2.1 Codon optimization of fpox gene
For the heterologous expression of recombinant FPOX in E. coliBL21 (DE3) and shuffle strains, the fpox gene sequence fromE. terrenum was retrieved from NCBI and the codons of fpoxsequence were optimized using Jcat server (http://www.jcat.de/). Different physicochemical properties of FPOX protein were investigated by protparam server (https://web.expasy.org/protparam/).The optimized fpox gene was evaluated using the GeneScript rare codon analysis server (https://www.genscript.com/tools/rarecodon-analysis). The codon-optimized sequence of FPOX,with NcoI and XhoIrestriction sites, was chemically synthesized sub-cloned in pET22b(+) and, transformed into E. coli shuffle and BL21 strains.The pET22b(+)-FPOX- E. coli construct was cultured on LB-agar plate containing ampicilin (100 µg/mL) at 37 C overnight. The positive clone selection, plasmid stability and glycerol stocks preservation were done.