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Construction of synthetic open reading frame encoding human interferon alpha 2b for high expression in Escherichia coli and characterization of its gene product.

January 7, 2012

J Biotechnol. 2010 Jan 15;145(2):193-8. Epub 2009 Dec 1.

Construction of synthetic open reading frame encoding human interferon alpha 2b for high expression in Escherichia coli and characterization of its gene product.

Retnoningrum DS, Ningrum RA, Kurniawan YN, Indrayati A, Rachmawati H.

Source

School of Pharmacy, Institut Teknologi Bandung, Ganesha 10, Bandung 40132, Indonesia. retnoningrum@indo.net.id

Abstract

The aim of this research was to obtain recombinant human interferon alpha 2b (rhIFNalpha2b) from a synthetic open reading frame (ORF) overexpressed in Escherichia coli. For gene assembly, oligonucleotides were designed by Thermodynamically Balanced Inside Out (TBIO) method using the published synthetic codon optimized hIFNalpha2b ORF for high expression in E. coli. The synthetic ORF was assembled by a two-step Polymerase Chain Reaction (PCR) and cloned into a pGEM-T vector. The two-step PCR resulted in a DNA band of 522 base pairs (bp) corresponding to the size of hIFNalpha2b ORF. Fifteen recombinant pGEM-Ts were obtained and the sequencing results showed that the ORFs contained one to ten mutations with an error rate of 8.3 per kilo base. An ORF carrying one mutation was cloned into a pET32b vector and site-directed mutagenesis was performed to correct the mutation. The hIFNalpha2b ORF was overexpressed as a thioredoxin-his-tag fusion protein in E. coli BL21. The rhIFNalpha2b fusion protein was isolated from inclusion bodies (IB), renatured, and purified using Nickel columns, and all steps were monitored by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). A rhIFNalpha2b fusion protein of 37kDa in size was produced in high expression levels relative to total protein, renatured and purified from IB with a yield of 3.46mg/l without any further optimization. The purified rhIFNalpha2b was confirmed by peptide analysis with nano-LC-MS/MS2 mass spectrometry. Our current research demonstrates for the first time that by using the TBIO method a synthetic ORF encoding hIFNalpha2b gene can be expressed at high levels in E. coli.

 

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Identification of sequence mutations affecting hemagglutinin specificity to sialic acid receptor in influenza A virus subtypes.

January 7, 2012

Bioinformation. 2010 Nov 27;5(6):244-9.

Identification of sequence mutations affecting hemagglutinin specificity to sialic acid receptor in influenza A virus subtypes.

Tambunan US, Ramdhan.

Source

Department of Chemistry, Faculty of Mathematics and Natural Science, University of Indonesia, Depok Campus, Depok 16424, Indonesia.

Abstract

The attachment of the hemagglutinin protein of the H1N1 subtype of the pandemic influenza A virus to the sialic acid receptor Sia(α2-6)Gal has contributed to the ability of the virus to replicate in the human body and transmit among humans. In view of the pandemic caused by the replication and transmission of the H1N1 virus, more studies on the specificity of hemagglutinin towards sialic acid and how it affects the replication and transmission ability of this virus among humans are needed. In this study, we have applied sequence, structural and functional analyses to the hemagglutinin protein of the pandemic H1N1 virus, with the aim of identifying amino acid mutation patterns that affect its specificity to sialic acid. We have also employed a molecular docking method to evaluate the complex formed between hemagglutinin protein and the sialic acid receptor. Based on our results, we suggest two possible mutation patterns, namely (1) positions 190 and 225 from glutamic acid and glycine to aspartic acid (E190D in A/Brevig Mission/1/18 (H1N1), A/New York/1/18(H1N1) and A/South Carolina/1/1918(H1N1) and G225D in A/South Carolina/1/1918(H1N1), A/South Carolina/1/1918(H1N1), and A/Puerto Rico/8/34(H1N1)), and (2) positions 226 and 228 from glutamine and glycine to leucine and serine, respectively (Q226L and G228S in A/Guiyang/1/1957(H2N2), A/Kayano/57(H2N2), A/Aichi/2/1968(H3N2), A/Hong Kong/1/1968(H3N2) and A/Memphis/1/68(H3N2)) that can potentially contribute to the specificity of hemagglutinin to Sia(α2-6)Gal, thereby enabling the replication and transmission of virus within and among humans.

