Supplementary Methods
Construction of plasmids
All of the primers used in this study are listed in supplementary Table 1. To
construct the expression plasmid for GST-TbPex14p or TbPex5p-His, the DNA
fragment encoding TbPex14p or TbPex5p were inserted into pGEX-6P-1 (GE
Healthcare) or pET24b (Novagen, Madison, WI), respectively. A fragment with the
1.1-kb
TbPEX14
coding
region
was
amplified
with
the
primer
set
TbPEX14-Bam-Fw/TbPEX14-Not-Rv using genomic DNA as the template. Similarly,
the
2.0-kb
TbPEX5
coding
region
was
amplified
with
the
primers
TbPEX5-Nde-Fw/TbPEX5-Xho-Rv. The PCR products were digested with BamHI/NotI
or NdeI/XhoI, then ligated into the BamHI–NotI site of pGEX-6P-1 or NdeI–XhoI site of
pET24b, respectively, to yield pGEX-6P-1/TbPEX14 and pET24b/TbPEX5. The
expression
plasmids
for
TbPex5p(a.a.47–60),
TbPex5p(a.a.196–208)
and
TbPex5p(a.a.310–333), which contained the deduced Pex14p binding motifs fused with
a His-tagged lipoyl domain at the N-terminus, were constructed as follow. First, the
0.9-kb TbPex5p(a.a.47–333) coding region was amplified with the primer set
Tb5motif1NdeI-Fw/Tb5motif3BamHI-Rv using pET24b/TbPEX5 as the template. The
PCR product was digested with NdeI and BamHI, then ligated into the NdeI–BamHI site
of pOPHLT to yield pOPHLT/TbPEX5(a.a.47–333). Then the 3.3-kb fragments
containing the TbPEX5(a.a.47–60) or TbPEX5(a.a.310–333) region was amplified by
inverse
PCR
using
the
primer
sets
Tb5M1inv-Fw/Tb5M1inv-Rv
or
Tb5M3inv-Fw/Tb5M3inv-Rv using pOPHLT/TbPEX5(a.a.47–333) as the template.
This fragment was self-ligated using T4 Polynucleotide Kinase (Toyobo) and Ligation
high
(Toyobo)
to
form
pOPHLT/TbPEX5(a.a.47–60)
1
and
pOPHLT/TbPEX5(a.a.310–333).
pOPHLT/TbPEX5(a.a.196–208)
and
pOPHLT/TbPEX5(a.a.310–333) were constructed from pOPHLT/TbPEX5(a.a.47–333)
according to the same procedure described above. The DNA fragment encoding
TbPex14p
(a.a.23–71)
was
amplified
Tb14domain-Nde-Fw/Tb14domain-Bam-Rv
using
with
the
primer
pGEX-6P-1/TbPEX14
set
as
the
template. The PCR product was digested with NdeI and BamHI, then ligated with the
NdeI/BamHI fragment of pOPHBL to yield pOPHBL/TbPEX14(a.a.23–71). All of the
cloned inserts were confirmed using the Big Dye V.3.1 Terminator Kit (Applied
Biosystems) in an ABI PRISM 3130 sequencer (Applied Biosystems).
Purification of TbPex5p-His and GST-TbPex14p
TbPex5p-His6 was overexpressed using E. coli BL21(DE3)pLysS cells (Novagen,
Darmstadt, Germany) as the host. The E. coli cells harboring pET-24b/TbPEX5 were
grown at 37°C in LB media containing 50 µg/ml kanamycin. At a cell density of 0.7
(OD600), protein expression was induced with 0.5 mM IPTG for 4 h at 37°C. The cells
were harvested by centrifugation at 4,000 x g for 20 min, resuspended in 3.5 ml of
buffer A (50 mM Hepes, pH7.5, 150 mM NaCl, 4 mM β-mercaptoethanol, 1% Triton
X-100, 5% glycerol and 5 mM imidazole) and disrupted 20 times for 20 s in an ice bath
using an Astrason XL-2020 ultrasonic processor (Misonix Inc., Farmingdale, NY). The
lysate was centrifuged at 20,000 x g for 20 min and TbPex5p-His in the supernatant was
immediately applied to 250 µl of cOmplete His-Tag Purification resin (Roche
Diagnostics GmbH, Mannheim, Germany) equilibrated with the lysis buffer A. After
extensive washing, the TbPex5p-His was eluted with buffer A containing 250 mM
imidazole. The TbPex5p-His mutants were purified with the same procedure.
