Notes: This paper describes an improved method for on-bead conjugation of antibodies that overcomes the limitations of solution-based protocols (requirement for purified, high-concentration antibodies and the need for multiple buffer changes). The method uses HaloTag technology to immobilize protein A and G onto high-capacity magnetic beads. Antibodies are then captured and labeled on-bead. The authors demonstrate that the method is compatible with amine- and thiol-based chemistries as well as with  mouse and human antibody isotypes, both purified and in cell media. Protein G and Protein A-HaloTag fusion proteins were constructed by cloning the coding sequences for the Fc-binding domains between N-terminal HQ and C-terminal HaloTag sequences. To create Protein A and G beads, the purified Protein G-HaloTag and Protein A-HaloTag constructs were covalently attached to Magne HaloTag Beads--magnetic cellulose beads activated with HaloTag ligands. (4586)

Notes: In this study, the L. pneumophila protein Lem3 was expressed as a HaloTag^® fusion protein and purified using HaloLink™ Resin. Lem3 was first cloned into the pFN22K HaloTag^® Vector and the resultant HaloTag-Lem3 protein was expressed in Single-Step (KRX) competent cells before purification using the HaloTag^® Protein Purification System. Lem3 was cleaved from the HaloLink™ Resin using TEV protease.

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Notes: The authors note that while many protein fusion tags are available to assist in purifying functional, recombinant proteins in soluble form and adequate amounts, none of the available tags are ideal when applied to the diversity of proteins studied. Available tags are often best-suited to specific aspects of the overall process, be it expression, solubilization, protein capture, etc. HaloTag was designed to address multiple features. The authors demonstrated that HaloTag provided enhanced expression and solubility in E. coli, with efficient protein purification and labeling for screening and quantitation. Enhanced solubility coupled with covalent capture gives HaloTag advantages over conventional affinity fusion tags, demonstrated by the successful purification of a wide variety of proteins, including low expressers, with higher yield and purity compared to the other tested tags. Covalent capture coupled with efficient tag removal to release the target protein, provides higher purity by eliminating tag contaminants. Using full-length cDNAs encoding 23 human proteins of varying size and function, the authors compared the efficacy of HaloTag as an expression and purification tag to the frequently used solubility enhancers MBP and GST. As reported previously, the three larger tags (HaloTag, MBP and GST) provided higher total and soluble expression compared to the smaller His6tag. In comparing soluble protein expression levels for the three larger tags, it was found that HaloTag solubilized 74% of the 23 human proteins examined, compared to 52% for MBP and 39% for GST. G2681 was one of the T7, bacterial promoter-based Flexi vectors used.
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