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The lysed protoplast solution was then treated with 4 % Ficoll buffer followed by vacuole isolation buffer (0.45 M mannitol, 5 mM sodium phosphate, 2 mM EDTA, pH 7.5)

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According to the above-mentioned results that Fusion 6 with L207A/L214A lost the cleavage activity completely, here we further replaced L207 with its isomer Ile to construct Fusion 9, trying to find a suitable substitution to avoid the alternative hydrolysis at L207

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