Publications by authors named "Natasa Skoko"

20 Publications

  • Page 1 of 1

Thioridazine reverts the phenotype in cellular and Drosophila models of amyotrophic lateral sclerosis by enhancing TDP-43 aggregate clearance.

Neurobiol Dis 2021 Dec 24;160:105515. Epub 2021 Sep 24.

International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149 Trieste, Italy. Electronic address:

Brain inclusions mainly composed of misfolded and aggregated TAR DNA binding protein 43 (TDP-43), are characteristic hallmarks of amyotrophic lateral sclerosis (ALS). Irrespective of the role played by the inclusions, their reduction represents an important therapeutic pathway that is worth exploring. Their removal can either lead to the recovery of TDP-43 function by removing the self-templating conformers that sequester the protein in the inclusions, and/or eliminate any potential intrinsic toxicity of the aggregates. The search for curative therapies has been hampered by the lack of ALS models for use in high-throughput screening. We adapted, optimised, and extensively characterised our previous ALS cellular model for such use. The model demonstrated efficient aggregation of endogenous TDP-43, and concomitant loss of its splicing regulation function. We provided a proof-of-principle for its eventual use in high-throughput screening using compounds of the tricyclic family and showed that recovery of TDP-43 function can be achieved by the enhanced removal of TDP-43 aggregates by these compounds. We observed that the degradation of the aggregates occurs independent of the autophagy pathway beyond autophagosome-lysosome fusion, but requires a functional proteasome pathway. The in vivo translational effect of the cellular model was tested with two of these compounds in a Drosophila model expressing a construct analogous to the cellular model, where thioridazine significantly improved the locomotive defect. Our findings have important implications as thioridazine cleared TDP-43 aggregates and recovered TDP-43 functionality. This study also highlights the importance of a two-stage, in vitro and in vivo model system to cross-check the search for small molecules that can clear TDP-43 aggregates in TDP-43 proteinopathies.
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http://dx.doi.org/10.1016/j.nbd.2021.105515DOI Listing
December 2021

High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells.

Biomolecules 2021 07 30;11(8). Epub 2021 Jul 30.

Biotechnology Development Unit, International Centre for Genetic Engineering and Biotechnology (ICGEB), 34149 Trieste, Italy.

CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs-miRNAs-is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells.
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http://dx.doi.org/10.3390/biom11081125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392531PMC
July 2021

Inhibitors of Protein Glycosylation Are Active against the Coronavirus Severe Acute Respiratory Syndrome Coronavirus SARS-CoV-2.

Viruses 2021 04 30;13(5). Epub 2021 Apr 30.

Laboratory of Molecular Virology, International Centre for Genetic Engineering and Biotechnology (ICGEB) Padriciano, 99-34149 Trieste, Italy.

Repurposing clinically available drugs to treat the new coronavirus disease 2019 (COVID-19) is an urgent need in the course of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) pandemic, as very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases, such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated in the micromolar range for SARS-CoV-2 in vitro. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious viruses. The mechanism resides in the inhibitory activity toward α-glucosidases that are involved in the early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. Indeed, the antiviral potential of protein glycosylation inhibitors against SARS-CoV-2 is further highlighted by the low-micromolar activity of the investigational drug Celgosivir. These data point to a relevant role of this approach for the treatment of COVID-19.
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http://dx.doi.org/10.3390/v13050808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144969PMC
April 2021

ITC for Characterization of Self-Assembly Process of Cationic Dendrons for siRNA Delivery.

Methods Mol Biol 2021 ;2282:245-266

Molecular Biology and Nanotechnology Laboratory ([email protected]), Department of Engineering and Architecture, University of Trieste, Trieste, Italy.

siRNAs are emerging as promising therapeutic agents due to their ability to inhibit specific genes in many diseases. However, these tools require specific vehicles in order to be safely delivered to the targeted site. Among different siRNA delivery systems, self-assembled nanomicelles based on amphiphilic cationic dendrons (ACDs) have recently outperformed nanovectors based on covalent carriers. This chapter describes how isothermal titration calorimetry (ITC) can be exploited as one of the best techniques to investigate the self-assembly process of ACDs. Specifically, ITC can provide, as such or via specific analysis methods, a full thermodynamic characterization of these nanomicelles, including their critical micellar concentration, micelle aggregation number, degree of counterion binding, Gibbs free energy of micellization, and its enthalpic and entropic components.
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http://dx.doi.org/10.1007/978-1-0716-1298-9_15DOI Listing
June 2021

High-throughput screening discovers antifibrotic properties of haloperidol by hindering myofibroblast activation.

