OPTIMASI ISOLASI PROTEIN NANOBODI DARI BAKTERI Escherichia coli BL21(DE3) DENGAN VARIASI WAKTU SONIKASI

DARA SYLVIA ANGELLYNE, - (2023) OPTIMASI ISOLASI PROTEIN NANOBODI DARI BAKTERI Escherichia coli BL21(DE3) DENGAN VARIASI WAKTU SONIKASI. Skripsi thesis, Sekolah Tinggi Farmasi Indonesia.

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Abstract

Nanobodi adalah antibodi yang ditemukan dengan ukuran lebih kecil serta mampu berikatan dengan kortisol, sehingga dapat diproduksi sebagai komponen kit diagnostik untuk pemantauan kadar kortisol dalam tubuh. Dalam produksi protein rekombinan pada bakteri Escherichia coli BL21(DE3), dihasilkan protein intraseluler sehingga dibutuhkan proses pemecahan sel untuk mengisolasi protein nanobodi. Penelitian ini bertujuan mengetahui pengaruh variasi waktu sonikasi dalam pengoptimalan terhadap proses isolasi protein intraseluler dan mendapatkan waktu sonikasi yang optimum. Ekspresi protein nanobodi mutan dan natif diberi pengindukasi IPTG 0,6 mM pada suhu 25˚C selama 18 jam kemudian diisolasi menggunakan sonikasi pada frekuensi 37 khz dengan variasi waktu 20; 30 dan 40 menit. Validasi dilakukan dengan menggunakan SDS PAGE. Hasil sonikasi pada waktu 30 menit menunjukan kadar protein bakteri mutan yaitu 82,4414 mg/dl dan natif 76,5855 mg/dl, serta tebal pita protein tertinggi pada nanobodi mutan dengan nilai AUC 28130.836 piksel dan nanobodi natif 27749.472 piksel. waktu isolasi protein yang optimum dengan metode sonikasi baik bakteri mutan maupun natif yaitu berada pada waktu 30 menit. --- Nanobodies are antibodies found in smaller sizes and able to bind to cortisol, so they can be produced as components of diagnostic kits for monitoring cortisol levels in the body. In the production of recombinant proteins in Escherichia coli BL21 (DE3) bacteria, intracellular proteins are produced so that a cell breakdown process is needed to isolate nanobody proteins. This study aims to determine the influence of sonication time variation in optimization of the intracellular protein isolation process and obtain the optimum sonication time. Expression of mutant and native nanobody proteins was given IPTG inducer 0.6 mM at 25°C for 18 h then isolated using sonication at a frequency of 37 kHz with a time variation of 20; 30 and 40 minutes. Validation is performed using SDS PAGE. The sonication results at 30 min showed mutant bacterial protein levels of 76,5855 mg/dl and native 82,4414 mg/dl, as well as the highest protein band thickness in mutant nanobodies with AUC values of 28130,836 pixels and native nanobodies of 27749,472 pixels. The optimum protein isolation time by sonication method of both mutant and native bacteria is within 30 min.

Item Type:	Thesis (Skripsi)
Uncontrolled Keywords:	Kortisol, nanobodi, Protein rekombinan, Isolasi sonikasi, SDS PAGE
Subjects:	Q Science > Q Science (General) R Medicine > RS Pharmacy and materia medica
Divisions:	Program Studi S1 Farmasi
Depositing User:	pustakawan - -
Date Deposited:	15 Jun 2024 01:27
Last Modified:	26 Jun 2024 01:29
URI:	http://repository.stfi.ac.id/id/eprint/212

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