Site-Specific N-Glycosylation on the AAV8 Capsid Protein

ABSTRACT Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database forStreptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo.

Download Full-text

Purificação e avaliação da interferência de BthMP (uma metaloprotease da peçonha de Bothrops moojeni) na coagulação sanguínea

Jornal Interdisciplinar de Biociências ◽

10.26694/2448-0002.vl1iss2pp7-16 ◽

2016 ◽

Vol 1 (2) ◽

pp. 7

Author(s):

Manuela Andrade Santos ◽

Luzia Aparecida Pando ◽

Veridiana De Melo Rodrigues ◽

Mariana De Souza Castro ◽

Mário Sérgio Rocha Rocha Gomes

Keyword(s):

Peptide Mass Fingerprinting ◽

Maldi Tof ◽

Peptide Mass ◽

Sds Page ◽

Bothrops Moojeni

Neste trabalho relatamos a purificação da metaloprotease BthMP, proveniente da peçonha da serpente Bothrops moojeni. Para a purificação desta protease, utilizaram-se os passos cromatográficos de troca iônica (DEAE-Sepharose) e de exclusão molecular (Sephadex G-75), sendo o produto desses processos uma banda proteica com elevado grau de pureza, visualizada em SDS-PAGE a 14%, denominada BthMP. Esta, por sua vez, quando analisada em MALDI-TOF revelou a massa molecular nativa de 23.050 Da e 23.872 Da na forma reduzida, e a partir dos fragmentos peptídicos obtidos por Peptide Mass Fingerprinting (PMF) em MS (MALDI-TOF/TOF) indicou alta similaridade com a metaloprotease BmooMPα-I. Em termos enzimáticos, BthMP mostrou atividade proteolítica sobre azocaseína e frente ao PMSF e benzamidina, enquanto que esta atividade foi inibida na presença de EDTA, 1,10-fenantrolina e β-mercaptoetanol, sendo portanto uma metaloprotease zinco dependente da classe P-I. Ainda com este propósito, verificou-se sua especificidade enzimática sobre as cadeias Aα e Bβ do fibrinogênio, e também o consumo de fibrinogênio in vivo. Foi constatado ainda sua ação em componentes da cascata de coagulação, devido ao prolongamento do Tempo de Protrombina (TP) e do Tempo de Tromboplastina Parcial ativada (TTPa). Desta forma, a acentuada atividade fibrinogenolítica e o alto consumo de fibrinogênio in vivo são resultados que indicam a ação anticoagulante da BthMP; além do mais, sua capacidade de interferir na cascata de coagulação sugere que esta protease é promissora para futuros estudos que possam indicar um novo modelo de fármaco antitrombótico. https://doi.galoa.com.br/doi/10.17648/jibi-2448-0002-1-2-5128

Download Full-text

Purificação e avaliação da interferência de BthMP (uma metaloprotease da peçonha de Bothrops moojeni) na coagulação sanguínea

10.17648/jibi-2448-0002-1-2-5128 ◽

2016 ◽

Vol 1 (2) ◽

Author(s):

Manuela Andrade Santos ◽

Luzia Aparecida Pando ◽

Veridiana de Melo Rodrigues ◽

Mariana de Souza Castro ◽

Mário Sérgio Rocha Gomes

Keyword(s):

Peptide Mass Fingerprinting ◽

Maldi Tof ◽

Peptide Mass ◽

Sds Page ◽

Bothrops Moojeni

Neste trabalho relatamos a purificação da metaloprotease BthMP, proveniente da peçonha da serpente Bothrops moojeni. Para a purificação desta protease, utilizaram-se os passos cromatográficos de troca iônica (DEAE-Sepharose) e de exclusão molecular (Sephadex G-75), sendo o produto desses processos uma banda proteica com elevado grau de pureza, visualizada em SDS-PAGE a 14%, denominada BthMP. Esta, por sua vez, quando analisada em MALDI-TOF revelou a massa molecular nativa de 23.050 Da e 23.872 Da na forma reduzida, e a partir dos fragmentos peptídicos obtidos por Peptide Mass Fingerprinting (PMF) em MS (MALDI-TOF/TOF) indicou alta similaridade com a metaloprotease BmooMPα-I. Em termos enzimáticos, BthMP mostrou atividade proteolítica sobre azocaseína e frente ao PMSF e benzamidina, enquanto que esta atividade foi inibida na presença de EDTA, 1,10-fenantrolina e β-mercaptoetanol, sendo portanto uma metaloprotease zinco dependente da classe P-I. Ainda com este propósito, verificou-se sua especificidade enzimática sobre as cadeias Aα e Bβ do fibrinogênio, e também o consumo de fibrinogênio in vivo. Foi constatado ainda sua ação em componentes da cascata de coagulação, devido ao prolongamento do Tempo de Protrombina (TP) e do Tempo de Tromboplastina Parcial ativada (TTPa). Desta forma, a acentuada atividade fibrinogenolítica e o alto consumo de fibrinogênio in vivo são resultados que indicam a ação anticoagulante da BthMP; além do mais, sua capacidade de interferir na cascata de coagulação sugere que esta protease é promissora para futuros estudos que possam indicar um novo modelo de fármaco antitrombótico.

