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.