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All Journal Jurnal Akademika Biologi Biogenesis: Jurnal Ilmiah Biologi Biopropal Industri
Wien Kusharyoto
Unknown Affiliation
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Immunology & microbiology Industrial & Manufacturing Engineering

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Strategy for Designing the Synthetic Gene Encoding Human papillomavirus Major Capsid L1 Protein for Heterologous Expression in Escherichia coli System Kartika Sari Dewi; Wien Kusharyoto
Biogenesis: Jurnal Ilmiah Biologi Vol 8 No 2 (2020)
Publisher : Department of Biology, Faculty of Sci and Tech, Universitas Islam Negeri Alauddin Makassar

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24252/bio.v8i2.15805

Abstract

DNA is widely used to construct heterologously expressed genes. The adaptation of the codons to the host organism is necessary in order to ensure sufficient production of proteins. The GC content, codon identity and the mRNA from the translation site are also important in the design of the gene construct. This study performed a strategy for the design of synthetic gene encoding HPV52 L1 protein and several analyses at the genetic level to optimize its protein expression in the Escherichia coli BL21(DE3) host. The determination of the codon optimization was performed by collecting 75 HPV52 L1 protein sequences in the NCBI database. Furthermore, all the sequences were analyzed using multiple global alignments by Clustal Omega web server. Once the model was determined, codon optimization was performed using OPTIMIZER and the web server of the IDT codon optimization tool based on the E. coli B. The generated open reading frame (ORF) sequence was analyzed using Restriction mapper web server to choose the restriction site for facilitating the cloning stage, which is adjusted for pJExpress414 expression vector. To maximize the protein expression level, the mRNA secondary structure analysis around the ribosome binding site (rbs) was performed. A slight modification at the 5’-terminal end waa carried out in order to get more accessible rbs and increasing mRNA folding free energy. Finally, the construction of the synthetic gene was confirmed to ensure that no mutation occurs in the protein and to calculate its Codon Adaptation Index (CAI) and GC content. The above strategy, which leads to a good ORF sequence with the value of the free mRNA folding energy around rbs, is -5.5 kcal / mol, CAI = 0.787 and GC content 49.5%. This result is much better than its original gene. This result is much better compared to its native gene. Theoretically it is possible that this synthetic gene construct generates a high level protein expression in E. coli BL21 (DE3) under the regulation of the T7 promoter.
POTENSI AKTIVITAS ANTIMALARIA DARI EKSTRAK AIR DAUN JUNG RAHAB (BAECKEA FRUTECENS) (Potency of Antimalarial Activity from Aqueaous Extract of Jung Rahab Leaves (Baeckea frutecens)) Yatri Hapsari; Eris Septiana; Fauzy Rachman; Syamsiah Syamsiah; Wien Kusharyoto; Leny Heliawati; Bustanussalam Bustanussalam; Siti Irma Rahmawati; Fauzia Nurul Izzati; Partomuan Simanjuntak
Biopropal Industri Vol 12, No 2 (2021)
Publisher : Balai Riset dan Standardisasi Industri Pontianak

