scholarly journals A baculoviral system for the production of human β-glucocerebrosidase enables atomic resolution analysis

2020 ◽  
Vol 76 (6) ◽  
pp. 565-580
Author(s):  
Rhianna J. Rowland ◽  
Liang Wu ◽  
Feng Liu ◽  
Gideon J. Davies
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Crystal Form ◽  
Lysosomal Storage ◽  
Binding Studies ◽  
Enzyme Replacement ◽  
Resolution Analysis ◽  
Comparable Activity ◽  
Hydrolysis Of ◽  
Medical Interest

The lysosomal glycoside hydrolase β-glucocerebrosidase (GBA; sometimes called GBA1 or GCase) catalyses the hydrolysis of glycosphingolipids. Inherited deficiencies in GBA cause the lysosomal storage disorder Gaucher disease (GD). Consequently, GBA is of considerable medical interest, with continuous advances in the development of inhibitors, chaperones and activity-based probes. The development of new GBA inhibitors requires a source of active protein; however, the majority of structural and mechanistic studies of GBA today rely on clinical enzyme-replacement therapy (ERT) formulations, which are incredibly costly and are often difficult to obtain in adequate supply. Here, the production of active crystallizable GBA in insect cells using a baculovirus expression system is reported, providing a nonclinical source of recombinant GBA with comparable activity and biophysical properties to ERT preparations. Furthermore, a novel crystal form of GBA is described which diffracts to give a 0.98 Å resolution unliganded structure. A structure in complex with the inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-D-glucopyranoside was also obtained, demonstrating the ability of this GBA formulation to be used in ligand-binding studies. In light of its purity, stability and activity, the GBA production protocol described here should circumvent the need for ERT formulations for structural and biochemical studies and serve to support GD research.

scholarly journals Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of Spodoptera litura with Insecticidal Crystal Protein Cry1C

2002 ◽  
Vol 68 (9) ◽  
pp. 4583-4592 ◽  
Author(s):  
Neema Agrawal ◽  
Pawan Malhotra ◽  
Raj K. Bhatnagar
Keyword(s):  
Insect Cell ◽  
Spodoptera Litura ◽  
Expression System ◽  
Insect Cell Line ◽  
Baculovirus Expression System ◽  
Aminopeptidase N ◽  
Receptor Protein ◽  
Binding Studies ◽  
Toxin Binding ◽  
Cry Toxin

ABSTRACT Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.

scholarly journals Recombinant human sulphamidase: expression, amplification, purification and characterization

1998 ◽  
Vol 329 (1) ◽  
pp. 145-150 ◽  
Author(s):  
Julie BIELICKI ◽  
J. John HOPWOOD ◽  
L. Elizabeth MELVILLE ◽  
S. Donald ANSON
Keyword(s):  
Cell Lines ◽  
Cho Cells ◽  
Expression System ◽  
Heparan Sulphate ◽  
Chinese Hamster ◽  
Lysosomal Storage ◽  
Dhfr Gene ◽  
Enzyme Replacement ◽  
High Level ◽  
Mps Iiia

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is a lysosomal storage disease that causes a profound neurological deterioration. The disorder is caused by a deficiency of the lysosomal enzyme sulphamidase which is a requisite for the degradation of heparan sulphate. To facilitate the development of enzyme-replacement strategies for MPS IIIA patients, we have constructed a high-level expression system for recombinant human sulphamidase in Chinese hamster ovary (CHO) cells. An expression construct containing a methotrexate-resistant dihydrofolate reductase (DHFR) gene allowed amplification of expression levels from less than 1 mg of sulphamidase per litre of culture medium to approx. 15 mg/l. Unlike many cell lines made by gene amplification in DHFR-deficient CHO cells, and utilizing the normal DHFR gene, these cell lines appeared to be stable in the absence of selective pressure. Recombinant human sulphamidase was purified from unamplified and amplified cell lines. The native enzyme was found to be a dimer of 115 kDa. Denaturing and reducing SDS/PAGE revealed a subunit size of 62 kDa. Kinetic analysis demonstrated that the recombinant enzyme had broadly similar kinetic characteristics to sulphamidase purified from liver. Recombinant human sulphamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.

scholarly journals NTP binding and phosphohydrolase activity associated with purified bluetongue virus non-structural protein NS2

2000 ◽  
Vol 81 (8) ◽  
pp. 1961-1965 ◽  
Author(s):  
Nigel J. Horscroft ◽  
Polly Roy
Keyword(s):  
Bluetongue Virus ◽  
Inclusion Bodies ◽  
Insect Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Structural Protein ◽  
Baculovirus Expression ◽  
Infected Cells ◽  
Viral Inclusion ◽  
Hydrolysis Of

