scholarly journals A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

10.3791/52911 ◽  
2015 ◽  
Author(s):  
Farokh Dotiwala ◽  
Isabelle Fellay ◽  
Luis Filgueira ◽  
Denis Martinvalet ◽  
Judy Lieberman ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
High Yield ◽  
Efficient Expression ◽  
Cost Efficient

scholarly journals Efficient Expression of Acetylcholine-Binding Protein fromAplysia californicain Bac-to-Bac System

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Bo Lin ◽  
Hailing Meng ◽  
Hui Bing ◽  
Dongting Zhangsun ◽  
Sulan Luo
Keyword(s):  
Cell Density ◽  
Lymnaea Stagnalis ◽  
Expression System ◽  
Volume Ratio ◽  
Baculovirus Expression System ◽  
High Yield ◽  
Factors Affecting ◽  
Functional Studies ◽  
Efficient Expression ◽  
Inoculum Volume

The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea haresAplysia californica(Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 106 cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP fromLymnaea stagnalissuccessfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

scholarly journals Expression of functional recombinant human procathepsin B in mammalian cells

1996 ◽  
Vol 319 (3) ◽  
pp. 793-800 ◽  
Author(s):  
Wei-Ping REN ◽  
Rafael FRIDMAN ◽  
James R. ZABRECKY ◽  
Leticia D. MORRIS ◽  
Nancy A DAY ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Mammalian Cells ◽  
Expression System ◽  
Molecular Size ◽  
Single Step ◽  
Surface Proteins ◽  
High Yield ◽  
Virus Expression ◽  
Substrate Inhibitor

Cathepsin B has been implicated in numerous pathobiological processes. In order to study its interactions with other proteins implicated in these processes, quantities of functional recombinant cathepsin B are needed. Therefore, we expressed recombinant human procathepsin B in mammalian cells (BSC-1 monkey kidney cells and HeLa human cervical carcinoma cells) using a vaccinia virus expression system. The recombinant human procathepsin B appeared to be authentic and expressed in its native conformation as indicated by: (1) N-terminal sequencing; (2) molecular size; (3) processing intracellularly to mature double-chain cathepsin B; (4) in vitro cleavage by pepsin to mature cathepsin B coincident with appearance of activity against a selective synthetic substrate; and (5) substrate/inhibitor profiles. This is the first report of the expression of functional recombinant human procathepsin B in mammalian cells. We also report a single-step immunoaffinity purification procedure for the isolation of electrophoretically pure proenzyme. By the methodologies described, human procathepsin B can now be obtained in high yield. This should facilitate studies of its interactions with protease inhibitors, other proteases, extracellular matrices, cell-surface proteins and biological substrates that may be of relevance to the pathobiological functions of this enzyme.

scholarly journals A Novel and Efficient High-Yield Method for Preparing Bacterial Ghosts

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 420
Author(s):  
Yi Ma ◽  
Liu Cui ◽  
Meng Wang ◽  
Qiuli Sun ◽  
Kaisheng Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Expression System ◽  
High Yield ◽  
Wild Type ◽  
High Quality ◽  
Efficient Protection ◽  
Bacterial Ghosts ◽  
Cell Envelopes ◽  
Extracellular Structures ◽  
First Time

Bacterial ghosts (BGs) are empty cell envelopes possessing native extracellular structures without a cytoplasm and genetic materials. BGs are proposed to have significant prospects in biomedical research as vaccines or delivery carriers. The applications of BGs are often limited by inefficient bacterial lysis and a low yield. To solve these problems, we compared the lysis efficiency of the wild-type protein E (EW) from phage ΦX174 and the screened mutant protein E (EM) in the Escherichia coli BL21(DE3) strain. The results show that the lysis efficiency mediated by protein EM was improved. The implementation of the pLysS plasmid allowed nearly 100% lysis efficiency, with a high initial cell density as high as OD600 = 2.0, which was higher compared to the commonly used BG preparation method. The results of Western blot analysis and immunofluorescence indicate that the expression level of protein EM was significantly higher than that of the non-pLysS plasmid. High-quality BGs were observed by SEM and TEM. To verify the applicability of this method in other bacteria, the T7 RNA polymerase expression system was successfully constructed in Salmonella enterica (S. Enterica, SE). A pET vector containing EM and pLysS were introduced to obtain high-quality SE ghosts which could provide efficient protection for humans and animals. This paper describes a novel and commonly used method to produce high-quality BGs on a large scale for the first time.

pH-sensing nano-crystals of carbonate apatite: Effects on intracellular delivery and release of DNA for efficient expression into mammalian cells

Gene ◽  
2006 ◽  
Vol 376 (1) ◽  
pp. 87-94 ◽  
Author(s):  
E.H. Chowdhury ◽  
A. Maruyama ◽  
A. Kano ◽  
M. Nagaoka ◽  
M. Kotaka ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Intracellular Delivery ◽  
Carbonate Apatite ◽  
Ph Sensing ◽  
Efficient Expression

scholarly journals Mitigating Future Respiratory Virus Pandemics: New Threats and Approaches to Consider

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 637
Author(s):  
Gregory C. Gray ◽  
Emily R. Robie ◽  
Caleb J. Studstill ◽  
Charles L. Nunn
Keyword(s):  
Pathogen Detection ◽  
Respiratory Virus ◽  
Emerging Infectious Diseases ◽  
Health Resources ◽  
Mitigation Strategies ◽  
High Yield ◽  
Animal Virus ◽  
Virus Surveillance ◽  
Human Animal ◽  
Cost Efficient

