2.3 Specialist
Reagents
and Equipment
- The protein purification protocol used here makes use of the
A ̈KTA purification system (GE Healthcare). While these sys-
tems are widely available in many laboratories, an alternative
chromatography system or stand-alone peristaltic pump could
be substituted. - Chromatography columns. Size exclusion chromatography,
Superdex HiLoad prep grade XK 16/600 (GE Healthcare).
Nickel affinity chromatography, HisTrap FF Sepharose, 5 mL
(GE Healthcare). - For gel analysis of protein samples. The NuPAGE mini-gel
system (Thermo Fisher Scientific) was used with 4–12% acryl-
amide gradient Bis-Tris gels and the MES SDS gel running
buffer system. The sample buffer was NuPAGE LDS sample
buffer with 1 mM DTT. Mark12 markers (Thermo Fisher
Scientific) were used to size the bands. InstantBlue (Expedeon)
Coomassie protein stain was used to visualize the bands. - Protein was concentrated using Vivaspin protein concentrator
columns (GE Healthcare). - The kinase CaMKKβwas prepared as described in Ref. [10].
- The AMPK constructs were prepared in pET47 plasmid as
described previously [4]. - The benzimidazole derivative small molecule referred to as
991 is an AMPK activator that binds at the ADaM site. 991 is
used in this protocol to stabilize full-length AMPK for crystal-
lization. Its chemical name is 5-[6-chloro-5-(1-methylindol-5-
yl)-1H-benzimidazol-2-yl]oxy-2-methyl-benzoic acid, and it
can be purchased as a custom synthesis from specialist chemical
supplies such as SpiroChem AG. As 991 is sparingly soluble in
aqueous solvents, a 10 mM solution in 100% dimethyl sulfox-
ide (DMSO) needs to be prepared and stored at 20 C. - As an alternative to 991, the commercially available small mol-
ecule ADaM-binding activator, A-769662 4-hydroxy-3-
[4-(2-hydroxyphenyl)phenyl]-6-oxo-7H-thieno[2,3-b]pyri-
dine-5-carbonitrile can be obtained from ApexBio Technology.
Crystals obtained with this ligand diffract less well possibly due
to the lower affinity of A-769662 compared to 991.
3 Methods
3.1 General Points The first part of the protocol below describes optimized methods
for expression and purification of crystallization-grade full-length
AMPKα 1 β 1 γ1; however, the protocol can equally be used to
produce the truncated core domain AMPK, which has also been
crystallized [2]. Full-length protein is prone to aggregation during
this process. This has been reduced by optimization of the process
4 Julia A. Hubbard et al.