Abstract
T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method:•Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays;•Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and•Incorporates a simplified expression and purification protocol for T4 lysozyme.
Highlights
T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function
Using the assay described above, we compared the activity of T4L, T4L-E11H, human lysozyme (HL), and hen egg white lysozyme (HEWL) using two different assay buffer conditions (Table 1)
HL and HEWL were equivalently active in both buffer conditions; T4L only showed measurable activity in 30 mM potassium phosphate pH 7.2 (Table 1)
Summary
Genes encoding T4 lysozyme (T4L) and an enzymatically inactive variant, T4L-E11H, were cloned into an expression system which allows easy expression in E. coli and purification. 1. Lyse cells by incubation in lysis buffer [30 mM potassium phosphate pH 7.6, 0.5 M NaCl, 5% glycerol, 5 mM Imidazole pH 8.0, 2 mM MgCl2, 1X HALT protease inhibitor (Thermo Scientific), 0.5 mg/mLÀ1 hen egg white lysozyme (HEWL; MP Biomedicals)] followed by sonication for 3 Â 10 s on ice. 6. Elute the protein with elution buffer (30 mM potassium phosphate pH 7.6, 0.5 M NaCl, 5% glycerol, 150 mM Imidazole pH 8.0), collected in 1 bed volume fractions. We solubilized lysozyme at 1.0 mg/mLÀ1 in 30 mM potassium phosphate pH 7.6, 200 mM NaCl, 5% glycerol, 1 mM TCEP, to be consistent with the buffer of the purified T4 lysozyme. Calculate final activity values and standard deviations for the average of each experiment for each experimental condition
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