OPTIMASI EKSPRESI PROTEIN NANOBODI KORTISOL PADA BAKTERI Escherichia coli BL21 (DE3) DENGAN VARIASI WAKTU INKUBASI

WIDIA OKTAPIANI, - (2023) OPTIMASI EKSPRESI PROTEIN NANOBODI KORTISOL PADA BAKTERI Escherichia coli BL21 (DE3) DENGAN VARIASI WAKTU INKUBASI. Skripsi thesis, Sekolah Tinggi Farmasi Indonesia.

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Abstract

Produksi protein rekombinan nanobodi kortisol sebanding dengan konsentrasi sel bakteri, yang dipengaruhi oleh waktu inkubasi. Waktu inkubasi yang optimum menghasilkan konsentrasi sel yang tinggi, sebanding dengan jumlah protein rekombinan yang dihasilkan. Penelitian ini bertujuan untuk menentukan waktu inkubasi optimum dan mengetahui pengaruh dari variasi waktu inkubasi terhadap produksi protein rekombinan nanobodi kortisol pada Escherichia coli BL21 (DE3). Plasmid pET-28a yang mengandung nanobodi natif dan mutan ditransformasikan kedalam inang bakteri dengan metode heat shock. Waktu inkubasi ekspresi protein digunakan dengan variasi 16 jam, 18 jam dan 24 jam pada suhu 25 C serta konsentrasi IPTG (Isoprophyl-ß-D-thiogalactoside) 0,6 mM. Validasi keberhasilan ekspresi protein dianalisis menggunakan metode SDS-PAGE. Hasil penelitian menunjukan konsetrasi sel pada waktu inkubasi 16 jam bakteri masih mengalami peningkatan, sedangkan pada waktu inkubasi 24 jam mengalami penurunan. Waktu inkubasi 18 jam didapatkan konsentrasi sel tertinggi yaitu pada natif 0,33 gram dan mutan 0,29 gram. Pada waktu inkubasi selama 16 jam dan 24 jam didapatkan ketebalan pita protein yang rendah, sedangkan pada waktu inkubasi 18 jam didapatkan ketebalan pita protein tinggi, pada natif 7.522,146 piksel dan mutan 8.796,167 piksel. Berdasakan hasil tersebut waktu inkubasi 18 jam ditetapkan sebagai waktu inkubasi ekspresi protein yang optimum. --- The production of cortisol nanobody recombinant protein is proportional to the concentration of bacterial cells, which is affected by the incubation time. The optimum incubation time results in a high concentration of cells, proportional to the amount of recombinant protein produced. This study aims to determine the optimal incubation time and determine the effect of incubation time variations on the production of cortisol nanobody recombinant protein in Escherichia coli BL21 (DE3). The pET-28a plasmids containing native and mutant nanobodies were transformed into the bacterial host by heat shock method. Incubation time of protein expression was used with variations of 16 hours, 18 hours and 24 hours at a temperature of 25 ͦC and an IPTG (Isoprophyl-ß-D thiogalactoside) concentration of 0.6 mM. Validation of successful protein expression was analyzed using the SDS PAGE method. The results showed that cell concentration at the 16-hour incubation time of bacteria still increased, while at the 24-hour incubation time decreased. The incubation time of 18 hours obtained the highest cell concentration, namely at native 0.33 grams and mutants 0.29 grams. At the incubation time of 16 hours and 24 hours, a low protein band thickness was obtained, while at an incubation time of 18 hours, a high protein band thickness was obtained, at native 7,522,146 pixels and mutant 8,796,167 pixels. Based on these results, an incubation time of 18 hours was determined as the optimal incubation time for protein expression.

Item Type:	Thesis (Skripsi)
Uncontrolled Keywords:	nanobodi, waktu inkubasi, E.coli BL21(DE3). nanobody, incubation time, E.coli BL21(DE3).
Subjects:	Q Science > Q Science (General) Q Science > QR Microbiology
Divisions:	Program Studi S1 Farmasi
Depositing User:	pustakawan - -
Date Deposited:	21 Jun 2024 01:29
Last Modified:	21 Jun 2024 01:29
URI:	http://repository.stfi.ac.id/id/eprint/263

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