Codon optimization and cloning of bovine prochymosin gene for proper expression in tobacco plant

Bovine chymosin enzyme is one of the most commonly used enzymes in the dairy industry. The production of this enzyme from its natural source does not meet the needs of this huge industry. The production of recombinant bovine chymosin in plants can be a good alternative to native enzyme. Insertion and expression of foreign genes in plants can occur in the nucleus and chloroplast organelles. The expression of foreign genes in eukaryotic heterologous hosts can be increased by optimizing its codons as well as translocation of the heterologous protein after expression to encapsulated organs such as chloroplasts. In this study, codons of the bovine prochymosin gene were optimized to be suitable for expression in the nucleus and chloroplasts of tobacco plants. After optimization, the CAI index of this gene for nucleus and chloroplasts was 0.929 and 0.947, respectively. The optimized sequence was cloned in the pUC57 basic vector after artificial synthesis, and was confirmed by sequencing. The function of the synthesized gene was confirmed by expression in E. coli and milk coagulation test. To provide a suitable expression vector for gene transfer to the plant genome, the GFP gene was replaced by the prochymosin gene in the p35S-TP-GFP expression vector. This vector is suitable for gene transfer by gene gun method, and with the presence of chloroplast signal peptide, it causes protein accumulation in chloroplasts. The gene was also replaced with the GUS gene in the pBI121 vector, which is suitable for gene transfer by the Agrobacterium method. The recombinant vectors constructed in this study can be used to effectively transfer and express this industrial enzyme in plants.