Despite many recent efforts to predict and control emerging infectious disease threats to humans, we failed to anticipate the zoonotic viruses which led to pandemics in 2009 and 2020. The morbidity, mortality, and economic costs of these pandemics have been staggering. We desperately need a more targeted, cost-efficient, and sustainable strategy to detect and mitigate future zoonotic respiratory virus threats. Evidence suggests that the transition from an animal virus to a human pathogen is incremental and requires a considerable number of spillover events and considerable time before a pandemic variant emerges. This evolutionary view argues for the refocusing of public health resources on novel respiratory virus surveillance at human–animal interfaces in geographical hotspots for emerging infectious diseases. Where human–animal interface surveillance is not possible, a secondary high-yield, cost-efficient strategy is to conduct novel respiratory virus surveillance among pneumonia patients in these same hotspots. When novel pathogens are discovered, they must be quickly assessed for their human risk and, if indicated, mitigation strategies initiated. In this review, we discuss the most common respiratory virus threats, current efforts at early emerging pathogen detection, and propose and defend new molecular pathogen discovery strategies with the goal of preempting future pandemics.

Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells

1983 ◽  
Vol 3 (6) ◽  
pp. 1123-1132
Author(s):  
Archibald S. Perkins ◽  
Paul T. Kirschmeier ◽  
Sebastiano Gattoni-Celli ◽  
I. Bernard Weinstein
Keyword(s):  
Mammalian Cells ◽  
High Efficiency ◽  
Leukemia Virus ◽  
Restriction Enzymes ◽  
Virus Genome ◽  
Opposite Polarity ◽  
High Yield ◽  
Animal Cells ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique Pst I site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt + colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR- gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt + phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

Expression of hepatitis B viral core region in mammalian cells

1986 ◽  
Vol 6 (5) ◽  
pp. 1393-1400
Author(s):  
M J Roossinck ◽  
S Jameel ◽  
S H Loukin ◽  
A Siddiqui
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Mammalian Cells ◽  
Expression System ◽  
Core Region ◽  
Core Antigen ◽  
Northern Blot Hybridization ◽  
The Core ◽  
B Virus ◽  
Nuclease Mapping

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.

scholarly journals Characterization and purification of human retinoic acid receptor-γ 1 overexpressed in the baculovirus-insect cell system

1992 ◽  
Vol 287 (3) ◽  
pp. 833-840 ◽  
Author(s):  
A P Reddy ◽  
J Y Chen ◽  
T Zacharewski ◽  
H Gronemeyer ◽  
J J Voorhees ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Retinoic Acid Receptor ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Sf9 Cells ◽  
Acid Receptor ◽  
Gel Retardation

The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.

scholarly journals A method for the generation of pseudotyped virus particles bearing SARS coronavirus spike protein in high yields

2021 ◽  
Author(s):  
Yoichiro Fujioka ◽  
Sayaka Kashiwagi ◽  
Aiko Yoshida ◽  
Aya O. Satoh ◽  
Mari Fujioka ◽  
...  
Keyword(s):  
Global Economy ◽  
Expression System ◽  
Antiviral Agents ◽  
Production Method ◽  
Spike Protein ◽  
High Yield ◽  
Pseudotyped Virus ◽  
Efficient Production ◽  
Live Virus ◽  
High Yields

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 Spike protein in high yield. We found that pseudovirions produced with the conventional transient expression system lacked coronavirus Spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus Spike protein allowed the efficient production of progeny pseudoviruses decorated with Spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.

scholarly journals Blocking Hepatitis C Virus Infection with Recombinant Form of Envelope Protein 2 Ectodomain

2009 ◽  
Vol 83 (21) ◽  
pp. 11078-11089 ◽  
Author(s):  
Jillian Whidby ◽  
Guaniri Mateu ◽  
Hannah Scarborough ◽  
Borries Demeler ◽  
Arash Grakoui ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Expression System ◽  
Developed Countries ◽  
Viral Envelope ◽  
Hcv Infection ◽  
Size Exclusion ◽  
Amino Terminal ◽  
Endosomal Acidification

ABSTRACT More than 120 million people worldwide are chronically infected with hepatitis C virus (HCV), making HCV infection the leading cause of liver transplantation in developed countries. Treatment is limited, and efficacy depends upon the infecting strain and the initial viral load. The HCV envelope glycoproteins (E1 and E2) are involved in receptor binding, virus-cell fusion, and entry into the host cell. HCV infection proceeds by endosomal acidification, suggesting that fusion of the viral envelope with cellular membranes is a pH-triggered event. E2 consists of an amino-terminal ectodomain, an amphipathic helix that forms a stem region, and a carboxy-terminal membrane-associating segment. We have devised a novel expression system for the production of a secreted form of E2 ectodomain (eE2) from mammalian cells and performed a comprehensive biochemical and biophysical characterization. eE2 is properly folded, as determined by binding to human CD81, blocking of infection of cell culture-derived HCV, and recognition by antibodies from patients chronically infected with different genotypes of HCV. The glycosylation pattern, number of disulfide bonds, oligomerization state, and secondary structure of eE2 have been characterized using mass spectrometry, size exclusion chromatography, circular dichroism, and analytical ultracentrifugation. These results advance the understanding of E2 and may assist in the design of an HCV vaccine and entry inhibitor.

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