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Production and Purification of Cysteine-Rich Leptospiral Virulence-Modifying Proteins with or Without mCherry Fusion

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Abstract

Recombinant fluorescent fusion proteins are fundamental to advancing many aspects of protein science. Such proteins are typically used to enable the visualization of functional proteins in experimental systems, particularly cell biology. An important problem in biotechnology is the production of functional, soluble proteins. Here we report the use of mCherry-fusions of soluble, cysteine-rich, Leptospira-secreted exotoxins in the PF07598 gene family, the so-called virulence modifying (VM) proteins. The mCherry fusion proteins facilitated the visual detection of pink colonies of the VM proteins (LA3490 and LA1402) and following them through lysis and sequential chromatography steps. CD-spectroscopy analysis confirmed the stability and robustness of the mCherry-fusion protein, with a structure comparable to AlphaFold structural predictions. LA0591, a unique member of the PF07598 gene family that lacks N-terminal ricin B-like domains, was produced without mCherry tag that strengthens the recombinant protein production protocol without fusion protein as well. The current study provides the approaches for the synthesis of 50–125 kDa soluble, cysteine-rich, high-quality fast protein liquid chromatography (FPLC)-purified protein, with and without a mCherry tag. The use of mCherry-fusion proteins enables a streamlined, efficient process of protein production and qualitative and quantitative downstream analytical and functional studies. Approaches for troubleshooting and optimization were evaluated to overcome difficulties in recombinant protein expression and purification, demonstrating biotechnology utility in accelerating recombinant protein production.

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Data Availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

We would like to thank Dr. Ewa Folta-Stogniew, Director of Biophysics Resource Keck Laboratory, and Dr. James W Murphy, Research Scientist, Yale University for their critical suggestions in CD-spectroscopy. We acknowledge the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University.

Funding

This work was supported by grants from the United States Public Health Service through National Institutes of Health grants R01AI108276 and U19AI115658, and the Americas Foundation.

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Authors and Affiliations

  1. Section of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA

    Reetika Chaurasia, Cathleen Liang, Kenneth How, Dielson S. Vieira & Joseph M. Vinetz

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  1. Reetika Chaurasia
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Contributions

Conceptualization: RC, JV. Data curation and formal analysis: RC. Funding acquisition: JV. Investigation: RC, CL, KH, DSV, JV. Methodology: RC, JV. Resources: JV. Supervision: JV. Visualization: RC, CL, KH, DSV, JV. Writing—original draft: RC. Writing—review and editing: RC, CL, KH, DSV, JV.

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Correspondence to Reetika Chaurasia or Joseph M. Vinetz.

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10930_2023_10152_MOESM1_ESM.tiff

Supplementary file1 Figure S1. Optimization of expression and purification of mCherry-LA3490. The pellet of clone expressing mCherry-LA3490 was solubilized using various approaches, and the purification was performed using the standard protocol mentioned in the experimental procedures. (a) Lane 1 shows induced supernatant which was obtained upon solubilization in CelLytic™ B Cell Lysis Reagent. Lane 2 shows induced supernatant solubilized in lysis buffer containing 20 mM Tris–HCl + 150 mM NaCl + 1% TritonX-100. Lanes 3- and 4 show induced supernatant solubilized in CelLytic™ B Cell Lysis Reagent containing 0.1% and 0.01% TritonX-100 respectively. Induced soluble fraction solubilized in CelLytic™ B Cell Lysis Reagent containing 0.1% TritonX-100, was used to purify the LA3490 proteins. The eluted fractions were analyzed on 4–12% bis–tris SDS-PAGE (right panel). F and W1-3 represent flow through and washes. E1-E5 is the eluate fractions. (b) The recombinant clone of mCherry-LA3490 producing pellet was solubilized in CelLytic™ B Cell Lysis Reagent containing 0.01% TritonX-100 and 1 mM CHAPS. IP and IS represent induced pellet and induced supernatant. F and W1-2 represent flow through and wash. E1-E10 is the eluate fractions. (c) The pellet was solubilized in lysis buffer 100 mM NaH2PO4, 10 mM Tris–HCl pH 7.4 containing 8 M Urea followed by sonication and separation of the pellet (IP) and supernatant (IS) fractions. In addition, the pellet was solubilized in lysis buffer 20 mM Tris–HCl + 150 mM NaCl containing 2% or 5% sarkosyl. The induced soluble fraction solubilized with 5% sarkosyl was subjected to purification using AKTA pure and eluted fractions were analyzed on 4–12% bis–tris SDS-PAGE (right panel). W shows wash, and E1-E8 represents eluted fractions. M represents the molecular weight marker. (TIFF 4423 KB)

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Chaurasia, R., Liang, C., How, K. et al. Production and Purification of Cysteine-Rich Leptospiral Virulence-Modifying Proteins with or Without mCherry Fusion. Protein J 42, 792–801 (2023). https://doi.org/10.1007/s10930-023-10152-2

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