Metallo-β-lactamases (MBLs) are class B β-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of β-lactam antibiotics, thus conferring antibiotics resistance to bacterial pathogens. The attempt to structurally characterize BLEG-1, an evolutionary divergent B3 metallo-β-lactamase (MBL) with dual activity from Bacillus lehensis G1, led to the optimization of its purification, post-purification and crystallization processes for X-ray diffraction purpose. The workflow, conditions used and dataset obtained from each stage of the processes are presented herein. The optimization workflow has enabled the obtainment of purified, active BLEG-1 in high yield for its activity assays, crystallization and structure determination via X-ray diffraction. This is the first step to gain a better insight into its dual activity and evolutionary divergence from a structural perspective. The complete research article, including BLEG-1 dual activity analysis, is published in the International Journal of Molecular Sciences (Au et al., 2021). • The method was optimized to increase the stability of BLEG-1 in purification, post-purification and crystallization processes. • Protein crystallization using the optimized conditions presented herein is able to produce and regenerate BLEG-1 protein crystals of medium-size, which is an advantage in X-ray diffraction. • The method can be used for relevant homologs and variants of BLEG-1 for structure-function and mechanistic studies of such proteins.
Keywords: B3 metallo-β-lactamase; BLEG-1; Protein crystallography; Protein production; Protein purification.
© 2022 The Author(s).