Download Full-text

Genetic and Functional Analysis of the Soluble Oxaloacetate Decarboxylase from Corynebacterium glutamicum

Journal of Bacteriology ◽

10.1128/jb.01678-09 ◽

2010 ◽

Vol 192 (10) ◽

pp. 2604-2612 ◽

Cited By ~ 19

Author(s):

Simon Klaffl ◽

Bernhard J. Eikmanns

Keyword(s):

Specific Activity ◽

Fold Increase ◽

Peptide Mass Fingerprinting ◽

Transcriptional Analysis ◽

Lysine Production ◽

Ionization Time ◽

Peptide Mass ◽

Methyltransferase Gene ◽

High Level

ABSTRACT Soluble, divalent cation-dependent oxaloacetate decarboxylases (ODx) catalyze the irreversible decarboxylation of oxaloacetate to pyruvate and CO2. Although these enzymes have been characterized in different microorganisms, the genes that encode them have not been identified, and their functions have been only poorly analyzed so far. In this study, we purified a soluble ODx from wild-type C. glutamicum about 65-fold and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and peptide mass fingerprinting for identification of the corresponding odx gene. Inactivation and overexpression of odx led to an absence of ODx activity and to a 30-fold increase in ODx specific activity, respectively; these findings unequivocally confirmed that this gene encodes a soluble ODx. Transcriptional analysis of odx indicated that there is a leaderless transcript that is organized in an operon together with a putative S-adenosylmethionine-dependent methyltransferase gene. Biochemical analysis of ODx revealed that the molecular mass of the native enzyme is about 62 ± 1 kDa and that the enzyme is composed of two ∼29-kDa homodimeric subunits and has a Km for oxaloacetate of 1.4 mM and a V max of 201 μmol of oxaloacetate converted per min per mg of protein, resulting in a k cat of 104 s−1. Introduction of plasmid-borne odx into a pyruvate kinase-deficient C. glutamicum strain restored growth of this mutant on acetate, indicating that a high level of ODx activity redirects the carbon flux from oxaloacetate to pyruvate in vivo. Consistently, overexpression of the odx gene in an l-lysine-producing strain of C. glutamicum led to accumulation of less l-lysine. However, inactivation of the odx gene did not improve l-lysine production under the conditions tested.

Download Full-text

Detection of Norovirus Capsid Protein in Authentic Standards and in Stool Extracts by Matrix-Assisted Laser Desorption Ionization and Nanospray Mass Spectrometry

Applied and Environmental Microbiology ◽

10.1128/aem.72.4.2749-2755.2006 ◽

2006 ◽

Vol 72 (4) ◽

pp. 2749-2755 ◽

Cited By ~ 24

Author(s):

David R. Colquhoun ◽

Kellogg J. Schwab ◽

Robert N. Cole ◽

Rolf U. Halden

Keyword(s):

Mass Spectrometry ◽

Capsid Protein ◽

Homeland Security ◽

Peptide Mass Fingerprinting ◽

Peptide Sequencing ◽

Intact Proteins ◽

Peptide Mass ◽

Assay Performance ◽

Bioterrorism Agent ◽

Human Stool

ABSTRACT Mass spectrometry (MS) represents a rapid technique for the identification of microbial monocultures, and its adaptation to the detection of pathogens in real-world samples is a public health and homeland security priority. Norovirus, a leading cause of gastroenteritis in the world, is difficult to monitor because it cannot be cultured outside the human body. The detection of norovirus capsid protein was explored using three common MS-based methods: scanning of intact proteins, peptide mass fingerprinting, and peptide sequencing. Detection of intact target protein was limited by poor selectivity and sensitivity. Detection of up to 16 target peptides by peptide mass fingerprinting allowed for the reproducible and confident (P < 0.05) detection of the 56-kDa norovirus capsid protein in the range of 0.1 × 10−12 to 50 × 10−12 mol in authentic standards of recombinant norovirus virus-like particles (VLPs). To explore assay performance in complex matrixes, a non-gel-based, rapid method (2 to 3 h) for virus extraction from human stool was evaluated (72% ± 12% recovery), and additional analyses were performed on norovirus-free stool extracts fortified with VLPs. Whereas peptide mass fingerprinting was rendered impractical by sample interferences, peptide sequencing using nanospray tandem MS facilitated unambiguous identification of ≥250 fmol of capsid protein in stool extracts. This is the first report on MS-based detection of norovirus, accomplished by using structurally identical, noninfective VLPs at clinically relevant concentrations. It represents an important milestone in the development of assays for surveillance of this category B bioterrorism agent.