JCI Insight 2019 04 18;4(8). Epub 2019 Apr 18.

Cardiovascular Biology.

Fibrosis is a hallmark in the pathogenesis of various diseases, with very limited therapeutic solutions. A key event in the fibrotic process is the expression of contractile proteins, including α-smooth muscle actin (αSMA) by fibroblasts, which become myofibroblasts. Here, we report the results of a high-throughput screening of a library of approved drugs that led to the discovery of haloperidol, a common antipsychotic drug, as a potent inhibitor of myofibroblast activation. We show that haloperidol exerts its antifibrotic effect on primary murine and human fibroblasts by binding to sigma receptor 1, independent from the canonical transforming growth factor-β signaling pathway. Its mechanism of action involves the modulation of intracellular calcium, with moderate induction of endoplasmic reticulum stress response, which in turn abrogates Notch1 signaling and the consequent expression of its targets, including αSMA. Importantly, haloperidol also reduced the fibrotic burden in 3 different animal models of lung, cardiac, and tumor-associated fibrosis, thus supporting the repurposing of this drug for the treatment of fibrotic conditions.
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http://dx.doi.org/10.1172/jci.insight.123987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538355PMC
April 2019

A Simplified and Efficient Process for Insulin Production in Pichia pastoris.

PLoS One 2016 1;11(12):e0167207. Epub 2016 Dec 1.

ICGEB, Trieste, Italy.

A significant barrier to insulin is affordability. In this manuscript we describe improvements to key steps in the insulin production process in Pichia pastoris that reduce cost and time. The strategy for recovery and processing of human insulin precursor has been streamlined to two steps from bioreactor to the transpeptidation reaction. In the first step the insulin precursor secreted during the methanol induction phase is recovered directly from the culture broth using Tangential Flow Filtration with a Prostak™ module eliminating the laborious and time-consuming multi-step clarification, including centrifugation. In the second step the protein is applied at very high loadings on a cation exchange resin and eluted in a mixture of water and ethanol to obtain a concentrated insulin precursor, suitable for use directly in the transpeptidation reaction. Overall the yield from insulin precursor to human insulin was 51% and consisted of three purification chromatography steps. In addition we describe a method for recovery of the excess of H-Thr(tBu)-OtBu from the transpeptidation reaction mixture, one of the more costly reagents in the process, along with its successful reuse.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167207PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131935PMC
June 2017

Antimycobacterial activity of new N(1)-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives.

Bioorg Med Chem Lett 2016 07 19;26(14):3287-3290. Epub 2016 May 19.

Department of Life and Enviromental Sciences, Via Porcell 4, University of Cagliari, 09124 Cagliari, Italy.

N(1)-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives were design, synthesized and tested for their in vitro antimycobacterial activity. The new compounds showed a moderate antimycobacterial activity against the tested strain of Mycobacterium tuberculosis H37Ra and a significant antimycobacterial activity against several mycobacteria other than tuberculosis strains.
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http://dx.doi.org/10.1016/j.bmcl.2016.05.053DOI Listing
July 2016

Absence of TDP-43 is difficult to digest.

EMBO J 2016 Jan 23;35(2):115-7. Epub 2015 Dec 23.

ICGEB, Trieste, Italy.

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http://dx.doi.org/10.15252/embj.201593603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718458PMC
January 2016

Aggregate formation prevents dTDP-43 neurotoxicity in the Drosophila melanogaster eye.

Neurobiol Dis 2014 Nov 31;71:74-80. Epub 2014 Jul 31.

