Gunawan, Ellen Fenix and Prof. Dr. Agustin Krisna Wardani, STP., M.Si. Ph.D and Dr.rer.nat Tri Yudani Mardining Raras, M.App.Sc. (2024) Upaya Peningkatan Kelarutan Protein Rekombinan YidRv dari Klebsiella pneumoniae Hipervirulen yang Dioverekspresikan pada Escherichia coli BL21(DE3). Sarjana thesis, Universitas Brawijaya.
Abstract
Protein rekombinan YidRv merupakan protein hasil overekspresi gen yidRv dalam Escherichia coli BL21(DE3) yang diisolasi dari Klebsiella pneumoniae hipervirulen. Protein YidRv diduga merupakan protein yang berada di membran luar dan memiliki berat moleul 44,9 kDa. Gen yidRv mengkode protein pengikat ATP/GTP dan mampu memediasi fenotip hiperadherensi. Gen yidRv terdiri dari 1227 pasangan basa dan memiliki homologi sebesar 99,75% dengan gen yidR pada Klebsiella pneumoniae klasik. Protein YidRv mengandung dua domain conserved berkaitan dengan translokasi colicin yang berperan dalam patogenesis Enterobacteriaceae, seperti Escherichia coli dan Klebsiella pneumoniae hipervirulen. Klebsiella pneumoniae hipervirulen merupakan bakteri yang bersifat lebih ganas dibandingkan jenis klasiknya dan resistan terhadap antibiotik. Bakteri ini menyebabkan infeksi serius pada individu usia muda yang sehat. Adapun upaya pencegahan yang dapat dilakukan melalui pengembangan vaksin berbasis protein rekombinan. Produksi protein rekombinan dalam Escherichia coli umumnya menghasilkan badan inklusi. Badan inklusi merupakan kondisi dimana protein yang dihasilkan mengalami ketidaktepatan pelipatan, sehingga protein tidak dapat berfungsi dengan baik dan tidak larut di dalam sel. Oleh karena itu, dibutuhkan strategi dalam peningkatan kelarutan protein YidRv. Beberapa strategi dapat dilakukan dalam peningkatan kelarutan protein YidRv, seperti optimasi konsentrasi isopropyl β-D-1-thiogalactopyranoside (IPTG) yang berperan sebagai induser dan penambahan osmolit netral berupa gliserol dan sorbitol. Pada eksperimen ini overekspresi gen yidRv dilakukan pada suhu 37oC dengan variasi konsentrasi IPTG (0,1 mM, 0,5 mM, dan 1 mM). Overeskpresi gen yidRv juga dilakukan pada suhu 28oC dengan penambahan gliserol (5 mM) dan sorbitol (200 mM dan 300 mM) untuk mengetahui pengaruhnya terhadap kelarutan protein YidRv. Metode Sulfate Polyacrylamide Gel Electrophoresis (SDSPAGE) dan western blotting digunakan untuk mengetahui keberadan protein YidRv dan mengetahui kelarutan protein YidRv. Hasil eksperimen menunjukkan overekspresi dengan konsentrasi IPTG rendah (0,1 mM) menghasilkan kelarutan protein YidRv delapan kali lebih tinggi dibandingkan konsentrasi IPTG yang tinggi (0,5 mM). Sementara itu, penambahan gliserol (5 mM) dan sorbitol (200 mM dan 300 mM) tidak berpengaruh terhadap peningkatan kelarutan protein YidRv. Penambahan gliserol dan sorbitol pada media pertumbuhan kultur justru menyebabkan penurunan kelarutan protein YidRv. Dengan demikian, dalam produksi protein YidRv penggunaan IPTG konsentrasi rendah (0,1 mM) lebih dianjurkan. Penggunaan IPTG konsentrasi rendah dapat menekan biaya produksi. Sementara itu, penambahan gliserol (5 mM) dan sorbitol (200 mM dan 300 mM) tidak dianjurkan karena tidak berpengaruh terhadap peningkatan kelarutan protein YidRv.
English Abstract
The YidRv recombinant protein results from overexpression of the yidRv gene in Escherichia coli BL21(DE3) isolated from hypervirulent Klebsiella pneumoniae. The YidRv protein is thought to be located in the outer membrane and has a molecular weight of 44.9 kDa. The yidRv gene encodes an ATP/GTP binding protein and can mediate the hyperadherence phenotype. The yidRv gene consists of 1227 base pairs and has 99,75% homology with the yidR gene in classic Klebsiella pneumoniae. The YidRv protein contains two conserved domains related to colicin translocation which plays a role in the pathogenesis of Enterobacteriaceae, such as Escherichia coli and hypervirulent Klebsiella pneumoniae. Hypervirulent Klebsiella pneumoniae is a more virulent bacteria than the classic type and is resistant to antibiotics. This bacterium causes serious infections in healthy young individuals. Prevention efforts can be carried out through the development of recombinant protein-based vaccines. Recombinant protein production in Escherichia coli generally produces inclusion bodies. Inclusion bodies are a condition where the protein produced experiences incorrect folding, so the protein cannot function properly and does not dissolve in the cell. Therefore, strategies are needed to increase the solubility of the YidRv protein. Several strategies can be used to increase the solubility of the YidRv protein, such as optimizing the concentration of isopropyl β-D-1- thiogalactopyranoside (IPTG) which acts as an inducer and adding neutral osmolytes in the form of glycerol and sorbitol. In this experiment, overexpression of the yidRv gene was carried out at 37oC with varying IPTG concentrations of (0,1 mM, 0,5 mM, and 1 mM)1. Overexpression of the yidRv gene was also carried out at 28oC with the addition of glycerol (5 mM) and sorbitol (200 mM and 300 mM) to determine its effect on the solubility of the YidRv protein. The Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and western blotting methods were used to determine the presence of YidRv protein and determine the solubility of YidRv protein. Experimental results showed that overexpression with a low IPTG concentration (0,1 mM) resulted in YidRv protein solubility eight times higher than a high IPTG concentration (0.5 mM). Meanwhile, the addition of glycerol (5 mM) and sorbitol (200 mM and 300 mM) did not affect increasing the solubility of the YidRv protein. The addition of glycerol and sorbitol to the culture growth medium caused a decrease in the solubility of the YidRv protein. Thus, in the production of YidRv protein, the use of low-concentration IPTG (0,1 mM) is more recommended. The use of low-concentration IPTG can reduce production costs. Meanwhile, the addition of glycerol (5 mM) and sorbitol (200 mM and 300 mM) is not recommended because it does not affect increasing the solubility of the YidRv protein
Item Type: | Thesis (Sarjana) |
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Identification Number: | 0524100213 |
Uncontrolled Keywords: | IPTG, Osmolit netral, SDS-PAGE, YidRv- IPTG, Neutral Osmolyte, SDS-PAGE, YidRv |
Divisions: | Fakultas Teknologi Pertanian > Keteknikan Pertanian |
Depositing User: | soegeng Moelyono |
Date Deposited: | 10 Sep 2024 03:35 |
Last Modified: | 10 Sep 2024 03:35 |
URI: | http://repository.ub.ac.id/id/eprint/225603 |
Text (DALAM MASA EMBARGO)
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