Plasmid and enzyme generation

To further investigate the oxidation capability and substrate selectivity of these reconstructed enzymes, the E. coli optimized gene sequences for the 6 chosen ancestors were purchased. Then, specially designed primers were used in conjunction with PCR techniques to obtain the gene sequences with unique overhangs on either side for ligation. The genes with overhangs were then cloned into pet28a C-term 6X His tag vectors using Gibson assembly. 

The assembled plasmids were transformed into competent DH10β E. coli cells and plated on LB+kanamycin agar plates, followed by plasmid minipreps of surviving colonies. Gene insertion into the plasmid was verified via Sanger sequencing.

The 6 ancestral VHPO plasmids were then heterologously expressed in BL21(DE3) E. coli cells and purified via Ni-NTA affinity chromatography. N177 and N220 had the highest yield, with 75 and 50 mg from 1 liter of culture, respectively. N153 and N160 had the second highest yields of 15-30 mg, while N241 and N291 had the lowest yields of <2 mg each. 

Initial testing was done using a thymol blue (TB) assay, which measures iodide oxidation via color change due to the reaction of hypoiodous acid with thymol blue, turning the solution from yellow to blue. Since selective bromo and chloroperoxidases are capable of oxidizing and releasing hypoiodous acid, this assay is good for a quick readout of oxidative capability. All 6 enzymes were able to cause a color shift from yellow to blue in the TB assay, indicating that at least some portion of the purified enzyme is folded and oxidatively active.