Judul: Ekspresi Enzim Rekombinan MMLV-Reverse Transcriptase di Sistem Ekspresi Escherichia coli BL21 Menggunakan Metode Autoinduksi
Abstrak: Moloney Murine Leukemia Virus (MMLV) memiliki enzim transkripsi balik atau reverse transcriptase yang dapat mengubah RNA menjadi cDNA. Selama pandemi Covid-19, terjadi peningkatan permintaan reverse transkriptase yang merupakan komponen penting dari alat pendeteksi virus corona. Oleh karena itu, penelitian ini bertujuan mempelajari ekspresi enzim rekombinan Moloney Murine Leukemia Virus-Reverse Transcriptase (MMLV-RT) serta mendapatkan waktu dan media inkubasi optimum menggunakan metode autoinduksi. Protein rekombinan MMLV-RT diproduksi di sistem ekspresi Escherichia coli yang telah disisipi plasmid pembawa gen MMLV-RT. Plasmid pD451-SR diregulasi dengan promotor T7 untuk mengekspresikan MMLV-RT. Beberapa penelitian terdahulu menggunakan isopropyl -D-thiogalactopyranoside (IPTG), namun dalam penelitian ini menggunakan laktosa yang memiliki harga lebih murah dibandingkan IPTG sehingga dapat mengurangi biaya produksi. Sel inang E. coli BL21 ditransformasi dan dikulturkan dalam empat media autonduksi, yaitu media Luria Bertani, 2XYT, Imperial College, dan Terrific Broth. Protein rekombinan MMLV-RT diekspresikan paling optimum di media Imperial College dengan waktu inkubasi 96 jam. Protein MMLV-RT memiliki kelarutan yang baik berdasarkan uji solubilitas dan sebagian besar protein MMLV-RT terlarut pada fraksi supernatan hasil ekstraksi. Protein rekombinan MMLV-RT dipurifikasi menggunakan Immobilized Metal Affinity Chromatography (IMAC) dan mendapatkan konsentrasi sebesar 10,117 mg/mL. Aktivitas enzim MMLV-RT yang diproduksi dalam studi ini diuji dan menunjukkan aktivitas spesifik sebesar 629,4 U/mg.
Keyword: Autoinduksi, Ekspresi, MMLV-RT, Purifikasi, Transkripsi balik

Judul: Pengembangan enzim rekombinan reverse transcriptase simian betaretrovirus serotipe-2 isolat indonesia asal monyet ekor panjang
Abstrak: Reverse transcriptase (RT) is a multifunctional enzyme that catalyzes the formation of double-stranded DNA from single-stranded RNA viral genomes. Reverse transcription is an essential and critical step in life cycle of all retroviruses replication. RT has become an important enzyme in molecular biology, genetics and medicine for the synthesis of complementary DNA (cDNA) from messenger RNA (mRNA). The application of this enzyme combining with PCR amplification technique (RT-PCR) has expanded our knowledge of numerous cellular regulatory mechanisms and has become a gold standard to facilitate the coding region cloning of any gene of interest. The development of biotechnology has been relying on the availability of this efficient RT. In biomedical fields, RT of human immunodeficiency virus (HIV), the virus causing acquired immunodeficiency syndrome (AIDS) has been the most studied enzyme due to the prime target for the development of antiretroviral drug therapy of HIV/AIDS. Simian betaretrovirus serotype-2 (SRV-2) is the causative agent of simian acquired immunodeficiency syndrome (SAIDS) in Asian macaques with varying severity. While SRV is an important pathogen in Asian macaque and causes a potential confounding variable in biomedical research, SRV also provides a valuable viral model to compare with other retrovirals for a better understanding of aspects of retroviral–host interactions, infection mechanism, retroviral structure, antiretroviral and vaccine development. As is characteristic of all retroviruses, SRV-2 can transcribe its own RNA genome into a double-stranded DNA by the action of reverse transcriptase enzyme. The presence of gene encoding RT enzyme in SRV-2 genome potentially be studied and utilized further as RT enzyme model for developing the anti RT drug of others retrovirus. On the other hands, the gene encoding SRV-2 RT can be isolated and cloned to produce RT recombinant enzyme and applied in molecular biology techniques. In this study, we isolated the gene encoding RT enzyme of SRV-2 that infected Indonesian cynomolgus monkey and predicted the three dimensional (3D) structure model using I-TASSER (Iterative Threading ASSEmbly Refinement) computational program and visualized with PyMol program. Gene expression was constructed in Escherichia coli expression system with Gateway technology methods using pENTR/SD/TOPO entry vector and pDEST17 destination vector. Purification was performed using Ni2+-NTA affinity chromatography. Expression was analyzed using SDS-PAGE and Western blot techniques; meanwhile enzyme activity was measured using RT colorimetric assay and RT-PCR technique. We isolated the gene encoding RT enzyme of SRV-2 Indonesian isolate at nucleotide position 3284-4925 consisted of 547 amino acids. The I-TASSER program has predicted the three dimensional structure model of RT SRV-2 enzyme that consisted of palm/finger, thumb, connection and RNase H domain. The polymerase active site located in the finger/palm subdomain characterized by three conserved catalytic aspartates (Asp90, Asp165, Asp166), and has a highly conserved YMDD motif as Tyr163, Met164, Asp165, and Asp166. This SRV-2 RT structure model has the highest homology to HIV-1 RT (2B6A.pdb). Meanwhile, the structure of SRV-2 RNase H closely related to its ortologs such as Escherichia coli RNase H (1GOA) and HIV-1 RNase H (3QIO, 3HYF). The catalytic active site of SRV-2 RNase H contains three amino acid carboxylateresidues as Asp436, Glu465 and Asp486. Analysis of expression using SDS
Keyword: reverse transcriptase, recombinant enzyme, SRV-2 RT,, Escherichia coli expression system, I-TASSER

Judul: Pengaruh Konsep Lanskap Keraton Ngayogyakarta Hadinigrat terhadap Lanskap Kota Yogyakarta
Abstrak: Kota Yogyakarta merupakan kota yang menjadi pusat kebudayaan Jawa dengan adanya Keraton Yogyakarta dan bekas Kerajaan Mataram Islam. Sebagai sebuah kota dengan latar historis kerajaan yang kuat dan legilimasi keraton yang sampai saat ini masih cukup kuat, maka kehidupan keraton mempengaruhi pula kehidupan rakyat Kota Yogyakarta. Keraton dan kawasan kerajaan dibangun berdasarkan konsep-konsep filosofis yang menghasilkan wujud fisik penuh makna dan merupakan cikaI bakaI Kota Yogyakarta. Namun dalam perkembangannya, dengan adanya pengaruh pendatang dan globalisasi, malta banyak terjadi pergeseran nilai dan kebudayaan serta filosofi yang melingkupinya Hal. ini dikhawatirkan akan semakin mengaburkan identitas Kota Yogyakarta.
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