The bluetongue virus ssRNA-binding protein, NS2, is a phosphoprotein that forms viral inclusion bodies in infected cells. Recombinant NS2 was expressed in the baculovirus expression system and purified to homogeneity from insect cells. Purified NS2 bound nucleosides. Further investigation revealed that the protein bound ATP and GTP and could hydrolyse both nucleosides to their corresponding NMPs, with a higher efficiency for the hydrolysis of ATP. The increased efficiency of hydrolysis of ATP correlated with a higher binding affinity of NS2 for ATP than GTP. Ca2+, Mg2+ and Mn2+ were able to function as the required divalent cation in the reactions. The phosphohydrolase activity was not sensitive to ouabain, an inhibitor of cellular ATPases, suggesting that this activity was not the result of a cellular contaminant.

scholarly journals A corynecaterium glutamicum expression system for glycoside hydrolase analysis

2021 ◽  
Author(s):  
Hirak Saxena
Keyword(s):  
Expression System ◽  
Lysosomal Storage Diseases ◽  
Industrial Applications ◽  
Glycoside Hydrolases ◽  
Cellulosic Biomass ◽  
Lysosomal Storage ◽  
Cellular Functions ◽  
Domains Of Life ◽  
Hydrolysis Of ◽  
Structure And Activity

The biological hydrolysis of glycosidic linkages in complex sugars is facilitated by glycoside hydrolases. These enzymes are ubiquitous across all domains of life, playing significant roles in important biological processes like the degradation of cellulosic biomass, viral pathogenesis, antibacterial defense, and normal cellular functions. The potential industrial applications of highly efficient glycoside hydrolases, as well as the fact that a number of lysosomal storage diseases have been attributed to deficiencies in these enzymes 43, 22, merits further study into their structure and activity. For this reason, a handful of novel glycoside hydrolases from Cellulomonas fimi, a Gram-positive Actinobacteria known for its ability to degrade cellulose 39, will be cloned, expressed and biochemically analyzed.

scholarly journals A corynecaterium glutamicum expression system for glycoside hydrolase analysis

2021 ◽  
Author(s):  
Hirak Saxena
Keyword(s):  
Expression System ◽  
Lysosomal Storage Diseases ◽  
Industrial Applications ◽  
Glycoside Hydrolases ◽  
Cellulosic Biomass ◽  
Lysosomal Storage ◽  
Cellular Functions ◽  
Domains Of Life ◽  
Hydrolysis Of ◽  
Structure And Activity

The biological hydrolysis of glycosidic linkages in complex sugars is facilitated by glycoside hydrolases. These enzymes are ubiquitous across all domains of life, playing significant roles in important biological processes like the degradation of cellulosic biomass, viral pathogenesis, antibacterial defense, and normal cellular functions. The potential industrial applications of highly efficient glycoside hydrolases, as well as the fact that a number of lysosomal storage diseases have been attributed to deficiencies in these enzymes 43, 22, merits further study into their structure and activity. For this reason, a handful of novel glycoside hydrolases from Cellulomonas fimi, a Gram-positive Actinobacteria known for its ability to degrade cellulose 39, will be cloned, expressed and biochemically analyzed.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

Hallazgos oftalmológicos en enfermedad de Fabry: relación de su severidad en una familia mexicana

2019 ◽  
Vol 1 (1) ◽  
pp. 43-49
Author(s):  
Araceli Borja Borja ◽  
Gabriela Salas Pérez ◽  
Pablo Radillo Díaz
Keyword(s):  
Premature Mortality ◽  
Retinal Vessel ◽  
Multiple Organ ◽  
High Morbidity ◽  
Lysosomal Storage ◽  
Enzyme Replacement ◽  
Non Invasive ◽  
High Incidence ◽  
Cornea Verticillata ◽  
Gla Gene

Introduction. Fabry disease (FD) is a lysosomal storage disorder associated with multiple organ dysfunction which eventually leads to high morbidity and premature mortality. Ophthalmologic findings in FD are very common and have been described extensively. We describe the ophthalmologic findings of a family diagnosed with FD at Hospital de Especialidades de Puebla and establish their relationship with other phenotypic findings. Cases Presentation. A renal, cardiac, audiological, neurological, and ophthalmologic evaluation was carried out. The disease was confirmed by GLA gene sequencing. The ophthalmologic assessment was focused on the changes described in the literature, as well as the search for other anomalies possibly related to the disease. All the patients had the c.260delA (P.Glu87Glyfs*34) mutation in the GLA gene. The main ophthalmologic finding in our patients was cornea verticillata (in 100 % of the female patients). Other ophthalmologic manifestations were dry eye, retinal vessel tortuosity, ametropia, chromatic vision disorders, ocular annexes, eyelids, and conjuntiva disorders. Conclusions. Most of the assessed patients showed ophthalmologic changes, consistent with the results described in the literature. A remarkable finding in the sample was the high incidence of changes in women, in whom one would not expect the disease to be as severe because they are heterozygous. Ophthalmologic abnormalities in FD require deeper evaluation to establish their possible use as markers of disease progression and/or enzyme replacement therapy initiation due to the benefit of the non-invasive nature of ophthalmologic evaluations.

