Human vasohibin-1 gene with optimized codons for Escherichia coli expression was cloned into pET-32 LIC/Xa (Novagen, Madison, WI). The resultant expression plasmid encoded vasohibin-1 with a sequence of GSNSPLAMAISDPNSSSVDKLAAALEHHHHHH at its C terminus, as a thioredoxin fusion protein. E. coli BL21(DE3) transformants were cultivated at 37°C in TB medium (2.4% yeast extract, 1.2% tryptone, 1.25% K2HPO4, 0.23% KH2PO4, 500 mg/mL polypropylene glycol 2000, and 50 μg/mL ampicillin [pH 7.0]) supplemented with 4% glycerol, and the expression was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (OD650 = 5). After 16 hours of cultivation, the cells were harvested and disrupted in 20 mM sodium phosphate buffer (pH 7.6), containing 0.5 M NaCl and 1 mM phenylmethylsulfonyl fluoride (PMSF) in a high-pressure homogenizer. The inclusion bodies were collected, washed with the same buffer, and solubilized in 20 mM sodium phosphate buffer (pH 8.0), containing 0.5 M NaCl, 1 mM PMSF, 5 mM 2-mercaptoethanol, 60 mM imidazole, and 7 M guanidine HCl. The soluble fraction was loaded onto a Ni chelating Sepharose column (GE Health care, Princeton, NJ) which was equilibrated with the solubilization buffer. Vasohibin-1 fusion protein was eluted with 20 mM sodium phosphate buffer (pH 8.0), containing 0.5 M NaCl, 1 mM PMSF, 5 mM 2-mercaptoethanol, 300 mM imidazole, and 8 M urea. The eluted protein fraction was then dialyzed against 20 mM glycine-HCl buffer (pH 3.5) and digested with blood coagulation factor Xa (Novagen) for 1 hour at 25°C after the addition of the reaction buffer. The released vasohibin-1 was collected as the insoluble fraction, solubilized, and purified with Ni chelating Sepharose by a method used for fusion proteins. Vasohibin-1 was then collected as an insoluble fraction after dialysis against 20 mM Tris-HCl buffer (pH 8.0), resolubilized in 25 mM sodium phosphate (pH 7.2) containing 4 M urea, loaded onto a Q Sepharose column (GE Health care), and eluted by linearly increasing the NaCl concentration to 1 M. After vasohibin-1 was dialyzed against 20 mM glycine-HCl buffer (pH 3.5), the protein fraction was recovered as an insoluble fraction by addition of 1 M Tris-HCl buffer (pH 8.0) to 20% volume of the above solution. Vasohibin-1 was resolubilized with 50 mM Tris-HCl buffer containing 50 mM NaCl, 5 mM Tris(2-carboxyethyl)phosphine, 0.5 mM EDTA, 5% glycerol, and 4.4% N-lauroylsarcosine (pH 8.0) and was dialyzed twice against 20 mM sodium phosphate buffer (pH 8.0) and once with PBS, each for at least a day.
The protein concentration was determined by the Bradford method with a protein assay kit (Bio-Rad Laboratories, Hercules, CA), with bovine serum albumin as the standard protein.