Abstract
Objective
We aimed to investigate the expression of a novel small cysteine-rich (SCR) effector protein SCR96 from the phytopathogenic oomycete Phytophthora cactorum in mammalian cells, its bioactivity and to exploit its polyclonal antibody.
Results
The gene encoding the SCR effector protein SCR96 was codon-optimized, custom-synthesized, cloned into pcDNA3.1(−) and overexpressed in human embryonic kidney (HEK) 293-6E cells. The recombinant protein SCR96 was prone to aggregation and purified with its monomer to homogeneity with a predicted molecular weight of 8.9 kDa. SCR96 exhibited strong phytotoxic activity on tomato seedlings at 24 h post treatment with 4.2 μg of the purified protein. An anti-SCR96 polyclonal antibody was prepared by immunization of New Zealand white rabbits. The good-titer antibody had a detection sensitivity at 6.25-ng level and could specifically detect the SCR96 protein expressed either in yeast, or in tomato leaves.
Conclusions
Transient production of the SCR effector protein SCR96 in mammalian cells is reliable, providing sufficient recombinant protein that can be utilized for analysis of its phytotoxic activity and preparation of its polyclonal antibody.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (Nos. 31671971, 31871907), Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. SJCX19_0895), the Open Project of Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China (No. JRK20180012), the Yangzhou University 2016 Project for Excellent Young Key Teachers, and High-Level Talent Support Program of Yangzhou University.
Supporting information
Supplementary Table 1—Primers used in the study. Supplementary Fig. 1—The gene cloning strategy, DNA and protein sequences of SCR96.
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Huang, SX., Zhang, ZH., Liu, W. et al. Expression of the small cysteine-rich protein SCR96 from Phytophthora cactorum in mammalian cells: phytotoxicity and exploitation of its polyclonal antibody. Biotechnol Lett 42, 125–133 (2020). https://doi.org/10.1007/s10529-019-02754-7
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DOI: https://doi.org/10.1007/s10529-019-02754-7