclease during ATR suppression. The results indicate that ATR suppression in wild-type L. lactis may serve as a better strategy than the use of an L. lactis htrA mutant for the production of secreted recombinant proteins.Secretion efficiencies of recombinant proteins for wild-type L. lactis and an L. lactis htrA mutant. The wild-type L. lactis strains used in this work were L. lactis MG1363 (6) and L. lactis

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“…Levels of heterologous proteins secreted by L. lactis and other lactic acid bacteria are generally low, and efforts have been made to improve the secretion efficiency by modifying secretion signal sequences (5,31), inactivating proteases (2,23,29), or supplying heterologous secretion machinery (26). Insights into protein secretion in L. lactis were gained when Nouaille et al identified 13 genes that affect the secretion efficiency of a staphylococcal nuclease reporter enzyme (NucT) by using random mutagenesis (25).…”

mentioning

confidence: 99%

Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis

Tan

Giffard

Barry

et al. 2008

Appl Environ Microbiol

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Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8-to 16.1-fold, for the llmg_0609 mutants. The lysostaphinoversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.

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Construction and characterization of a Lactococcus lactis strain deficient in intracellular ClpP and extracellular HtrA proteases

Cited by 45 publications

References 22 publications

Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of Shigella dysenteriae Type-1 in Lactococcus lactis Using Nisin Inducible Controlled Expression (NICE)

Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of Shigella dysenteriae Type-1 in Lactococcus lactis Using Nisin Inducible Controlled Expression (NICE)

HtrA Is Essential for Efficient Secretion of Recombinant Proteins by Lactococcus lactis

Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis

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