Molecular cloning, protein sample preparation and
crystallization
A cDNA plasmid that contains a DNA fragment encoding the full-length
human PPA1 (GenBank NC_000010.11) was purchased from DNASU Plasmid
Repository. The protein product of this cDNA corresponds to the human
PPA1 protein with GenBank accession No. NP_066952. The DNA encoding the
full length PPA1 (residues 1-289) was amplified by PCR from the plasmid
with the forward primer
5’-CAAGGACCGAGCAGCCCCTCA atgagcggcttcagcaccg-3’ and the reverse
primer 5’-ACCACGGGGAACCAACCCTTA CTAGTTTTTCTGGTGATGGAAC-3’. Each of
these primers contains a 5’-end specific sequence (21 nucleotides,
underlined) for ligation-independent cloning (LIC). The PCR product was
purified by agarose gel electrophoresis and processed by T4 DNA
polymerase in the presence of 2.5 mM dATP to generate 5’-overhangs at
both ends. To find an appropriate
way to express the PPA1 protein in E. coli in a soluble form, we
had tested a series of in-house developed LIC cloning vectors containing
different fusion tags, including the MBP, GST, TRX, SUMO, HALO, NusA,
and Fc tags, etc. The cloning vector with a HALO tag [51] gave the
best result. The cloning vector encodes the Halo tag, His tag, and an
eight amino-acid recognition sequence for human rhinovirus (HRV) 3C
protease in front of a specific LIC sequence. The vector was linearized
by SmaI digestion and subsequently processed by T4 DNA polymerase in the
presence of 2.5 mM dTTP to generate 5’-overhangs at both ends. The
processed PCR insert and vector were mixed and incubated at room
temperature for 20 minutes. The mixture was used to transform NEB-turboEscherichia coli competent cells. The recombinant plasmid was
transformed into NiCo21(DE3) E. coli cells (New England Biolabs).
Cell culture was grown in LB media until it reached an
OD600 of 0.6-0.8. Isopropyl β-D-1-thiogalactopyranoside
(IPTG) was added to the culture (at 0.4 mM final concentration) to
induce protein expression. After induction, the temperature was lowered
to 288 K and the culture was allowed to grow overnight before harvest.
The PPA1 protein was expressed in E. coli as a fusion protein
with an N-terminal Halo tag and His tag, and a HRV 3C protease
recognition for cleavage motif. The fusion protein was purified by NTA
affinity resin. The fusion tags were then cleaved by HRV 3C protease.
After removal of the tags, the target protein contains an artificial
sequence GPSSP at the N-terminus as a cloning artifact. The cleaved tags
were separated from the target protein by a reverse IMAC (immobilized
metal-affinity chromatography) process with NTA resin. The purified
target proteins were concentrated to ~10 mg/ml in a
buffer containing 25 mM Tris (pH 7.5), 200 mM NaCl, and 1 mM DTT.
Crystallization trials were carried out using 96-well-format plates. Ten
sets of in-house prepared screening solutions (each containing 96
different conditions with various PEGs as the precipitants) were used in
the trials. Multiple crystallization conditions were identified within a
few days. After optimizing the crystallization conditions, diffracting
crystals of the PPA1 were obtained at 295 K by hanging-drop vapor
diffusion against 50 µl well solution using 96-well format
crystallization plates. The crystallization drops contained 2 µl protein
sample mixed with 2 µl well solution. The well solution contains 20%
PEG 3350, 100 mM Bis-Tris propane (pH 7.5), 200 mM KBr, and 10% (v/v)
glycerol. Crystals were flash-cooled in liquid nitrogen.