Similar to E.coli, now a days Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae have also emerged as a promising heterologous expression system for prokaryotic and eukaryotic candidate genes. 


There are several factors which govern the production of recombinant therapeutic proteins in
B. subtilis. The factors are as follows:
a) Well understood transcription and translation machinery including different regulatory factors responsible for extracellular product.
b) Generation of stable recombinant plasmids.
c) Development of novel recombinant B. subtilis strains with reduced nuclease and protease contents.
d) Better systematic understanding of the protein secretion method to elucidate the factors responsible for the secretion of intracellular proteins.

To get the soluble extracellular protein product from the gene, it can be linked to a DNA fragment coding for B. subtilis signal peptide for extra-cellular secretion. The signal sequence may be preceded by a efficient translation initiator sequence or ribosome binding site (RBS) which is followed by mRNA stability-enhancing sequences (SES) at 5’ end of mRNA. The ‘strong’ promoter for this gene consist a cluster of other efficient promoters which would be regulated temporally by the growth condition or growth medium components. Thus genes possess suitable properties not only for efficient transcription, translation but would be under temporal control avoiding other exogenous induction. Thus generated mRNA would be stabilized by 3’SES which protects mRNA from degradation by 3’ exonucleases. The protein product consists of a typical B. subtilis signal peptide linked to N-terminus and have to be further processed with proteases to recover only the desired portion.