OPTIMASI ISOLASI PROTEIN NANOBODI DARI Escherichia coli BL21 (DE3) DENGAN VARIASI KONSENTRASI BUFFER LISIS

NOVI OKTAVIANI, - (2023) OPTIMASI ISOLASI PROTEIN NANOBODI DARI Escherichia coli BL21 (DE3) DENGAN VARIASI KONSENTRASI BUFFER LISIS. Skripsi thesis, Sekolah Tinggi Farmasi Indonesia.

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Abstract

Nanobodi dapat diekspresikan oleh Escherichia coli sebagai protein intraseluler, sehingga untuk memperolehnya harus dengan pemecahan sel. Untuk menghasilkan protein nanobodi dari bakteri inang E. coli, dilakukan penentuan konsentrasi buffer Tris HCl yang optimal untuk membantu menstabilkan protein dan pH pada saat sel dipecah menggunakan sonikator, Tujuan penelitian ini untuk mengetahui pengaruh variasi konsentrasi buffer Tris HCl dalam pengoptimalan proses isolasi protein dan divalidasi dengan SDS PAGE. Sampel yang digunakan adalah plasmid pET-28a yang ditransformasikan ke dalam bakteri Escherichia coli dan diekspresikan pada kondisi suhu optimum 25oC dengan penambahan IPTG 0,6 mM hasil ekspresi diisolasi dengan penambahan buffer Tris HCl. Variasi konsentrasi buffer Tris HCl yang digunakan yaitu 5 mM, 10 mM, dan 20 mM menggunakan sonikasi selama 30 menit. Hasil penelitian menunjukkan pada konsentrasi 10 mM dihasilkan kadar protein total tertinggi pada mutan dan natif yaitu 19,6036 mg/dl dan 68,4324 mg/dl. Dengan validasi SDS PAGE terbukti pada ketebalan pita tertinggi tercatat pada nanobodi natif, dengan nilai AUC 31455,990 piksel, dan nanobodi mutan, dengan nilai AUC 36773,153 piksel. Dapat disimpulkan bahwa penambahan buffer Tris HCl pada konsentrasi 10 mM merupakan yang optimal baik untuk nanobodi mutan maupun natif. --- Nanobodies can be expressed by Escherichia coli as intracellular proteins, so to acquire them must be by cell breakdown. To produce protein nanobodies from the host bacterium E. coli, determination of the optimal concentration of Tris HCl buffer was carried out to help stabilize the protein and pH at the time of the cell being broken down using a sonicator, The aim of this study to determine the effect of variations in Tris HCl buffer concentration in optimization of the protein isolation process and validated with SDS PAGE. The sample used was a PET-28A plasmid transformed into the bacterium Escherichia coli and expressed at optimum temperature conditions of 25oC with the addition of 0.6 mM IPTG the expression result was isolated by addition of Tris HCl buffer. Variations in Tris HCl buffer concentrations used were 5 mM, 10 mM, and 20 mM using sonication for 30 min. The results showed that at a concentration of 10 mM the highest total protein levels in mutants and natives were 19,6036 mg/dl and 68.4324 mg/dl. With SDS validation PAGE proved at the highest band thickness recorded on native nanobodies, with AUC values of 31455,990 pixels, and mutant nanobodies, with an AUC value of 36773,153 pixels. It can be concluded that the addition of Tris HCl buffer at a concentration of 10 mM is optimal for both mutant and native nanobodies.

Item Type:	Thesis (Skripsi)
Uncontrolled Keywords:	Nanobodi, kortisol, isolasi protein, buffer Tris HCl, SDS-PAGE. Nanobody, cortisol, protein isolation, Tris HCl buffer, SDS-PAGE.
Subjects:	Q Science > Q Science (General) Q Science > QD Chemistry
Divisions:	Program Studi S1 Farmasi
Depositing User:	pustakawan - -
Date Deposited:	17 May 2024 06:53
Last Modified:	06 Jun 2024 07:39
URI:	http://repository.stfi.ac.id/id/eprint/174

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