Novel affinity tag system using structurally defined antibody‐tag interaction: Application to single‐step protein purification

Abstract

Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody-peptide interaction, opening opportunities for further improvements/modifications.

Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region

“…Protein Expression, Immunoprecipitation, and Western Blotting-The expression vector for the N-terminally tagged full-length reelin was prepared as described previously (23). The substitutions of cysteine to alanine were introduced into the expression vectors by using the QuikChange strategy (Stratagene).…”

“…Transient expression of the full-length reelin (wild type and mutants) in 293T cells was performed using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. Cell culture supernatant was mixed with P20.1 IgG-immobilized Sepharose 4 Fast Flow (GE Healthcare) (23) and rotated at 4°C for 1 h. After washing with 20 mM Tris, pH 7.5, 150 mM NaCl (Tris-buffered saline (TBS)), proteins retained on beads were eluted with 2ϫ SDS-PAGE sample buffer without reducing reagents followed by heat treatment at 95°C for 3 min. Eluates were divided into two equal parts, further incubated with or without 100 mM DTT, and subjected to SDS-PAGE (5% acrylamide gel) followed by electrophoretic transfer onto PVDF membranes.…”

“…The cell culture supernatant was concentrated by using Acquacide II (Calbiochem) prior to the following purification procedure. Proteins were purified using the P20.1-immobilized Sepharose as described previously (23). Eluted proteins were dialyzed against TBS containing 2 mM CaCl 2 and concentrated by ultrafiltration (100-kDa molecular mass cutoff).…”

“…To circumvent these difficulties, we developed a purification protocol for recombinant reelin using an affinity tag system developed in our laboratory (23). Moreover, we utilized monoclonal antibodies recognizing the central portion of reelin established by Goffinet and co-workers (24).…”

Direct binding of parotid secretory protein to phosphatidylinositol phosphates.

“…There are several fusion tags available, including polyhistidine tags [76], FLAG tags [77], thioredoxin, Protein A, strep tag and maltose binding protein [76]. Glutathione-S-transferase [53] has been widely used as a fusion tag for protein expressed in E.coli.…”