ICGEB - International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy. Electronic address:

TDP-43 inclusions are an important histopathological feature in various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis and Fronto-Temporal Lobar Degeneration. However, the relation of these inclusions with the pathogenesis of the disease is still unclear. In fact, the inclusions could be toxic themselves, induce loss of function by sequestering TDP-43 or a combination of both. Previously, we have developed a cellular model of aggregation using the TDP-43 Q/N rich amino acid sequence 331-369 repeated 12 times (12xQ/N) and have shown that these cellular inclusions are capable of sequestering the endogenous TDP-43 both in non-neuronal and neuronal cells. We have tested this model in vivo in the Drosophila melanogaster eye. The eye structure develops normally in the absence of dTDP-43, a fact previously seen in knock out fly strains. We show here that expression of EGFP 12xQ/N does not alter the structure of the eye. In contrast, TBPH overexpression is neurotoxic and causes necrosis and loss of function of the eye. More important, the neurotoxicity of TBPH can be abolished by its incorporation to the insoluble aggregates induced by EGFP 12xQ/N. This data indicates that aggregation is not toxic per se and instead has a protective role, modulating the functional TBPH available in the tissue. This is an important indication for the possible pathological mechanism in action on ALS patients.
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http://dx.doi.org/10.1016/j.nbd.2014.07.009DOI Listing
November 2014

Dual role of dextran sulfate 5000 Da as anti-apoptotic and pro-autophagy agent.

Mol Biotechnol 2013 Jun;54(2):711-20

International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.

Dextran sulfate 5,000 Da (DS), a sulfated polysaccharide, has been used in recombinant mammalian cell cultures to prevent cell aggregation, thereby increasing cell viability. Previous studies using Chinese hamster ovary (CHO) suspension cultures had shown that low concentrations of DS are related to an inhibition of apoptosis. In this study, DS was used on anchorage-dependent CHO cells producing erythropoietin (EPO), in order to investigate the effect of this molecule on anti-apoptotic and pro-survival cellular pathways. DS 5,000 Da treatment was shown to prolong the life of cells and increase productivity of EPO by 1.8-fold comparing with controls, in standard batch conditions. At a molecular level, we show that DS inhibits apoptosis by DNA fragmentation delay and decrease of annexin V-labeled cells, causes a G0/G1 cell cycle arrest, decreases p53 expression and increases the pro-survival factor Hsc70 expression. DS treatment also resulted in an enhanced LC3-I to LC3-II conversion and increased autophagosomes formation employing tagged-LC3. Our data show, for the first time, that low doses of DS may promote autophagy in different cell lines. These findings suggest that a better understanding and manipulation of phenomenon of autophagy could be of crucial importance in the bio-pharmaceutical industry, in particular in the field of protein production.
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http://dx.doi.org/10.1007/s12033-012-9620-xDOI Listing
June 2013

Complexities of 5'splice site definition: implications in clinical analyses.

RNA Biol 2012 Jun 23;9(6):911-23. Epub 2012 May 23.

International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.

In higher eukaryotes, the 5' splice site (5'ss) is initially recognized through an RNA-RNA interaction by U1 small nuclear ribonucleoprotein (U1 snRNP). This event represents one of the key steps in initial spliceosomal assembly and many disease-associated mutations in humans often disrupt this process. Beside base pair complementarity, 5'ss recognition can also be modified by additional factors such as RNA secondary structures or the specific binding of other nuclear proteins. In this work, we have focused on investigating a few examples of changes detected within the 5'ss in patients, that would not be immediately considered "disease causing mutations". We show that the splicing outcome of very similar mutations can be very different due to variations in trans-acting factor(s) interactions and specific context influences. Using several NF1 donor sites and SELEX approaches as experimental models, we have examined the binding properties of particular sequence motifs such as GGGU found in donor sites, and how the sequence context can change their interaction with hnRNPs such as H/F and A1/A2. Our results clearly show that even minor differences in local nucleotide context can differentially affect the binding ability of these factors to the GGGU core. Finally, using a previously identified mutation in KCNH2 that resulted in intron retention we show how very similar 5'ss mutations found in patients can have a very different splicing outcome due to the neighbouring sequence context, thus highlighting the general need to approach splicing problems with suitable experimental approaches.
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http://dx.doi.org/10.4161/rna.20386DOI Listing
June 2012

InTRONs in biotech.

Mol Biotechnol 2011 Jul;48(3):290-7

International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy.