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Prediction of conformational changes by single mutation in the hepatitis B virus surface antigen (HBsAg) identified in HBsAg-negative blood donors.

January 7, 2012

Virol J. 2010 Nov 18;7:326.

Prediction of conformational changes by single mutation in the hepatitis B virus surface antigen (HBsAg) identified in HBsAg-negative blood donors.

Ie SI, Thedja MD, Roni M, Muljono DH.

Source

Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta, Indonesia.

Abstract

BACKGROUND:

Selection of hepatitis B virus (HBV) by host immunity has been suggested to give rise to variants with amino acid substitutions at or around the ‘a’ determinant of the surface antigen (HBsAg), the main target of antibody neutralization and diagnostic assays. However, there have never been successful attempts to provide evidence for this hypothesis, partly because the 3 D structure of HBsAg molecules has not been determined. Tertiary structure prediction of HBsAg solely from its primary amino acid sequence may reveal the molecular energetic of the mutated proteins. We carried out this preliminary study to analyze the predicted HBsAg conformation changes of HBV variants isolated from Indonesian blood donors undetectable by HBsAg assays and its significance, compared to other previously-reported variants that were associated with diagnostic failure.

RESULTS:

Three HBV variants (T123A, M133L and T143M) and a wild type sequence were analyzed together with frequently emerged variants T123N, M133I, M133T, M133V, and T143L. Based on the Jameson-Wolf algorithm for calculating antigenic index, the first two amino acid substitutions resulted in slight changes in the antigenicity of the ‘a’ determinant, while all four of the comparative variants showed relatively more significant changes. In the pattern T143M, changes in antigenic index were more significant, both in its coverage and magnitude, even when compared to variant T143L. These data were also partially supported by the tertiary structure prediction, in which the pattern T143M showed larger shift in the HBsAg second loop structure compared to the others.

CONCLUSIONS:

Single amino acid substitutions within or near the ‘a’ determinant of HBsAg may alter antigenicity properties of variant HBsAg, which can be shown by both its antigenic index and predicted 3 D conformation. Findings in this study emphasize the significance of variant T143M, the prevalent isolate with highest degree of antigenicity changes found in Indonesian blood donors. This highlights the importance of evaluating the effects of protein structure alterations on the sensitivity of screening methods being used in detection of ongoing HBV infection, as well as the use of vaccines and immunoglobulin therapy in contributing to the selection of HBV variants.

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Isolation and characterization of antioxidant protein fractions from melinjo (Gnetum gnemon) seeds.

January 7, 2012

J Agric Food Chem. 2011 May 25;59(10):5648-56. Epub 2011 Apr 27.

Isolation and characterization of antioxidant protein fractions from melinjo (Gnetum gnemon) seeds.

Siswoyo TA, Mardiana E, Lee KO, Hoshokawa K.

Source

Department of Agronomy, Faculty of Agriculture, University of Jember, Jember, Indonesia. siswoyo.triagus@gmail.com

Abstract

The protein from the seeds of melinjo ( Gnetum gnemon ) was purified using a precipitation method and ion exchange chromatographic techniques to identify the potent antioxidant and free radical scavenging activities. Two antioxidant protein fractions were isolated from G. gnemon seed with molecular weights of approximately 30 kDa (Gg-AOPI) and 12 kDa (Gg-AOPII) by SDS-PAGE. The N-terminal amino acid sequence of Gg-AOPII is Gly-Asn-Gly-Lys-Ala-Thr-Val-Ala-Ile-Leu-Val-Lys-Glu-Lys-Val-Glu-Tyr-Gly-Glu-Glu, and the result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that they were distinct from each other; no protein in database matching was found to both Gg-AOPI and Gg-AOPII. The antioxidant or free radical scavenging activities of Gg-AOPs were investigated by employing in vitro assay systems including the inhibition of linoleic acid autoxidation, scavenging effect on α,α-diphenyl-β-picrylhydrazyl free radical (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), reducing power, chelating abilities of metal ions Cu(2+) and Fe(2+), and protections against hydroxyl radical-mediated DNA damages. The result showed that two protein fractions exhibited significant (p < 0.05) antioxidant activities against free radicals such as DPPH, ABTS, and superoxide anion and showed activities similar to those of glutathione (G-SH) and BHT in a linoleic acid emulsion assay system. Moreover, Gg-AOPI and Gg-AOPII also exhibited notable reducing power and strong chelating effect on Fe(2+) and protected hydroxyl radical induced oxidative DNA damage. The data obtained by the in vitro systems obviously established the antioxidant potency of Gg-AOPs.

