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Affinity Chromatography pp 169–190Cite as

Amylose Affinity Chromatography of Maltose-Binding Protein

Purification by both Native and Novel Matrix-Assisted Dialysis Refolding Methods

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 421))

Summary

Maltose-binding protein (MBP) is a carrier protein for high level recombinant protein and peptide production from either the cytoplasm or periplasm of Escherichia coli. The affinity matrix for purifying MBP-passenger proteins utilizes amylose covalently attached to magnetic beads, agarose, or a chemically inert fast protein liquid chromatography (FPLC) matrix – exploiting the natural affinity of MBP for α-(1→4)-maltodextrins in the stationary phase. A fundamental problem is the expression and purification failure of as much as 30% of all constructs, which is limiting for one of the best solubilizing carrier proteins available for recombinant expression. In this chapter, we have discussed aspects of MBP biology that can impact upon binding to the amylose affinity matrix including cloning considerations, structural complications, hydrophobic buffer additives and the presence of cellular biomolecules that bind or modify the matrix during purification. Chromatography conditions are presented for purification at very small scales of less than 0.5 mL using amylose magnetic beads, a batch and semi-batch method for small to moderate scale purifications up to approximately 35 mg and larger scale FPLC methods. A novel matrix-assisted dialysis refolding method is also described whereby MBP-passenger proteins can be refolded in the presence of amylose matrix in instances where native purification methods fail to bind the amylose matrix.

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Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Monash University, Clayton Victoria, Australia

    Leonard K Pattenden

  2. Baker Heart Research Institute, Melbourne, Victoria, Australia

    Walter G Thomas

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  1. Leonard K Pattenden
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  2. Walter G Thomas
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Editors and Affiliations

  1. BioMarin Pharmaceutical Inc., CA

    Michael Zachariou (Director Project Management) (Director Project Management)

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© 2008 Humana Press, a part of Springer Science+Business Media, LLC

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Pattenden, L.K., Thomas, W.G. (2008). Amylose Affinity Chromatography of Maltose-Binding Protein. In: Zachariou, M. (eds) Affinity Chromatography. Methods in Molecular Biology™, vol 421. Humana Press. https://doi.org/10.1007/978-1-59745-582-4_12

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  • DOI: https://doi.org/10.1007/978-1-59745-582-4_12

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-659-7

  • Online ISBN: 978-1-59745-582-4

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