Abstract
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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Acknowledgement
This work was conducted in the lab of Dr. Lawrence Wackett at the University of Minnesota. I thank him for his support and mentorship. Financial support came from the NIH’s Biotechnology Training Grant and internal University of Minnesota funds. Many others at the University of Minnesota made this research possible. Additionally, I thank Dr. Matthew Jensen, Troy Biernath, and Jordan Holthe for helpful comments in the revision process.
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Christenson, J. (2022). Recombinant Expression and Purification of Large Bacterial Multienzyme Assemblies for Biosynthetic Processes. In: Stamatis, H. (eds) Multienzymatic Assemblies. Methods in Molecular Biology, vol 2487. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2269-8_1
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DOI: https://doi.org/10.1007/978-1-0716-2269-8_1
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-2269-8
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