Eukaryotic gene expression relies on several complex molecular machineries that act in a highly coordinated fashion. These machineries govern all the different steps of mRNA maturation, from gene transcription and pre-mRNA processing in the nucleus to the export of the mRNA to the cytoplasm and its translation. In particular, the pre-mRNA splicing process consists in the joining together of sequences (known as "exons") that have to be differentiated from their intervening sequences commonly referred to as "introns." The complex required to perform this process is a very dynamic macromolecular ribonucleoprotein assembly that functions as an enzyme, and is called the "spliceosome." Because of its flexibility, the splicing process represents one of the main mechanisms of qualitative and quantitative regulation of gene expression in eukaryotic genomes. This flexibility is mainly due to the possibility of alternatively recognizing the various exons that are present in a pre-mRNA molecule and therefore enabling the possibility of obtaining multiple transcripts from the same gene. However, regulation of gene expression by the spliceosome is also achieved through its ability to influence many other gene expression steps that include transcription, mRNA export, mRNA stability, and even protein translation. Therefore, from a biotechnological point of view the splicing process can be exploited to improve production strategies and processes of molecules of interest. In this work, we have aimed to provide an overview on how biotechnology applications may benefit from the introduction of introns within a sequence of interest.
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http://dx.doi.org/10.1007/s12033-011-9390-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090800PMC
July 2011

Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin.

Microb Cell Fact 2010 May 12;9:31. Epub 2010 May 12.

Helmholtz Centre for Infection Research, Braunschweig, Germany.

Background: The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.

Results: A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae alpha-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of approximately 3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to approximately 1.5 g of 99% pure recombinant human insulin per liter of culture broth.

Conclusions: A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.
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http://dx.doi.org/10.1186/1475-2859-9-31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882349PMC
May 2010

Improving human interferon-beta production in mammalian cell lines by insertion of an intronic sequence within its naturally uninterrupted gene.

Biotechnol Appl Biochem 2009 Mar;52(Pt 3):191-8

International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.

Human beta-interferon is used extensively as a therapeutic agent in a wide variety of diseases, ranging from multiple sclerosis to viral infections. At present, the most common source of interferon-beta is derived from CHO (Chinese-hamster ovary) cells. Interestingly, however, the IFNB gene is characterized by a lack of intronic sequences and therefore does not undergo splicing during its expression pathway. As nuclear processing of pre-mRNA molecules has often been demonstrated to improve production yields of recombinant molecules, we have inserted a heterologous intronic sequence at different positions within the IFNB gene and analysed its effects on protein production. The results obtained in the present study show that the position of intron insertion has profound effects on the expression levels of the IFNB gene and on the nuclear/cytoplasm distribution levels of its mRNA as determined by FISH (fluorescent in situ hybridization) analysis of stably transfected clones. In conclusion, our results provide additional evidence that insertion of intronic sequences may be used to improve protein expression efficiency also in molecules that do not normally undergo any splicing process.
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http://dx.doi.org/10.1042/BA20080046DOI Listing
March 2009

The pathological splicing mutation c.6792C>G in NF1 exon 37 causes a change of tenancy between antagonistic splicing factors.

FEBS Lett 2008 Jun 27;582(15):2231-6. Epub 2008 May 27.

International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, Trieste, Italy.

We have previously identified an ESE in NF1 exon 37 whose disruption by the pathological mutation c.6792C>G caused aberrant splicing. We now investigate the RNA-protein complexes affected by the c.6792C>G mutation observing that this concurrently decreases the affinity for the positive splicing factor YB-1 and increases the affinity for the negative splicing factors, hnRNPA1, hnRNPA2 and a new player in these type of complexes, DAZAP1. Our findings highlight the complexity of the interplay between positive and negative factors in the exon inclusion/skipping outcome. Furthermore, our observations stress the role of a wide genomic context in NF1 exon 37 definition.
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http://dx.doi.org/10.1016/j.febslet.2008.05.018DOI Listing
June 2008

Binding of DAZAP1 and hnRNPA1/A2 to an exonic splicing silencer in a natural BRCA1 exon 18 mutant.

Mol Cell Biol 2008 Jun 7;28(11):3850-60. Epub 2008 Apr 7.

International Center for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy.

A disease-causing G-to-T transversion at position +6 of BRCA1 exon 18 induces exclusion of the exon from the mRNA and, as has been suggested by in silico analysis, disrupts an ASF/SF2-dependent splicing enhancer. We show here using a pulldown assay with an internal standard that wild-type (WT) and mutant T6 sequences displayed similar ASF/SF2 binding efficiencies, which were significantly lower than that of a typical exonic splicing enhancer derived from the extra domain A exon of fibronectin. Overexpression or small interfering RNA (siRNA)-mediated depletion of ASF/SF2 did not affect the splicing of a WT BRCA1 minigene but resulted in an increase and decrease of T6 exon 18 inclusion, respectively. Furthermore, extensive mutation analysis using hybrid minigenes indicated that the T6 mutant creates a sequence with a prevalently inhibitory function. Indeed, RNA-protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a splicing factor-dependent silencer. This sequence specifically binds to the known repressor protein hnRNPA1/A2 and to DAZAP1, the involvement of which in splicing inhibition we have demonstrated. Our results indicate that the binding of the splicing factors hnRNPA1/A2 and DAZAP1 is the primary determinant of T6 BRCA1 exon 18 exclusion.
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http://dx.doi.org/10.1128/MCB.02253-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423284PMC
June 2008

NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region.

