CONSTRUCTION OF TWO SEPARATE PLASMIDS CARRYING ccd TOXIN-ANTIDOTE STABILITY SYSTEM FOR THERAPEUTIC RECOMBINANT PROTEIN PRODUCTION

• Main Author: A. A. ABDOSSALAM (NIM: 30712701), SAAD
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/30751
• Institution: Institut Teknologi Bandung

<p align="justify">Stability of expression vector is the cornerstone for therapeutic recombinant proteins production. Since expression vector is a key element to carry and express the therapeutic gene in certain host cell. Normally, stabilization of a vector utilizes its selection system. Most of commercialized expression vectors apply antibiotic-based selection system due to its simplicity. Nevertheless, World Health Organization and United States Food and Drug Administration highly recommend avoiding the using of antibiotic-based selection system due to its risk on spreading of resistance gene and hypersensitivity reaction of some patients to ?-lactam antibiotics. Hence, <br /> <br /> alternative selection system should be developed. This research was conducted to develop a ccd toxin-antidote stabilization system for production of recombinant <br /> <br /> proteins that is more flexible in terms of using any strains of Escherichia coli by separating them into two plasmids. An antidote gene (ccdA) with its natural promoter and terminator was placed in a plasmid named pDCSAsod. The plasmid carried an expression cassette containing sodA Staphylococcus equorum directed under the <br /> <br /> isopropyl thiogalactose (IPTG) inducible T7 promoter regulated by lac operator. The toxin gene (ccdB) was located in a separate plasmid named pDCSB and its expression is under L-arabinose inducible PBAD promoter. Furthermore, cer region was inserted to pDCSB plasmid to increase the plasmid stability to result pDCSBcer. All <br /> <br /> constructed plasmids were confirmed by restriction analysis for their sizes, by polymerase chain reaction (PCR) method for their components, and DNA sequencing <br /> <br /> for their sequence. To evaluate the function of this ccd toxin-antidote system, growth of cells harboring each and both plasmids was observed in the absent and presence of <br /> <br /> L-arabinose in the Luria Bertani medium. Even though the antibiotic resistance gene was still present in the plasmids in this study, all the experiments were conducted in antibiotic-free condition. Yield and activity of the sodA gene product, rMnSODSeq, produced from expression cassette in E. coli BL21(DE3) carrying both plasmids were <br /> <br /> compared with those from expression cassette in cells carrying pDCSA only by SDS-PAGE analysis and zymography. Performance of plasmid stabilization of the system was examined by plasmid retention test conducted at the end of protein production time i.e. four hours after IPTG induction. The plasmids constructed in this study were <br /> <br /> confirmed for the identity. The suppression of growth profile was observed when E. coli carrying pDCSB plasmid series induced by L-arabinose. The growth of E. coli <br /> <br /> harboring both pDCSAsod and pDCSB plasmid series was similar with that of E. coli with no plasmid, even with L-arabinose induction. rMnSODSeq production and activity were not affected by ccd toxin-antidote system. Without any optimization, the yield of rMnSODSeq was 2.3 mg/ml. Plasmid stability of pDCSAsod increased to 94% when the plasmid coexisted with pDCSB or pDCSBcer but only 90.5% stability when it was present alone. The addition of cer fragment in pDCSB also increased significantly the stability from 68% to 75%. In conclusion, ccd toxin-antidote system in this study works well even when it is separated into two plasmids. It has potency for stability system in production of recombinant therapeutic proteins and provides more flexibility in choosing the E. coli strains compared to commercially available stability system based on chromosomally integrated ccdB gene. <p align="justify"> <br />