#TITLE_ALTERNATIVE#

• Main Author: FIRDHA SUPARNINGTYAS (NIM : 20715033), JUNIZA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/22737
• Institution: Institut Teknologi Bandung

Autoinducible promoter is one of the molecular genetic development to produce recombinant protein from recombinant vector that can active in correlation with the host cell growth phase. Laboratorium of Pharmaceutical Biotechnology ITB has developed several recombinant vector using autoinucible promoter that active in stastionary phase such as pMCD using gadA promoter. Unfortunately, the protein model, MnSOD recombinant (rMnSODSeq), could not be produced from this vector in Escherichia coli BL21(DE3) yet. This promoter naturally works in stationary phase, minimum nutrient content, and acidic media. This research was aimed to get the optimum condition for producing rMnSODSeq using pMCD_sod in E. coli TOP10 with some parameters i.e., growth phase, pH media, and medium variation. E. coli TOP10 that carry pMCD_sod was confirmed by isolating the plasmid and analyzed its migration. The substantial component of gadA promoter in pMCD_sod was examined by DNA sequencing. The growth phase effect, medium acidity and the variety of media on rMnSODSeq production was performed by growing E. coli TOP10-pMCD_sod in LB or TB medium and then observed the cell density at OD600, change of media acidity, and also the protein production at 3, 6, 12, 24, 36, and 48 hour of incubation time. The rMnSODSeq amount relative to total intracellular protein was analyzed by SDS PAGE on the lysate samples of cell pellet. The rMnSODSeq protein was produced by pMCD_sod from the late of exponential until the stationary phase, in the pH range 5.0 up to 10, also in LB and TB media. Medium refreshment in initial growing phase of high density cell was able to increase the rMnSODSeq production. Produced rMnSODSeq shown dismutase activity by zymography analysis. Production of rMnSODSeq has been succesfully done optimally at stationary phase in the range of pH 5.0-10.0 with the highest yield at pH 5.5 in 11.5h incubation time as much as 32,47 ± 5,03% from the total protein and had an activity of dismutation.