Current Chemical Genomics and Translational Medicine

2012, 6 : 8-17
Published online 2012 September 20. DOI: 10.2174/1875397301206010008
Publisher ID: CCGTM-6-8

RESEARCH ARTICLE
 The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

Scott N Peterson and Keehwan Kwon, *
J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850, USA

* Address correspondence to this author at the J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850: Tel: 301-795-7647; Fax: (301)294-3142; E-mail: kkwon@jcvi.org

ABSTRACT

Technological and methodological advances have been critical for the rapidly evolving field of proteomics. The development of fusion tag systems is essential for purification and analysis of recombinant proteins. The HaloTag is a 34 KDa monomeric protein derived from a bacterial haloalkane dehalogenase. The majority of fusion tags in use today utilize a reversible binding interaction with a specific ligand. The HaloTag system is unique in that it forms a covalent linkage to its chloroalkane ligand. This linkage permits attachment of the HaloTag to a variety of functional reporters, which can be used to label and immobilize recombinant proteins. The success rate for HaloTag expression of soluble proteins is very high and comparable to maltose binding protein (MBP) tag. Furthermore, cleavage of the HaloTag does not result in protein insolubility that often is observed with the MBP tag. In the present report, we describe applications of the HaloTag system in our ongoing investigation of protein-protein interactions of the Y. pestis Type 3 secretion system on a custom protein microarray. We also describe the utilization of affinity purification/mass spectroscopy (AP/MS) to evaluate the utility of the Halo Tag system to characterize DNA binding activity and protein specificity.

Keywords:

HaloTag, protein-protein interactions, protein-DNA interactions, expression, immobilization, Type 3 secretion factors, RpoA.