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CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli Kusdianawati, Kusdianawati; Mustopa, Apon Zaenal; Suharsono, Suharsono; Budiarto, Bugi Ratno; Fatimah, Fatimah; Danuri, Hasim
Indonesian Journal of Agricultural Science Vol 16, No 1 (2015): April 2015
Publisher : Indonesian Center for Agricultural Library Technology Dissemination - IAARD

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21082/ijas.v16n1.2015.31-38

Abstract

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from Lactobacillus plantarum S34 in Escherichia coli. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. plantarum S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. plantarum S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. coli BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.
ANTIBACTERIAL ACTIVITY OF EXTRACELLULAR PROTEASE ISOLATED FROM AN ALGICOLOUS FUNGUS XYLARIA PSIDII KT30 AGAINST GRAM-POSITIVE BACTERIA Indarmawan, Taufik; Mustopa, Apon Zaenal; Budiarto, Bugi Ratno; Tarman, Kustiariyah
HAYATI Journal of Biosciences Vol. 23 No. 2 (2016): April 2016
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1051.929 KB) | DOI: 10.4308/hjb.23.2.73

Abstract

Infectious diseases became more serious problem for public health in recent years. Although existing antibacterial drugs have been relatively effective, they do not rule out the emergence of resistance to the drug. Therefore, the intensive exploration of new bioactive compounds from natural, especially peptide compounds began in recent decades in order-handling infection. This study aimed to isolate, purify and test the potential application of Xylaria psidii KT30 extracellular protease as antibacterial agent against Gram-positive bacteria. X. psidii KT30, a marine fungus isolated from red seaweed Kappaphycus alvarezii showed antibacterial activity against Bacillus subtilisand Staphylococcus aureus. Antibacterial compounds of this fungus were predicted as a group of proteases. Extracellular protease exhibited an optimum activity when potato dextrose broth was used as cultivation medium. Furthermore, the highest activity of these proteases was found on fungal extract after day 15 of cultivation with value of 2.33 ± 0.19 U/mL. The partial purification of proteases using G-75 column chromatography resulted in 2 groups of fractions and showed protease activity based on zymogram assay. The extracellular proteases obtained from those fractions have 3 patterns of molecular mass based on sodium dodecyl sulfate?polyacrylamide gel electrophoresis which are 56.62, 89.12, 162.18 kDa.
PREPARASI FRAGMEN GEN DENGAN TEKNIK ALLELE-SPECIFIC POLYMERASE CHAIN REACTION (AS-PCR) UNTUK STANDAR MARKER SNPs Human Epidermal Growth Factor 2 (HER2I655V) PADA PENDERITA KANKER PAYUDARA Wahyuningsih, Arika; Budiarto, Bugi Ratno
BIOTROPIC The Journal of Tropical Biology Vol 1 No 1 (2017): Biotropic, Volume 1, Nomor 1, 2017
Publisher : Program Studi Biologi, Fakultas Sains dan Teknologi, Universitas Islam Negeri Sunan Ampel Surabaya

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (19.495 KB)

Abstract

HER2/neu merupakan anggota dari keluarga protein EerbB. Single nukleotida polimorfisme (SNP) pada gen HER2 terutama posisi kodon 655 diduga terkait dengan resiko kanker payudara. Deteksi SNP ini dapat menggunakan teknik AS-PCR (Allele Specifik Polymerase Chain Reaction) yang cukup sensitif dan spesifik namun teknik deteksi post AS-PCR selama ini yang diterapkan belum menggunakan fragmen standar gen yang menjadi target. Oleh karena itu dalam penelitian ini perlu dilakukan preparasi fragmen gen dengan teknik Allele Specific PCR untuk standar marker SNP HER2I655V. Tujuan penelitian ini untuk menyediakan standar marker DNA untuk deteksi gen SNP HER2I655V. Metode yang digunakan adalah melalui penapisan DNA, AS-PCR, elektroforesis, UV trasluminator dan spektrofotometri. Penapisan HER2I655V pada beberapa sample pGEM rekombinan dengan AS-PCR didapatkan fragmen gen HER2 genotipe AA (142 bp) dan GG (168 bp). Purifikasi fragmen menghasilkan konsentrasi akhir sebesar 37 ng/μl. Validasi fragmen HER2 dilakukan dengan menguji SNP pada 5 sampel DNA sel bukal melalui metode AS-PCR. Varian SNP pada sampel memiliki ukuran yang sama dengan standar marker HER2 untuk genotipe AA yaitu 142 bp. preparasi standar marker gen HER2I655V dengan teknik AS-PCR berhasil.
Improvement of HER2I655V TARMS-PCR Performance by DNA Quality Analysis Budiarto, Bugi Ratno; Azamris, Azamris; Desriani, Desriani
ANNALES BOGORIENSES Vol 21, No 2 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ab.v21i2.308

