Codon Optimization for Subtilisin QK-2 Expression

Combined with lysozyme, genomic DNA and/or plasmids were extracted from B. subtilis and L. lactis strains using a rapid bacterial genomic DNA isolation kit (Sangon Biotech, Shanghai, China) and a rapid mini plasmid kit (Tiangen Biotech, Beijing, China), respectively. To study the effect of codon bias on the expression of Subtilisin QK-2 in L. lactis, codon optimization was performed using the JCat program [33 (link)] according to the protein sequence of Subtilisin QK-2 (GenBank: AJ579472.2). The codon-optimized gene of Subtilisin QK-2 (qk′, GenBank accession number KT725198) was synthesized by TsingKe Biological Technology (Wuhan, China) and was ligated into the pUC19 plasmid, resulting in pUC19-qk′. On the basis of the qk′, qkpro′ (GenBank accession number KT991841) containing the codon-optimized propeptide sequence only was obtained by overlap extension (SOE)-PCR. KOD Dash DNA polymerase (TOYOBO) was used to maintain the veracity of the PCR process. Restriction enzymes and T4 DNA ligase were purchased from TaKaRa and were used according to the manufacturer’s instructions. General molecular biological procedures were performed according to [34 ].