4.1 Cloning, expression, and purification of L-hIGFBP2
The primers were designed for L-h IGFBP2 [residues 97–191 of
the full-length protein] and its L-h IGFBP mutants
(L-h IGFBP2[desK150-E161]) lacking the C-terminal tag and
residues 150-161. Oligonucleotide strands 5’ CAT GGT ACC GAT GAT GAT GAT
AAA AAG CGC CGG GAC GCC GAG TAT G 3’ with enterokinase cleavage site and
5’ GAC GAA TTC TTA GGG AGT CCT GGC AGG GGG TGG TCG CA 3’ were used as
forward and reverse primers for L-h IGFBP2[desK150-E161]. The
insert was cloned between Kpn1 and Xho1 restriction sites of the pET 32a
vector having a thioredoxin tag fused to the protein.
The oligonucleotide strands 5’ ATTG GGA TCC GAG AAG CGC CGG 3’ and 3’
ATTG GAA TTCTTA CAG GGA GTC CTG 5’ with BamH1 and EcoR1 restriction
sites on forward and reverse primers, respectively, were synthesized by
MWG-Eurofins and used to amplify L-h IGFBP2 at 65 °C in a
thermo-cycler. Following amplification, PCR products were purified using
a Sigma Gel elution kit after analyzing on 1% agarose gel in 1X TAE
buffer. The vector used was an IPTG-inducible pGEX6P-1 with a GST tag.
The vector and L-h IGFBP2 were digested with BamH1 and EcoR1,
producing staggered ends, and treated with ligase at 16οC for 16 h. The ligated product was used to transformE. coli Top10 cells, and the desired clone was obtained using a
Sigma miniprep plasmid isolation kit. The clone was confirmed by
sequencing. During the process of cloning the following C-terminal tag
was introduced: KNSRVDSSGRIVTD. This tag did not affect the binding of
IGF-1 as verified using the construct
L-h IGFBP2[desK150-E161], which did not contain these
additional residues. At the N-terminal end, cleavage by HRV 3C protease
to separate the GST tag from the protein resulted in the following
residues added to the N-terminus: GPLGS.
The L-h IGFBP2 construct was transformed into E. coliBL21(DE3) competent cells. The transformed colony was inoculated in 50
mL of primary culture (LB broth with 100 µg/mL ampicillin) and incubated
at 37 °C for 16 h, 200 rpm. The cells were transferred to minimal media
and grown to the mid-log phase (optical density at 600 nm
(OD600) ~ 0.6). At this OD, the
expression of L-h IGFBP2 was induced with 1 mM isopropyl
β-D-1-thiogalactopyranoside (IPTG) for 5 h at 37 °C, 200 rpm.
Cells were harvested by centrifugation at 6000 rpm and resuspended in
phosphate-buffered saline (PBS) [150 mM NaCl, 2.5 mM KCl, 10 mM
Na2HPO4, 2 mM
KH2PO4, pH 7.4], and 1 mM PMSF. Lysis
by sonication was carried out on the ice for 10 min (30 s pulse) and the
lysate was centrifuged at 8000 rpm for 40 min. The supernatant was
collected and allowed to bind to the pre-equilibrated GST bind resin for
2 h at 4 ο C on a rotator. The resin was washed three
times with each of 10 bed volume of PBS buffer, high salt [25 mM
HEPES, 0.05% NaN3, 0.5M NaCl, 0.1% Triton X-100, pH
7.5] and low salt [25 mM HEPES, 0.5% NaN3, 0.1 M
NaCl, 0.1% TritonX-100, pH 7.5] buffer. The cleavage buffer (PBS) was
used to wash the beads before adding HRV 3C protease to perform
on-column cleavage at 4 º C for 16 h.
L-h IGFBP2[desK150-E161] was expressed in E. coli BL21
cells at 30 °C and induced for 4 h with 0.5 mM IPTG. For the
purification of thioredoxin fused L-h IGFBP2[desK150-E161],
the cell pellet was resuspended in buffer A (20 mM Tris pH 8.0, 100 mM
NaCl, 10 mM imidazole, and 10% glycerol) containing 0.3 mg/mL lysozyme
and incubated for 20 min in the presence of EDTA-free protease inhibitor
cocktail from Roche. The cell resuspension was sonicated, and the cell
lysate was clarified by centrifuging at 14,000 rpm for 30 min at 4 °C.
The supernatant was loaded onto a pre-equilibrated Ni-NTA HisTrap column
(GE Healthcare, 5ml) with a flow rate of 0.25 mL /min. Unbound proteins
were removed by extensive washing with buffer A followed by a wash with
buffer A containing 0.5 M NaCl. The fusion protein was eluted with
buffer A containing 250 mM imidazole and exchanged with enterokinase
cleavage buffer (50 mM Tris-Cl, pH 8.0, 1mM CaCl2, and
50 mM NaCl). The thioredoxin tag was removed by incubating the fusion
protein in enterokinase at 23 °C for 16 h and passing the mixture back
onto the Ni-NTA HisTrap column (GE Healthcare, 5ml). Unbound protein was
eluted in buffer A. The protein was concentrated using a Millipore
centricon with a 3 kDa molecular weight cut-off.