Download Full-text

Genetic Engineering of AAV Capsid Gene for Gene Therapy Application

Current Gene Therapy ◽

10.2174/1566523220666200930105521 ◽

2020 ◽

Vol 20 (5) ◽

pp. 321-332

Author(s):

Yunbo Liu ◽

Xu Zhang ◽

Lin Yang

Keyword(s):

Gene Therapy ◽

Neutralizing Antibodies ◽

Human Subjects ◽

Genetic Diseases ◽

Capsid Gene ◽

Human Populations ◽

Stable Gene ◽

Efficient Gene ◽

Gene Therapy Application

Adeno-associated virus (AAV) is a promising vector for in vivo gene therapy because of its excellent safety profile and ability to mediate stable gene expression in human subjects. However, there are still numerous challenges that need to be resolved before this gene delivery vehicle is used in clinical applications, such as the inability of AAV to effectively target specific tissues, preexisting neutralizing antibodies in human populations, and a limited AAV packaging capacity. Over the past two decades, much genetic modification work has been performed with the AAV capsid gene, resulting in a large number of variants with modified characteristics, rendering AAV a versatile vector for more efficient gene therapy applications for different genetic diseases.

Download Full-text

Proteomic Analysis of Medicinal Plant Calotropis Gigantea by insilico Peptide Mass Fingerprinting

Current Computer - Aided Drug Design ◽

10.2174/1573409916666200219114531 ◽

2020 ◽

Vol 16 ◽

Author(s):

Saad Ur Rehman ◽

Muhammad Rizwan ◽

Sajid Khan ◽

Azhar Mehmood ◽

Anum Munir

Keyword(s):

Medicinal Plant ◽

Structure Prediction ◽

3D Structure ◽

Peptide Mass Fingerprinting ◽

Protein Docking ◽

Receptor Protein ◽

Human Leukemia ◽

Protein Database ◽

Calotropis Gigantea ◽

Peptide Mass

: Medicinal plants are the basic source of medicinal compounds traditionally used for the treatment of human diseases. Calotropis gigantea a medicinal plant belonging to the family of Apocynaceae in the plant kingdom and subfamily Asclepiadaceae usually bearing multiple medicinal properties to cure a variety of diseases. Background: The Peptide Mass Fingerprinting (PMF) identifies the proteins from a reference protein database by comparing the amino acid sequence that is previously stored in a database and identified. Method: The calculation of insilico peptide masses is done through the ExPASy PeptideMass and these masses are used to identify the peptides from MASCOT online server. Anticancer probability is calculated from the iACP server, docking of active peptides is done by CABS-dock the server. Objective: The purpose of the study is to identify the peptides having anti-cancerous properties by in-silico peptide mass fingerprinting. Results : The anti-cancerous peptides are identified with the MASCOT peptide mass fingerprinting server, the identified peptides are screened and only the anti-cancer are selected. De novo peptide structure prediction is used for 3D structure prediction by PEP-FOLD 3 server. The docking results confirm strong bonding with the interacting amino acids of the receptor protein of breast cancer BRCA1 which shows the best peptide binding to the Active chain, the human leukemia protein docking with peptides shows the accurate binding. Conclusion : These peptides are stable and functional and are the best way for the treatment of cancer and many other deadly diseases.

Download Full-text

Covalent protein display on Hepatitis B core-like particles in plants through the in vivo use of the SpyTag/SpyCatcher system

Scientific Reports ◽

10.1038/s41598-020-74105-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hadrien Peyret ◽

Daniel Ponndorf ◽

Yulia Meshcheriakova ◽

Jake Richardson ◽

George P. Lomonossoff

Keyword(s):

Hepatitis B ◽

Cost Saving ◽

Capsid Protein ◽

Recombinant Proteins ◽

Nicotiana Benthamiana ◽

Vaccine Development ◽

Binding Partner ◽

Gfp Fluorescence ◽

Display Systems

Abstract Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.

Download Full-text

Development of a potent embryonic chick lens model for studying congenital cataracts in vivo

Communications Biology ◽

10.1038/s42003-021-01849-0 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Zhen Li ◽

Sumin Gu ◽

Yumeng Quan ◽

Kulandaiappan Varadaraj ◽

Jean X. Jiang

Keyword(s):

Light Transmission ◽

Genetic Diseases ◽

Gene Mutations ◽

Underlying Mechanism ◽

Dominant Negative ◽

Lens Model ◽

Embryonic Chick ◽

Congenital Cataracts ◽

Chick Lens

AbstractCongenital cataracts are associated with gene mutations, yet the underlying mechanism remains largely unknown. Here we reported an embryonic chick lens model that closely recapitulates the process of cataract formation. We adopted dominant-negative site mutations that cause congenital cataracts, connexin, Cx50E48K, aquaporin 0, AQP0R33C, αA-crystallin, CRYAA R12C and R54C. The recombinant retroviruses containing these mutants were microinjected into the occlusive lumen of chick lenses at early embryonic development. Cx50E48K expression developed cataracts associated with disorganized nuclei and enlarged extracellular spaces. Expression of AQP0R33C resulted in cortical cataracts, enlarged extracellular spaces and distorted fiber cell organization. αA crystallin mutations distorted lens light transmission and increased crystalline protein aggregation. Together, retroviral expression of congenital mutant genes in embryonic chick lenses closely mimics characteristics of human congenital cataracts. This model will provide an effective, reliable in vivo system to investigate the development and underlying mechanism of cataracts and other genetic diseases.

Download Full-text