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

Abstract

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.

Immunization with r-Lactococcus lactisexpressing outer membrane protein A ofShigella dysenteriaetype-1: evaluation of oral and intranasal route of administration

“…pSEC:OmpA was constructed and confirmed as reported earlier (Yagnik et al . ). pET28a:OmpA vector was constructed as described in Materials and methods and confirmed by restriction enzyme analysis of isolated plasmid from a transformed clone of E. coli BL21 (DE3) (Fig.…”

Intracellular Expression of Antifreeze Peptides in Food Grade Lactococcus lactis and Evaluation of Their Cryoprotective Activity

“…This culture was used to inoculate fresh media at 1:100, which was incubated for 2 to 3 hr at 30 °C until logarithmic growth (OD = 0.4 to 0.5). After referring to a number of relevant references (Berlec et al., ; Mierau, Olieman, Mond, & Smid, ; Yagnik et al., ) and performing sufficient preliminary experiments, to determine the optimal culture conditions, the recombinant strain SF‐P was incubated with different concentrations of inducer Nisin (25, 50, or 100 ng/mL) at different culture medium pHs (4, 5, or 6), temperatures (20, 30, or 40 °C), for different incubation time (6, 12, or 24 hr) (Zhou, Pan, Wang, & Li, ). The cultures were centrifuged at 6000 rpm for 10 min at 4 °C.…”

Oral immunization with LacVax® OmpA induces protective immune response against Shigella flexneri 2a ATCC 12022 in a murine model

“…In this regard, our group has explored food grade Lactococcus lactis as an antigen and DNA vaccine delivery vehicle against enteric pathogens such as Shigella [8][9][10][11]. We have developed non-invasive and invasive r-L. lactis harbouring DNA vaccine reporter plasmid and evaluated DNA delivery potential of these strains in vitro as well as in vivo [9,11].…”