Abstract

Reliable TARMS-PCR is a prerequisite in constructing a solid conclusion in genetic diagnostics. The validity of data generated by this molecular technique is hampered by a false positive result. In attempt to develop a  TARMS-PCR for HER2I655V genotyping with no interfering of bias we used DNase I to eliminate DNA contaminant resided in PCR reagent. TARMS-PCR without enzyme treatment using recombinant plasmids that contained HER2I655V gene with represented its alleles was used to evaluate the presence of false positive  result while DNase I treated-PCR reagent was used in TARMS-PCR to evaluate the effective dose of the enzyme and further to adjust the TARMS-PCR conditions.  PCR master mix kit used in this study produced a false positive result on HER2I655V TARMS-PCR as proven by the presence of multiple PCR products in Non-Template Control (NTC) and 0.1 U of the enzyme could eliminate this DNA contaminant effectively, although this pretreatment altered the specificity of HER2I655V TARMS-PCR genotyping on certain genotype. Combination of touchdown TARMS-PCR with another allele-specific primer recovered specificity of detection on this model system. Interestingly, this optimized HER2I655V TARMS-PCR can only be used for genotyping the clinical samples if only further optimization was done using genomic DNA as template
ILE655VAL Genotyping Study of HER2 - Positive Breast Cancer of Patients from Padang - Indonesia With High Resolution Melting Technique Wulandari, Dwi; Azamris, Azamris; Nurhayati, Isna; Warisman, M Ali; Budiarto, Bugi Ratno; Desriani, Desriani
ANNALES BOGORIENSES Vol 21, No 2 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (282.104 KB) | DOI: 10.14203/ab.v21i2.310

Abstract

Transtuzumab has proven to be a great improvement in the treatment of HER2 + of breast cancer patients, but it is associated with relevant adverse cardiac events and with significantly elevated cost of treatment.  One of the risk factors for cardiotoxicity due to transtuzumab is the I655V HER2 polymorphism (GTC> ATC mutation) in which the allele mutant (Ile val or val / val) has a higher risk than the wild type (Ile/Ile). The detection of specific alleles is very important for therapeutic decision-making. In this study, our group have developed a HRM with EvaGreen dye method to discriminate specific allele of I655V HER2 (wild type, heterozygote mutant or homozygote mutant) in 66 frozen section samples of HER2+ of breast cancer patients. Our group reported the wild type is the most prevalent allele (77,27%), whereas heterozygous mutation is significantly present in this research (21,21%) and only 1,52% of sample detected as minor allele.  It showed that only one sample detected as a minor allele (val/val) and may have relatively low abundance in the population. This method has been compared with sanger sequencing and giving 100% of validity.
Inventarisasi Jenis Pohon di Kawasan Pusat Pembinaan, Pendidikan, dan Pelatihan (Pusbindiklat) Peneliti - LIPI untuk Menunjang Faktor Keselamatan DAMAYANTO, I PUTU GEDE P.; NARAKUSUMO, RADEN PRAMESA; KINTAMANI, ENDANG; PUTRI, ADE LIA; ROSYIDAH, A’LIYATUR; OCTAVIANA, SENLIE; BUDIARTO, BUGI RATNO; POHAN, FIQOLBI NURO; PUTRIE, RAHAYU FITRIANI WANGSA; RACHMADIYANTO, ARIEF NOOR
Jurnal Arsitektur Lansekap Vol.3, No.2, Oktober 2017
Publisher : Prodi Arsitektur Pertamanan, Fakultas Pertanian, Universitas Udayana

