Cloning and investigating GFP (green fluorescent protein) allowing higher expression in Bacillus subtilis

Abstract

Gfp gene coding for protein GFP (green fluorescent protein) from the jellyfish Aequorea victoria is the most popular indicator protein, due to (i) easy to recognize the expression through the fluorescent ability, (ii) without substrate or cofactor, (iii) less impact on the fusion protein target form. A lot of modifications of GFP have been studied and improved, in which GFP superfolder containing beneficial properties such as rapid folding, structural stability and strong fluorescence showed great applicability. However, GFP superfolder has just been developed in Escherichia coli. It is difficult to apply this version for all other strains since codon usage and the folding ability of each host species. In this study, with the aim of generating a GFP gene which is suitable for Bacillus subtilis, we mutated in the gene gfp+ to delete general BamHI, XhoI restriction enzyme site for facilitating in cloning step; as well asmodified Y39N, N105T, I171V codon that is compatible with B. subtilis. The results showed that the mutations have made the new version of GFP stronger in fluorescence and sequences of restriction enzymes have also been removed.