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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)

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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 ΔhtrA) 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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Acknowledgments

This work was financially supported by the Grant of Indian Council of Medical Research (ICMR) and B.V. Patel PERD Centre. Bhrugu Yagnik is recipient of Lady Tata Memorial Trust’s Fellowship. We gratefully acknowledge Dr. Luis Bermudez-Humaran, INRA, France for providing the backbone vector pSEC:Nuc and L. lactis NZ9000 and Dr. Isabelle Poquet, INRA, France for providing the L. lactis VEL1153 (NZ9000 ΔhtrA). We also express our gratitude to Mr. Jignesh Parmar, NIPER-Ahmedabad, India for contributing in pSEC:OmpA construction.

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  1. Department of Cell and Molecular Biology, B. V. Patel PERD Centre, Ahmedabad, Gujarat, 380054, India

    Bhrugu Yagnik, Shivangi Patel, Maitree Dave, Drashya Sharma, Harish Padh & Priti Desai

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  1. Bhrugu Yagnik
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  2. Shivangi Patel
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  3. Maitree Dave
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Correspondence to Priti Desai.

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Yagnik, B., Patel, S., Dave, M. et al. Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of Shigella dysenteriae Type-1 in Lactococcus lactis Using Nisin Inducible Controlled Expression (NICE). Indian J Microbiol 56, 80–87 (2016). https://doi.org/10.1007/s12088-015-0556-2

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