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Recombinant Expression and Purification of Large Bacterial Multienzyme Assemblies for Biosynthetic Processes. | LitMetric
Many biosynthetic transformations have strict spatial and temporal requirements that necessitate the physical association of multiple enzymes for proper function. Here, we describe protocols for obtaining large multienzyme assemblies (>500 kDa) by recombinant expression in Escherichia coli. We focus on assemblies from stand-alone enzymes joined by intermolecular forces rather than multiple catalytic domains from a single polypeptide chain. Details are given for strategies to optimize protein expression and to design a multi-affinity tag purification scheme for large multienzyme assemblies. These insights are drawn from our study of bacterial hydrocarbon biosynthesis.
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