vAL1 cloning
Plasmid Construction
To construct an Escherichia coli expression system for a fusion protein comprising glutathione S-transferase (GST), vAL-1, and histidine-tagged sequences, the vAL-1 gene (1,047 bp) was inserted into the expression vector pET42b (Novagen) encoding GST and two histidine-tagged sequences as follows. The vAL-1 gene was amplified by PCR by using KOD-Plus polymerase (Toyobo), pGEX_4T_3-vAL-1 as the template (1), and two synthetic oligonucleotides (Supplemental Table S1) as primers containing NcoI and XhoI sites at their 5' regions. After amplification, the PCR product was digested with NcoI and XhoI and then ligated with NcoI-XhoI-digested pET42b by using Ligation High (Toyobo). For gene cloning of the vAL-1(S) catalytic module including Thr-106 to Glu-349 of vAL-1, site-directed mutagenesis was carried out in order to delete the NdeI site from the vAL-1 gene. Adenine-879 was substituted with thymine by using the plasmid pET42b-vAL-1 as the template and two synthetic oligonucleotides (Supplemental Table S1) as the sense and antisense primers with a Quick Change site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The resultant plasmid was designated as pET42b-vAL-1_A879T. For construction of an expression system for vAL-1(S), the vAL-1(S) gene (732 bp) was inserted into pET21b (Novagen) encoding a histidine-tagged sequence, as follows. PCR was performed using KOD-Plus polymerase, pET42b-vAL-1_A879T as the template, and two synthetic oligonucleotides (Supplemental Table S1) as primers containing NdeI and XhoI sites at their 5' regions. The amplified PCR fragment was digested with NdeI and XhoI and then ligated with NdeI-XhoI-digested pET21b by using Ligation High. The resultant plasmid was designated as pET21b-vAL-1(S). DNA manipulations were carried out according to the standard method (2).
Overexpression and Purification
E. coli strain BL21(DE3) was transformed with the constructed plasmids pET42b-vAL-1 and pET21b-vAL-1(S). E. coli cells harboring pET42b-vAL-1 were grown in Luria-Bertani medium (2), collected by centrifugation at 6,000 X g at 4ºC for 5 min, and suspended in 0.5 M NaCl and 20 mM tris(hydroxymethyl)aminomethane-HCl (pH 7.5) (buffer A) including 0.5% Triton X-100. The fusion protein consists of GST, a linker including a Factor Xa site, vAL-1, and two histidine-tagged sequences in the linker and C-terminus. The cells were ultrasonically disrupted (Insonator Model 201M, Kubota) at 0ºC and 9 kHz for 20 min. After sonication, the clear supernatant obtained on centrifugation at 20,000 X g and 4ºC for 20 min was applied onto TALON metal affinity resin equilibrated with buffer A. The fusion protein GST-vAL-1 was retained on the resin after washing with buffer A, and it was then eluted with buffer A supplemented with 100 mM imidazole. The enzyme solution was dialyzed against 0.1 M NaCl, 5 mM CaCl2, and 50 mM glycine-NaOH (Gly) (pH 8.4) (buffer B). The dialysate was reacted with Factor Xa at a concentration of 1.2 mg-fusion protein/units at 20ºC for 16 h and then applied onto a Mono S 10/100 GL column equilibrated with buffer B. vAL-1 was retained on the column after washing with buffer B and then eluted with a gradient of 100–150 mM NaCl in buffer B. The eluted protein was collected and dialyzed against 20 mM Gly (pH 8.4). After centrifugation at 20,000 X g and 4oC for 20 min, the dialysate was used as purified vAL-1. As described above, E. coli cells harboring the plasmid pET21b-vAL-1(S) were grown in LB medium, collected, suspended in buffer A containing 0.5% Triton X-100, and ultrasonically disrupted. These cells produced a protein consisting of vAL-1(S) and a histidine-tagged sequence at the C-terminus. The protein was purified on TALON metal affinity resin as described above. The enzyme solution was dialyzed against 20 mM Gly (pH 8.4). After centrifugation at 20,000 X g and 4oC for 20 min, the dialysate was used as purified vAL-1(S).