title: High yield purification of nanobodies from the periplasm of E. coli as fusions with the maltose binding protein.
abstract: 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. 12-15kDa), 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 (His6) 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. coli based on their fusion to maltose binding protein (MBP) in the N-terminus and His6 tag in the C-terminus (MBP-NbHis6). Soluble MBP-NbHis6 fusions were consistently expressed at high levels (⩾12mg/L of induced culture in shake flasks) in the periplasm of E. coli HM140, a strain deficient in several periplasmic proteases. Highly pure MBP-NbHis6 fusions and free NbHis6 (after site specific proteolysis of the fusions), were recovered by amylose and metal affinity chromatography steps. The monomeric nature of the purified NbHis6 was determined by gel filtration chromatography. Lastly, we demonstrated by ELISA that both monomeric NbHis6 and MBP-NbHis6 fusions retained antigen binding activity and specificity, thus facilitating their direct use in antigen recognition assays.
authors.affiliation: Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain.
authors.forename: Valencio
authors.initials: V
authors.lastname: Salema
authors.forename: Luis Ángel
authors.initials: LÁ
authors.lastname: Fernández
mesh_terms: D000595:Amino Acid Sequence; D000941:Antigens / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000941:Antigens; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; C461619:MalE protein, E coli; D009842:Oligopeptides; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D006639:Histidine
keywords: Affinity chromatography; E. coli; MBP; Nanobodies; Recombinant antibodies