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.