OPTIMASI EKSPRESI ENZIM REKOMBINAN PFU DNA POLIMERASE PADA SISTEM EKSPRESI Escherichia coli BL21 DENGAN AGEN PENGINDUKSI ISOPROPIL-β-D-TIOGALAKTOPIRANOSIDA (IPTG)

Arfena Rizqi Amalia, NIM.: 20106030060 (2025) OPTIMASI EKSPRESI ENZIM REKOMBINAN PFU DNA POLIMERASE PADA SISTEM EKSPRESI Escherichia coli BL21 DENGAN AGEN PENGINDUKSI ISOPROPIL-β-D-TIOGALAKTOPIRANOSIDA (IPTG). Skripsi thesis, UIN SUNAN KALIJAGA YOGYAKARTA.

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	Text (OPTIMASI EKSPRESI ENZIM REKOMBINAN PFU DNA POLIMERASE PADA SISTEM EKSPRESI Escherichia coli BL21 DENGAN AGEN PENGINDUKSI ISOPROPIL-β-D-TIOGALAKTOPIRANOSIDA (IPTG)) 20106030060_BAB-II_sampai_SEBELUM-BAB-TERAKHIR.pdf - Published Version Restricted to Registered users only Download (3MB) | Request a copy

Abstract

Pfu polymerase is a DNA polymerase enzyme derived from Pyrococcus furiosus. This enzyme has a high fidelity rate, making it ideal for use in PCR processes that require high accuracy. This study aimed to determine the optimal IPTG conditions for maximizing recombinant Pfu polymerase protein expression in the Escherichia coli BL21 Star (DE3) expression system using the pD451-SR plasmid. Optimization of Pfu polymerase expression began with optimizing IPTG concentration, followed by optimizing induction time and cell density prior to induction. The IPTG concentrations used were 0,1 mM; 0,2 mM; 0,4 mM; 0,8 mM; and 1,6 mM. The induction times tested were 0 hours; 6 hours; 12 hours; and 24 hours. Meanwhile, the cell density variations before induction were OD 0,4; OD 0,6; OD 0,8; and OD 1,0. The optimization of Pfu polymerase enzyme expression was evaluated based on SDS-PAGE analysis. The thickness of the bands observed on the SDS-PAGE gel reflected the concentration of recombinant protein, with thicker bands indicating higher protein concentrations. The visualized bands were then analyzed using Gel Analyzer software to determine the IPTG concentration that resulted in the highest protein expression. The results showed that Pfu polymerase was successfully expressed optimally using 0.4 mM IPTG, an induction time of 12 hours, and a cell density before induction of OD 0.4, with an average protein yield concentration of 0.1323 g/L.

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