Steps to producing soluble VEGF-A₁₆₅-HBD.

<p>The gene sequence encoding the VEGF-A₁₆₅-HBD protein was adapted to that of <i>E. coli</i> K12 genome and the 5′ and 3′ restriction enzyme sequences of <i>Nde</i>I and <i>Xho</i>I were incorporated, respectively. The gene was then synthesised and subcloned into pET14b by Eurofins. The recombinant plasmid was transformed into Origami 2 and SHuffle cells and tested for expression. (a) VEGF-A₁₆₅-HBD was over-produced and purified from both cell types; however, VEGF-A₁₆₅-HBD produced from SHuffle cells showed a greater yield of presumably folded protein based on absorbance at 280 nm which was solely dependent on cysteines having formed cystines (disulphide bonds). Even though relative staining of the protein bands on the gel would suggest that the two protein samples are at similar concentrations, absorbance measurements at 280 nm indicate that the sample from SHuffle cells had a concentration of 3.1 mg/ml of disulphide-bonds-containing protein compared to 1.7 mg/ml in Origami cells. (b) VEGF-A₁₆₅-HBD protein from SHuffle cells was further purified on a preparative Superdex 75 size exclusion column. (c) His₆-tag was successfully removed using thrombin and resulting VEGF-A₁₆₅-HBD retained solubility and stability as assessed by absence of precipitation and further proteolytic cleavage.