New Advanced Strategies for the Treatment of Lysosomal Diseases Affecting the Central Nervous System

2019 ◽  
Vol 25 (17) ◽  
pp. 1933-1950 ◽  
Author(s):  
Maria R. Gigliobianco ◽  
Piera Di Martino ◽  
Siyuan Deng ◽  
Cristina Casadidio ◽  
Roberta Censi
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Polymeric Nanoparticles ◽  
Genetic Diseases ◽  
Point Of View ◽  
Lysosomal Storage ◽  
Enzyme Replacement ◽  
Lysosomal Diseases ◽  
The Central Nervous System ◽  
Enzyme Delivery

Lysosomal Storage Disorders (LSDs), also known as lysosomal diseases (LDs) are a group of serious genetic diseases characterized by not only the accumulation of non-catabolized compounds in the lysosomes due to the deficiency of specific enzymes which usually eliminate these compounds, but also by trafficking, calcium changes and acidification. LDs mainly affect the central nervous system (CNS), which is difficult to reach for drugs and biological molecules due to the presence of the blood-brain barrier (BBB). While some therapies have proven highly effective in treating peripheral disorders in LD patients, they fail to overcome the BBB. Researchers have developed many strategies to circumvent this problem, for example, by creating carriers for enzyme delivery, which improve the enzyme’s half-life and the overexpression of receptors and transporters in the luminal or abluminal membranes of the BBB. This review aims to successfully examine the strategies developed during the last decade for the treatment of LDs, which mainly affect the CNS. Among the LD treatments, enzyme-replacement therapy (ERT) and gene therapy have proven effective, while nanoparticle, fusion protein, and small molecule-based therapies seem to offer considerable promise to treat the CNS pathology. This work also analyzed the challenges of the study to design new drug delivery systems for the effective treatment of LDs. Polymeric nanoparticles and liposomes are explored from their technological point of view and for the most relevant preclinical studies showing that they are excellent choices to protect active molecules and transport them through the BBB to target specific brain substrates for the treatment of LDs.

scholarly journals Expression of functional G protein beta gamma dimers of defined subunit composition using a baculovirus expression system.

1992 ◽  
Vol 267 (19) ◽  
pp. 13123-13126 ◽  
Author(s):  
S.G. Graber ◽  
R.A. Figler ◽  
V.K. Kalman-Maltese ◽  
J.D. Robishaw ◽  
J.C. Garrison
Keyword(s):  
G Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Subunit Composition ◽  
Baculovirus Expression

scholarly journals Characterization of New Proteomic Biomarker Candidates in Mucopolysaccharidosis Type IVA

2020 ◽  
Vol 22 (1) ◽  
pp. 226
Author(s):  
Víctor J. Álvarez ◽  
Susana B. Bravo ◽  
Maria Pilar Chantada-Vazquez ◽  
Cristóbal Colón ◽  
María J. De Castro ◽  
...  
Keyword(s):  
Keratan Sulfate ◽  
Bone Lesions ◽  
Mucopolysaccharidosis Type ◽  
Lysosomal Storage ◽  
Protein Biomarkers ◽  
Enzyme Replacement ◽  
Endurance Tests ◽  
Mps Iva ◽  
Mucopolysaccharidosis Type Iva ◽  
Potential Biomarkers

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Skeletal dysplasia and the related clinical features of MPS IVA are caused by disruption of the cartilage and its extracellular matrix, leading to a growth imbalance. Enzyme replacement therapy (ERT) with recombinant human GALNS has yielded positive results in activity of daily living and endurance tests. However, no data have demonstrated improvements in bone lesions and bone grow thin MPS IVA after ERT, and there is no correlation between therapeutic efficacy and urine levels of keratan sulfate, which accumulates in MPS IVA patients. Using qualitative and quantitative proteomics approaches, we analyzed leukocyte samples from healthy controls (n = 6) and from untreated (n = 5) and ERT-treated (n = 8, sampled before and after treatment) MPS IVA patients to identify potential biomarkers of disease. Out of 690 proteins identified in leukocytes, we selected a group of proteins that were dysregulated in MPS IVA patients with ERT. From these, we identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: lactotransferrin, coronin 1A, neutral alpha-glucosidase AB, and vitronectin. Further studies of cartilage and bone alterations in MPS IVA will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.

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