2
GST-TbPex14p was also overexpressed using E. coli BL21(DE3)pLysS cells as
the host. The E. coli cells harboring pGEX-6P-1/TbPEX14 were grown at 37°C in LB
media containing 100 µg/ml ampicillin. At a cell density of 0.6 (OD600) protein
expression was induced with 0.5 mM IPTG for 10 h at 20°C. The cells were harvested
by centrifugation at 4,000 x g for 20 min, resuspended in 35 mL of buffer B (50 mM
Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1% Triton X-100 and 10%
glycerol) and disrupted 20 times for 20 s in an ice bath with an Astrason XL-2020
ultrasonic processor. The lysate was centrifuged at 20,000 x g for 20 min and the
GST-TbPex14p in the supernatant was immediately applied to 10 ml of
glutathione-Sepharose 4B resin (GE Healthcare, Little Chalfont, UK) equilibrated with
buffer B. After extensive washing, the GST-TbPex14p was eluted with buffer B
containing 20 mM glutathione.
Purification
of
His-Lipoyl-TbPex5p(a.a.47–60),
-TbPex5p(a.a.196–208),
-TbPex5p(a.a.310–333), -TbPex14p (a.a.23–71), and TbPex14p (a.a.23–71)
E.
coli
C41(DE3)RIPL
cells
were
pOPHLT/TbPEX5(a.a.47–60),
pOPHLT/TbPEX5(a.a.310–333)
transformed
with
the
plasmids,
pOPHLT/TbPEX5(a.a.196–208),
or
pOPHLT/TbPEX14(a.a.23–71)
according
to
standard procedures. The E. coli cells harboring these plasmids were grown at 37°C in
LB media containing 0.1 mg/mL ampicillin. At a cell density of 0.5 (OD600) protein
expression was induced with 0.3 mM IPTG for 3 h at 37°C. The cells were harvested by
centrifugation at 3,765 x g for 10 min, resuspended in buffer C (20 mM Tris-HCl, pH
7.5, 100 mM NaCl and 1 mM dithiothreitol) and lysed by sonication on ice. The lysate
was centrifuged at 100,000 x g for 20 min, then the supernatant was passed through the
3
0.45 µm filter. The supernatant was applied to HisTrap resin (GE Healthcare)
equilibrated with lysis buffer C. After extensive washing, the recombinant protein was
eluted using a linear gradient with an increasing imidazole concentration (0 to 500 mM)
in elution buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 500 mM imidazole).
The recombinant proteins were further purified by anion exchange chromatography (by)
with an HiTrap Q HP column (GE Healthcare) followed by gel filtration
chromatography with Superdex 75 16/60 (GE Healthcare). TEV protease was used for
His-Lipoyl-TbPex14p (a.a.23–71). TbPex14p (a.a.23–71) was purified by gel filtration
chromatography.
4
Supplementary Table 1 List of oligonucleotide primer sequences.
