CONSTRUCTION OF EXPRESSION VECTOR BASED ON pBR322 FOR THERAPEUTIC PROTEIN PRODUCTION WITH gadA PROMOTOR AUTOINDUCTION SYSTEM IN Escherichia coli

• Main Author: Wayan Martadi Santika, I
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45361
• Institution: Institut Teknologi Bandung

Production of recombinant therapeutic protein usually use expression vector that has high copy number and strong promoter. Nevertheless, this vector is less profitable for therapeutic protein production in industrial scale. High copy number can decrease the efficiency of therapeutic protein production, while strong promoter induction usually needs some addition of inducer which can raise production cost. The aim of this research is to construct expression vector that has low-medium copy number and carries autoinduction promoter gadA. Gene that encoded SOD (superoxide dismutase) was used as model for its expression level study under control of tested promotors. This research began with construction of pBR322 mini plasmid which has ?- lactamase (bla) gene and origin of replication (ori) from pBR322. The sod expression cassette, which carries T7 promoter from pJExpress_sod, was amplified by PCR and then cloned into pBR322 mini to construct pBM_sod plasmid. The synthetic autoinduction gadA promoter was inserted into pBM_sod plasmid to construct pMCD plasmid. The characteristic of these recombinant plasmids was determined by migration analysis, restriction site analysis, and nucleotide sequencing. The results showed that pBR322 mini, pBM_sod and pMCD have been successfully constructed and characterized. Comparison of sod expression level from pBM_sod, pMCD and pJExpress414_sod was analyzed by SDS-PAGE. The results showed that sod expression from pBM_sod and pJExpress414_sod are 14-fold and 10-fold stronger, respectively, as compared to sod expression from pMCD plasmid.