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.