TbPEX14-Bam-Fw
5’-AAATTTATAGGATCCATGTCTTTGCTGCTGTCGGG-3’
TbPEX14-Not-Rv
5’-AAATTTATAGCGGCCGCTCAAGCTGCCTCGCCGCCAA-3’
underbar shows BamHI and NotI site, respectively
TbPEX5-Nde-Fw
5’-AAATTTATACATATGATGGACTGCGGCGCCGG-3’
TbPEX5-Xho-Rv
5’-AAATTTATACTCGAGCCGGCGCCGCAGTCCAT-3’
underbar shows NdeI and XhoI site, respectively
Tb5W52A/F56A-Fw
5'-AGCACGCTGCTGCCCATCAACATCACCAC-3'
Tb5W52A/F56A-Rv
5'-GAGCCGCGTCTTCCATGGGCCCGGTGGG-3'
Tb5W200A/Y204A-Fw
5'-AGGATGCCAAGGATGTTGAGGTGCATACG-3'
Tb5W200A/Y204A-Rv
5'-GTCCCGCCTCCGCCTGGTGCAATTTCTC-3'
Tb5W318A/Y322A-Fw
5'-AGGAAGCCGCACAAATGCAGGCCATGCAG-3'
Tb5W318A/Y322A-Rv
5'-GTGCCGCCTGCTCAACGTCGGCGCTGGG-3'
Tb5motif1NdeI-Fw
5’-GGAGATATACATATGGGGCCCATGGAAGAC-3’
Tb5motif3BamHI-Rv
5’-CGCGGATCCTCACTGCAGACGCTCCTG-3’
underbar shows NdeI and BamHI site, respectively
Tb5M1inv-Fw
5'-TGAGGATCCAGATCTAAGCTTGGTACC-3'
Tb5M1inv-Rv
5'-TTGATGGGCAGCAAAGTGCTG-3'
Tb5M3inv-Fw
5'-GACCCCAGCGCCGACGTTGAG-3'
Tb5M3inv-Rv
5'-CATATGGGAGCCCTGGAAATACAGGTT-3'
Tb5M2short inv-Fw
5'-CACCAGGCGGAGTGGGGACAG-3'
Tb5M2short inv-Rv
5'-GCACCTCAACATCCTTGTAATCCTG-3'
Tb14domain-Fw
5'-AAATTTATACATATGTCTGAACGTGAGAAACGTGTT-3'
Tb14domain-Rv
5'-TATAAATTTGGATCCTCATCCAACTTTGGTGAACGC-3'
Tb5E321A-Fw
5'-CATACGCACAAATGCAGGCCATG-3'
Tb5E321A-Rv
5'-CCTGTGCCCACTGCTCAACGTC-3'
Tb5E321H-Fw
5'-ACTACGCACAAATGCAGGCCATG-3'
Tb5E321H-Rv
5'-GCTGTGCCCACTGCTCAACGTC-3'
5
Supplementary Table 2 Data collection, structure determination and refinement
statistics for TbPex14p.
TbPex14p
Data collection *
Space group
P6122
Cell dimensions
a, b, c (Å)
53.6, 53.6 342.8
(°)
90.0, 90.0, 120.0
Wavelength (Å)
1.000
27.57 - 1.65 (1.74 -
Resolution (Å)
1.65)
Rsym
0.061 (0.478)
I / I
24.3 (6.2)
Completeness (%)
100.0 (100.0)
Redundancy
17.4 (18.3)
Refinement
Resolution (Å)
27.57 - 1.65
No. reflections
37018
Rwork / Rfree
0.20 /0.23
No. atoms
Protein
1951
Ligand (Sulfate)
15
Water
195
B-factors
Protein
33
Ligand (Sulfate)
39
Water
40
R.m.s. deviations
Bond length (Å)
0.006
Bond angles (°)
0.899
a
Highest-resolution shell is shown in parenthesis.
Rmerge = ∑hkl∑i |Ii(hkl) - <I(hkl)>| / ∑hkl∑i Ii(hkl).
c
Rcryst and Rfree = ∑ ||Fobs| - |Fcalc|| / ∑|Fobs|; Rfree = R-factor for a selected subset (5%) of
reflections that was not included in prior refinement calculations.
d
r.m.s. deviations for bond angles and lengths in regard to Engh and Huber parameters [1]
b
6
1. Engh, R.A. and Huber, R. (1991) Accurate bond and angle parameters for X-ray
protein structure refinement. Acta Crystallogr. A 47, 392-400.
7