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Immunoinformatics analysis of H5N1 proteome for designing an epitope-derived vaccine and predicting the prevalence of pre-existing cellular-mediated immunity toward bird flu virus in Indonesian population.

January 7, 2012

Immunome Res. 2011 Nov 30;7(3):1-11.

Immunoinformatics analysis of H5N1 proteome for designing an epitope-derived vaccine and predicting the prevalence of pre-existing cellular-mediated immunity toward bird flu virus in Indonesian population.

Gustiananda M.

Source

Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta 10430, Indonesia.

Abstract

The persistence of influenza A virus H5N1 among poultry in Indonesia, along with increasing numbers of human infections by this virus, point to risk of a reassortment event with other prevalent influenza A sub-types in Indonesia. In the absence of cross-protective antibody to the emerging strain, the presence of cel-lular immunity might reduce the influenza illness to subclinical level and could dampen the pandemic. This study evaluated the degree of likely cross-protective T-cell mediated immunity against such hypothetical emerging influenza A strains. All 11 protein sequences from Indonesian H5N1 isolates were evaluated for the presence of T-cell epitopes restricted by Indonesian HLA types.Among 4433 possible nonamer peptides, 225 were predicted as good CTL epitopes (score less than 1%) by NetCTLpan and had strong binding affinity (IC50 less than 50 nM) toward HLA molecules by IEDBann and netMHCann. Epitope conservation analysis revealed that at least 60% of the H5N1 Indonesian isolates contained the identical sequences of this 225 peptide set. Sixty-nine peptides were specific for H5N1 and 156 were cross-reactive with other subtypes. Significant numbers (184) of peptides bound to more than one HLA allele (promiscuous). Blast analysis showed that the majority (213) of peptides did not have similarity with the human self peptides likely to induce autoimmunity if used for vaccination. Certain peptides can act as a core for HLA Class II molecules, and some superimposed with the putative linear B-cell epitopes. Eighteen peptides emerged as likely vaccine components optimized to an Indonesian population but likely also to be effective among most other ethnic groups. Routine exposure to seasonal influenza A likely ensures some level of cross-protective immunity to H5N1 and most reassortment mutants. Identifying of such likely cross-reactive T-cell epitopes may aid in gauging the threat of potential outbreaks and composing the vaccines that could contain them.

© 2011 Gustiananda M, licensee Petrovsky Publishing. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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DLBS1033, a protein extract from Lumbricus rubellus, possesses antithrombotic and thrombolytic activities.

January 7, 2012

J Biomed Biotechnol. 2011;2011:519652. Epub 2011 Mar 3.

DLBS1033, a protein extract from Lumbricus rubellus, possesses antithrombotic and thrombolytic activities.

Trisina J, Sunardi F, Suhartono MT, Tjandrawinata RR.

Source

Division of Protein Biochemistry and Molecular Pharmacology, Dexa Laboratories of Biomolecular Sciences, PT Dexa Medica, Industri Selatan V Block PP no. 7, Kawasan Industri Jababeka II, Cikarang 17550, Indonesia.

Abstract

The medicinal value of earthworm has been widely known since the history of Asian ancient medicine. This present study aims to determine the mechanism of action and effect of a standardized extract of Lumbricus rubellus named as DLBS1033. The fibrinogen degradation, antiplatelet aggregation, and ex vivo antithrombotic assay using human blood were performed to study antithrombotic activity. Fibrin plate and clot lysis assay were also done to examine thrombolytic properties. DLBS1033 was found to possess fibrinogenolytic activity on α-, β-, and γ-chain of fibrinogen. It also induced antiplatelet aggregation and prolonged blood clotting time, which further confirmed its antithrombotic properties. In addition, thrombolytic properties of DLBS1033 were shown with its fast and long-acting fibrinolytic activity, as well as its effective blood clot lysis activities. In conclusion, DLBS1033 conferred antithrombotic and thrombolytic action which could be used as a safe and promising oral thrombolytic drug.

 

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