#TITLE_ALTERNATIVE#

• Main Author: IMELDA NIM : 10714030, WIDYA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/31587
• Institution: Institut Teknologi Bandung

Auto-inducible promoter usage is one of efficiency strategy for recombinant protein production <br /> <br /> <br /> without inducer. pMCD_sod plasmid is a gadA auto-inducible promoter based expression vector <br /> <br /> <br /> developed in Laboratory of Pharmaceutical Biotechnology ITB which has coding sequence of <br /> <br /> <br /> superoxide dismutase from Staphyloccocus equorum (rMnSODSeq) for expression in Escherichia coli. <br /> <br /> <br /> gadA promoter requires stationary phase, low pH, or salt stress to be activated. In previous research, <br /> <br /> <br /> optimal protein production was accomplished by keeping the pH at 5.5 by adding HCl periodically. The <br /> <br /> <br /> purpose of this research is to observe the effect of buffer and media modification in protein <br /> <br /> <br /> production using pMCD_sod in Escherichia coli. pMCD_sod plasmid was confirmed by migration and <br /> <br /> <br /> restriction analysis. rMnSODSeq protein production was analysed by SDS-PAGE method. Buffers that <br /> <br /> <br /> had been used are succinate, acetate, and citrate buffer. The most suitable buffer was succinate <br /> <br /> <br /> buffer and was used for overproduction of rMnSODSeq protein. Modification of Luria Bertani (LB) <br /> <br /> <br /> media was performed by using LB media with ¼ of LB component. Effect of buffer to accelerate <br /> <br /> <br /> protein production was observed by the presence of rMnSODSeqamount of protein produced at early <br /> <br /> <br /> overproduction time. rMnSODSeq protein can be produced using succinate buffer at pH of 5.5 in LB <br /> <br /> <br /> media with protein amount equal to LB media with HCl. The amount of protein produced with ¼LB <br /> <br /> <br /> media was equal to overproduction with LB media. Optimal rMnSODSeq protein production can be <br /> <br /> <br /> done with ¼ LB-succinate media for 11.5 hours resulting in 35.275±1.47% rMnSODSeq from total <br /> <br /> <br /> protein with 4.05±0.69 mg rMnSODSeq in one g of wet cells and had activity of dismutasion. <br />