Expression, One-Step Purification, and Immobilization
of HaloTag
TM
Fusion Proteins on Chloroalkane-
Functionalized Magnetic Beads
Hassan Motejadded
&
Bertolt Kranz
&
Sonja Berensmeier
&
Matthias Franzreb
&
Josef Altenbuchner
Received: 28 January 2010 / Accepted: 26 April 2010 /
Published online: 15 May 2010
#
Springer Science+Business Media, LLC 2010
Abstract The presented work introduces a novel method to immobilize enzymes either
purified or directly out of a crude extract onto magnetic particles in the micrometer range.
This method is based on the creation of a fusion protein consisting of the enzyme of choice
and a mutant dehalogenase. The dehalogenase gene is commercially available from the
company Promega under the name HaloTag
TM
. When the fusion protein is contacted with
magnetic beads having chemically synthesized, chloroalkane ligands on their surface, the
dehalogenase and the ligand undergo a covalent coupling leading to stable and spatially
defined immobilization. The principle was proved with a lipase fused to the HaloTag
TM
gene and magnetic poly(methyl)methacrylate beads as carriers. The solubility of the tagged
lipase was strongly increased by fusion of the malE gene at the N-terminal end of the
HaloTag
TM
lipase gene. This tripartite protein was purified on amylose resin and used for
immobilization. About 13 µg protein could be immobilized per 1 mg of beads within a few
minutes. Due to the defined binding site, no activity loss was observed in the course of the
immobilization. The resulting enzyme carrier was tested with the same beads up to six
times for lipase activity over a storage period of 36 days at 8 °C. No loss of activity was
found during this time.
Keywords Enzyme immobilization
.
HaloTag™
.
Fusion protein
.
Magnetic bead
.
Dehalogenase
.
Expression
Appl Biochem Biotechnol (2010) 162:2098–2110
DOI 10.1007/s12010-010-8985-1
H. Motejadded
:
J. Altenbuchner
Institute of Industrial Genetics, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
B. Kranz
Institute for Food Science and Biotechnology, Department of Biotechnology, University of Hohenheim,
Garbenstr. 25, 70599 Stuttgart, Germany
S. Berensmeier
:
M. Franzreb (*)
Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT),
Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
e-mail: matthias.franzreb@kit.edu