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.36974/jbi.v12i2.7074

Abstract

Malaria is a disease caused by Plasmodium parasite transmitted by female Anopheles mosquito. Jung rahab plant (Baeckea frutecens) is known have an ability to inhibit the growth of Plasmodium falciparum. However, the inhibition of the parasite growth is still unknown. This research aimed to determine fractionation of aqueaous extract of jung rahab leaves that is contain antimalaria compound with a potential inhibition activity. Jung rahab plant was extracted using decoctition. The analysis included phytochemical screening, column chromatography fractionation, antimalaria activity, and compound identification by using UV-Vis, FT-IR, and GCMS. Jung rahab aqueous extract fraction resulted a potential antimalaria activity with IC50 105,9 ppm and fractionation result based on UV-Vis, FT-IR, and GCMS as pyrogallol.Keywords: antimalarial, β-hematin, Baeckea frutecens, extraction, IC50ABSTRAKMalaria merupakan penyakit yang disebabkan oleh parasit Plasmodium yang ditransmisikan oleh nyamuk Anopheles betina. Daun jung rahab (Baeckea frutecens) diketahui memiliki kemampuan untuk menghambat pertumbuhan Plasmodium falciparum. Akan tetapi, mekanisme penghambatan pertumbuhan parasit tanaman ini masih belum diketahui. Penelitian ini bertujuan untuk mencari fraksi lain dari ekstrak air daun jung rahab yang mengandung senyawa antimalaria dengan aktivitas penghambatan yang lebih tinggi. Ektraksi daun jung rahab dilakukan dengan metode dekoktasi, sedangkan analisis yang dilakukan adalah skrining fitokimia, fraksinasi menggunakan kromatografi kolom, uji aktivitas antimalaria, dan identifikasi senyawa hasil fraksinasi dengan UV-Vis, FT-IR, dan GCMS. Berdasarkan hasil penelitian ini, fraksi ekstrak air daun jung rahab memiliki aktivitas antimalaria potensial dengan nilai IC50 sebesar 105,9 ppm dan hasil fraksinasi senyawa berdasarkan UV-Vis, FT-IR, dan GCMS sebagai pirogalol.Kata kunci: antimalaria, β-hematin, Baeckea frutecens, ekstraksi, IC50­
TRANSFORMASI DAN KLONING PLASMID PJ804:77539 PADA E.coli TOP’10 Siu S.S Langden; Anto Budiharjo; w wijanarka; Wien Kusharyoto
Jurnal Akademika Biologi Vol. 6 No. 1 Januari 2017
Publisher : Departemen Biologi, Fakultas Sains dan Matematika Undip

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (125.848 KB)

Abstract

Kloning dan transformasi vektor PJ804:77539 dilakukan dengan tujuan perbanyakan vektor pRHA pada sel bakteri E.coli TOP’10. Ekpresi vektorpRHA diharapkan dapat terjadi pada periplasma E.coli dan memberikan ekspresi berupa kemampuan resistensi terhadap Ampicillin. Ekspresi pada periplasma bertujuan untuk meminimalisir kerugian yang timbul pada sistem ekspresi di sitoplasma di antaranya tingkat ekspresi yang rendah, protein terpotong atau resiko kontaminasi. Sekresi protein rekombinan pada periplasma dapat meningkatkan aktivitas biologis serta tingkat kestabilan produk menjadi lebih tinggi. Proses isolasi protein yang diekspresikan pada periplasma  dapat dilakukan dengan perlakuan stress osmotik ringan sehingga menurunkan resiko kontaminasi protein sitoplasma. Ekspresi protein pada periplasma diarahkan oleh peptida sinyal pelB. Peptida sinyal bekerja menarik produk protein ke periplasma dengan cara berfusi dengan ujung N-terminal pada peptida yang terekspresi. Penanda selektif (selectable marker) yang terdapat pada PJ804::77539 merupakan Ampr, suatu penanda yang memampukan bakteri untuk resisten pada keberadaan antibiotik Ampicillin. Transformasi dilakukan sesuai dengan metode heat – shock dan diseleksi pada medium LB agar dan LB cair yang mengandung antibiotik Ampicillin dengan konsentrasi 100 mg/mL. Diperoleh koloni tumbuh pada medium yang mengandung Ampicillin dan dilakukan isolasi plasmid. Visualisasi hasil elektroforesis memperlihatkan adanya pita plasmid yang diisolasi dari E.coli TOP’10 pada gel elektroforesis.Kata kunci : Ampicillin, E.coli, pelB, periplasm dan pRHA
Co-Authors Anto Budiharjo Asep Muhammad Ridwanuloh Bustanussalam Bustanussalam Eris Septiana Fauzia Nurul Izzati Fauzy Rachman Kartika Sari Dewi Leny Heliawati Martha Sari Partomuan Simanjuntak Rahmawati, Siti Irma Siu S.S Langden Syamsiah Syamsiah w wijanarka Yatri Hapsari