Nanobodies (Nbs) are single domain antibodies based on the variable domains of heavy chain only antibodies (HCAbs) found in camelids, also referred to as VHHs. Their small size (ca. 12-15 kDa), superior biophysical and antigen binding properties have made Nbs very attractive molecules for multiple biotechnological applications, including human therapy. The most widely used system for the purification of Nbs is their expression in the periplasm of Escherichia coli with a C-terminal hexa-histidine (His(6)) tag followed by immobilized metal affinity chromatography (IMAC). However, significant variability in the expression levels of different Nbs are routinely observed and a single affinity chromatography step is often not sufficient to obtain Nbs of high purity. Here, we report an alternative method for expression and purification of Nbs from the periplasm of E. coil based on their fusion to maltose binding protein (MBP) in the N-terminus and His6 tag in the C-terminus (MBP-Nb-His6). Soluble miw-Nb-His6 fusions were consistently expressed at high levels (>= 12 mg/L of induced culture in shake flasks) in the periplasm of E. coil HM140, a strain deficient in several periplasmic proteases. Highly pure MBP-Nb-His6 fusions and free Nb-His6 (after site specific proteolysis of the fusions), were recovered by amylose and metal affinity chromatography steps. The monomeric nature of the purified Nb-His6 was determined by gel filtration chromatography. Lastly, we demonstrated by ELISA that both monomeric Nb-His6 and MBP-Nb-His6 fusions retained antigen binding activity and specificity, thus facilitating their direct use in antigen recognition assays. (C) 2013 Elsevier Inc. All rights reserved.