The nisin-controlled gene expression system: Construction, application and improvements

Zhou, Xu Xia; Li, Wei Fen; Guo, Xia; Pan, Yijie

doi:10.1016/j.biotechadv.2005.11.001

Cited by 70 publications

(43 citation statements)

References 77 publications

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“…These reports strengthen the observation obtained with OmpA. Along with the aggregation, degraded protein product was also found which may be the consequence of the presence of proteases in host strain NZ9000 [11,17] and/or degradation of highly expressed protein [18].…”

Section: Discussionsupporting

confidence: 87%

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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: Discussionsupporting

confidence: 87%

“…In 2006, Zhou et al [17] reported the linear dose dependent relationship suggesting elevated protein expression at higher nisin concentration. Further, overexpression of membrane proteins also induces stress response in L. lactis, resulting in protein degradation by chaperons and proteases [19].…”

Section: Discussionmentioning

confidence: 99%

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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“…lactis is widely used as a model organism for heterologous expression of bacterial as well as eukaryotic proteins or viral antigens (Zhou et al, 2006). Here we showed that virulence factors such as PspC can be expressed in lactococci and that these recombinant clones have similar effects on host epithelial cells to S. pneumoniae.…”

Section: Discussionmentioning

confidence: 86%

“…L. lactis was further reported to be more advantageous an expression host than Escherichia coli (Mierau & Kleerebezem, 2005;Zhou et al, 2006) and, with regard to pneumococcal PspC, the use of lactococci is preferred as PspC is again decorated on a Gram-positive cell surface. Thus, we expressed PspC on the surface of L. lactis and explored the role of PspC as an adhesin, and its role on bacterial ingestion in the absence and presence of Factor H, respectively, by 'professional' and 'non-professional' phagocytes.…”

Section: Introductionmentioning

confidence: 99%

Heterologous expression of pneumococcal virulence factor PspC on the surface of Lactococcus lactis confers adhesive properties

et al. 2012

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Lactococcus lactis is a non-pathogenic bacterium that is used in the food industry but is also used as a heterologous host to reveal protein functions of pathogenic bacteria. The adhesin PspC from Streptococcus pneumoniae is a choline-binding protein that is non-covalently anchored to the bacterial cell wall. To assess the exclusive impact of pneumococcal surface protein C (PspC) on the interplay with its host we generated recombinant L. lactis producing a nisin-inducible and covalently anchored variant of PspC on the lactococcal cell surface. A translational fusion of the 59-end of pspC3.4 with the 39-end of hic (pspC11.4) was designed to decorate the surface of L. lactis with a chimeric PspC. The PspC3.4 part comprises the first 281 aa residues of PspC3.4, while the Hic sequence consists of the proline-rich and sortase-anchored domain. The results demonstrated that PspC is sufficient for adhesion and subsequent invasion of host epithelial cells expressing the human polymeric Ig receptor (hpIgR). Moreover, invasion via hpIgR was even more pronounced when the chimeric PspC was produced by lactococci compared with pneumococci. This study shows also for the first time that PspC plays no significant role during phagocytosis by macrophages. In contrast, recruitment of Factor H via the PspC chimer has a dramatic effect on phagocytosis of recombinant but not wild-type lactococci, as Factor H interacts specifically with the amino-terminal part of PspC and mediates the contact with phagocytes. Furthermore, L. lactis expressing PspC increased intracellular calcium levels in pIgR-expressing epithelial cells, thus resembling the effect of pneumococci, which induced release of Ca 2+ from intracellular stores via the PspC-pIgR mechanism. In conclusion, expression of the chimeric PspC confers adhesive properties to L. lactis and indicates the potential of L. lactis as a suitable host to study the impact of individual bacterial factors on their capacity to interfere with the host and manipulate eukaryotic epithelial cells. INTRODUCTIONStreptococcus pneumoniae (pneumococci) is the aetiological agent of community-acquired pneumonia, septicaemia and bacterial meningitis, all associated with high morbidity and mortality (Cartwright, 2002). Pneumococcal infections commence at the nasopharynx, where the bacteria reside as commensals without harming the host niche. Colonization of the respiratory mucosal epithelium is a prerequisite for invasive infections, which are most likely accompanied by dramatic changes in the nasopharyngeal physical barriers and the expression of pneumococcal virulence factors (Orihuela et al., 2004). The repertoire of uptake systems for essential nutrients enables pneumococci to adapt to and survive in different physiological conditions. More importantly, pneumococci have a variety of virulence factors facilitating adherence to host cells and evasion from the host immune system. Pneumococcal adherence to host cells is, similar to other Gram-positive pathogens including Staphylococcus aureus and Streptococcus...

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“…lactis has been developed as an excellent tool for the expression and study of integral membrane proteins of both prokaryotes and eukaryotes (Kunji et al, 2003;Monné et al, 2009). Furthermore, L. lactis are increasingly used as oral/nasal delivery vehicles for therapeutic and prophylactic proteins (Mierau et al, 2005a,b;Le Loir et al, 2005;Zhou et al, 2006). It can elicit more effective immunological responses and reduce the risk of eliciting immunologic tolerance compared with persistent strains.…”

Section: Discussionmentioning

confidence: 99%

Construction and expression of a heterologous protein in Lactococcus lactis by using the nisin-controlled gene expression system: the case of the PRRSV ORF6 gene

Wang¹,

Wang²,

Zeng³

2014

Genet. Mol. Res.

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The nisin-controlled gene expression system: Construction, application and improvements

Cited by 70 publications

References 77 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)

Heterologous expression of pneumococcal virulence factor PspC on the surface of Lactococcus lactis confers adhesive properties

Construction and expression of a heterologous protein in Lactococcus lactis by using the nisin-controlled gene expression system: the case of the PRRSV ORF6 gene

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