FEBS Lett 2006 Aug 14;580(18):4449-56. Epub 2006 Jul 14.

International Centre for Genetic Engineering and Biotechnology, ICGEB, Padriciano 99, 34012 Trieste, Italy.

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.
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http://dx.doi.org/10.1016/j.febslet.2006.07.018DOI Listing
August 2006

Construction of Saccharomyces cerevisiae strain FAV20 useful in detection of immunosuppressants produced by soil actinomycetes.

J Microbiol Methods 2005 Apr;61(1):137-40

Institute of Molecular Genetics and Genetic Engineering, P.O. Box 23, Vojvode Stepe 444a, 11 001 Belgrade, Serbia and Montenegro.

The screening of microbial natural products continues to represent an important route to the discovery of novel chemicals for development of new therapeutic agents. The aim of this work was to develop an efficient method for the detection of immunosuppressive compounds produced by soil actinomycetes. Mutant strain of Saccharomyces cerevisiae, named FAV20, sensitive to FK506 was constructed by disrupting VMA22 gene using the selectable marker kanMX4 which allowed detection of integration events. Actinomycetes were isolated from different soil samples and in a newly developed test with S. cerevisiae FAV20, six strains have been identified that produce bioactive compounds with the same mechanism of action as FK506. S. cerevisiae FAV20 can be easily used as a test strain in drug screening programs based on inhibition of the calcineurin phosphatase dependent signaling pathway in the cell.
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http://dx.doi.org/10.1016/j.mimet.2004.11.007DOI Listing
April 2005

Expression and characterization of human interferon-beta1 in the methylotrophic yeast Pichia pastoris.

Biotechnol Appl Biochem 2003 Dec;38(Pt 3):257-65

Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, 11001 Belgrade, Serbia and Montenegro.

We describe the heterologous expression of a human interferon-beta1 in the methylotrophic yeast Pichia pastoris. Biologically active recombinant human interferon-beta1 (rHuIFN-beta1) was secreted from shake-flask-grown P. pastoris cells into the medium using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence at the level of (1-3) x 10(5) i.u. (international units)/ml (6-12 mg/litre). An rHuIFN-beta1 with an N-terminal sequence identical with that of native HuIFN-beta1 was purified and the specific activity was determined (2-3 x 10(7) i.u./mg). It was found that the secreted recombinant protein was partially N-glycosylated.
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http://dx.doi.org/10.1042/BA20030065DOI Listing
December 2003

Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris.

FEMS Yeast Res 2002 Jan;1(4):271-7

Institute of Molecular Genetics and Genetic Engineering, Belgrade, FR Yugoslavia.

Penicillin G amidase from Providencia rettgeri is a heterodimer of 92 kDa. We have previously expressed the Pr. rettgeri pac gene coding for this enzyme in Saccharomyces cerevisiae, and now we report the expression and characterization in the methylotrophic yeast Pichia pastoris. The recombinant catalytically active enzyme (rPAC(Pr)) was secreted from shake flask-grown P. pastoris cells into the medium at a level of approximately 0.18 U ml(-1). This yield of rPAC(Pr) was higher, by two orders of magnitude, than that obtained using a single-copy expression plasmid in S. cerevisiae. In addition, the secreted recombinant enzyme was entirely N-glycosylated. The recombinant PAC(Pr) was further characterized in terms of specific activity, kinetic parameters and thermostability. Except the significantly higher thermostability of the glycosylated rPAC(Pr) produced in P. pastoris, the other parameters were very similar to those of the corresponding non-glycosylated enzymes produced in bacteria or in S. cerevisiae. The higher thermostability of this recombinant enzyme has a clear industrial advantage.
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http://dx.doi.org/10.1111/j.1567-1364.2002.tb00045.xDOI Listing
January 2002
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