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24843/JAL.2017.v03.i02.p03

Abstract

The area within Pusbindiklat Peneliti-LIPI (The National Training and Education Center for Researchers Development-Indonesian Institute of Sciences) has been planted with many trees as an effort for reforestration and increasing aesthetic value. Unfortunately, the management of Pusbindiklat Peneliti-LIPI paid less attention to the trees potency and its characteristic as well as inventorizing all trees. This study aim to inventorize tree species based on their morphological characteristic in order to know the potency concerned with safety. Research was conducted through inventorizing and scoring the suitability factors of all trees, determining the tree plantation points, which is converted to the map of Pusbindiklat Peneliti-LIPI. In addition, direct interviews were also conducted to the civitas of Pusbindiklat Peneliti-LIPI. Trees that have been found in the area of Pusbindiklat Peneliti-LIPI consist of 20 family, 42 species, and 217 individuals. From the inventory of the trees, there were 11 species which were unsuitable with the criteria of urban forest vegetation parameter. Moreover, based on the map of trees species at Pusbindiklat Peneliti-LIPI area, there are 85 individual from 20 species of trees that are not suitable to be planted in Pusbindiklat Peneliti, since they were planted near the parapet wall and buildings of Pusbindiklat Peneliti-LIPI.
IMPROVEMENT OF HER2I655V TARMS-PCR PERFORMANCE BY DNA QUALITY ANALYSIS Budiarto, Bugi Ratno; Azamris, Azamris; Desriani, Desriani
ANNALES BOGORIENSES Vol 21, No 2 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2017.v21.n2.52-62

Abstract

Reliable TARMS-PCR is a prerequisite in constructing a solid conclusion in genetic diagnostics. The validity of data generated by this molecular technique is hampered by a false positive result. In attempt to develop a  TARMS-PCR for HER2I655V genotyping with no interfering of bias we used DNase I to eliminate DNA contaminant resided in PCR reagent. TARMS-PCR without enzyme treatment using recombinant plasmids that contained HER2I655V gene with represented its alleles was used to evaluate the presence of false positive  result while DNase I treated-PCR reagent was used in TARMS-PCR to evaluate the effective dose of the enzyme and further to adjust the TARMS-PCR conditions.  PCR master mix kit used in this study produced a false positive result on HER2I655V TARMS-PCR as proven by the presence of multiple PCR products in Non-Template Control (NTC) and 0.1 U of the enzyme could eliminate this DNA contaminant effectively, although this pretreatment altered the specificity of HER2I655V TARMS-PCR genotyping on certain genotype. Combination of touchdown TARMS-PCR with another allele-specific primer recovered specificity of detection on this model system. Interestingly, this optimized HER2I655V TARMS-PCR can only be used for genotyping the clinical samples if only further optimization was done using genomic DNA as template
ILE655VAL GENOTYPING STUDY OF HER2 - POSITIVE BREAST CANCER OF PATIENTS FROM PADANG - INDONESIA WITH HIGH RESOLUTION MELTING TECHNIQUE Wulandari, Dwi; Azamris, Azamris; Nurhayati, Isna; Warisman, M Ali; Budiarto, Bugi Ratno; Desriani, Desriani
ANNALES BOGORIENSES Vol 21, No 2 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2017.v21.n2.69-75

Abstract

Transtuzumab has proven to be a great improvement in the treatment of HER2 + of breast cancer patients, but it is associated with relevant adverse cardiac events and with significantly elevated cost of treatment.  One of the risk factors for cardiotoxicity due to transtuzumab is the I655V HER2 polymorphism (GTC> ATC mutation) in which the allele mutant (Ile val or val / val) has a higher risk than the wild type (Ile/Ile). The detection of specific alleles is very important for therapeutic decision-making. In this study, our group have developed a HRM with EvaGreen dye method to discriminate specific allele of I655V HER2 (wild type, heterozygote mutant or homozygote mutant) in 66 frozen section samples of HER2+ of breast cancer patients. Our group reported the wild type is the most prevalent allele (77,27%), whereas heterozygous mutation is significantly present in this research (21,21%) and only 1,52% of sample detected as minor allele.  It showed that only one sample detected as a minor allele (val/val) and may have relatively low abundance in the population. This method has been compared with sanger sequencing and giving 100% of validity.