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J. Immunol. Methods 426, 95–103. On-bead antibody-small molecule conjugation using high-capacity magnetic beads. 2015

Nath, N., Godat, B., Benink, H. and Urh, M.

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-teminal 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)

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Protein Expr. Purif. 89, 62–72. Application of HaloTag technology to expression and purification of cannabinoid receptor CB2. 2013

Locatelli-Hoops, S., Sheen, Fangmin C., Zoubak, L., Gawrisch, K. and Yeliseev, A.A.

Notes: The HaloTag® Protein Tag was used to tag, immobilize and purify functional cannabinoid receptor CB2, a GPCR,  after induction of expression in E. coli.  (4263)

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Protein Expr. Purif. 68(1), 110-120. HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification 2009

Ohana, R.F., Encel, L.P., Zhao, K., Simpson, D., Slater, M.R., Urh, M., Wood, K.V.

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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Anal. Biochem. 392, 45-53. Protein-protein interaction studies on protein arrays: effect of detection strategies on signal-to-background ratios. 2009

Hurst, R., Hook, B., Slater, M.R., Hatrnett, J., Storts, D.R., and Nath, N.

Notes: These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)35S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use. (3999)

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DNA Research 15, 137-149. Exploration of human ORFeome: High-throughput preparation of ORF clones and efficient characterization of their protein products. 2008

Nagase, T., Yamakawa, H., Tadokoro, S., Nakajima, D., Inoue, S., Yamaguchi, K., Itokawa, Y., Kikuno, R.F., Koga, H. and Ohara, O.

Notes: These authors used the Flexi® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi® System and Gateway® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

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J. Proteome Res. 7(10), 4475-82. Improving protein array performance: focus on washing and storage conditions. 2008

Nath, N., Hurst, R., Hook, B., Meisenheimer, P., Zhao, K.Q., Nassif, N., Bulleit, R.F., and Storts, D.R.

Notes: These authors tested the effect of different washing, drying and storage conditions on the stability of various protein:protein interaction arrays. They tested five different interacting protein pairs and three enzymes, monitoring stability and activity under various processing conditions. They found that addition of 5% glycerol to the wash buffer helped retain enzyme activity during washing and drying. There was significant loss of enzyme activity when slides were stored dry at 4oC but slides stored at -20oC in 50% glycerol retained enzyme activity. HaloTag-fused enzymes in cell-free extracts could undergo multiple freeze-thaw cycles without significant effect on enzyme activity, indicating that such extracts could be frozen at -70oC and used to print small batches of arrays at various intervals over a period of weeks. (3890)

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Hum. Mol. Genet. 15, R31-R43. From genome to proteome: developing expression clone resources for the human genome. 2006

Temple, G., Lamesch, P., Milstein, S., Hill, D.E., Wagner, L., Moore, T. and Vidal, M.

Notes: The Kazusa cDNA Project is constructing a library of more than 1,000 "full ORF" (F-ORF) clones in the Flexi® Vector System to characterize the function of proteins that are larger than 50kD. (3393)

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