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 Purification of cathepsin B by a new form of affinity chromatography

1986 ◽  
Vol 235 (3) ◽  
pp. 731-734 ◽  
Author(s):  
D H Rich ◽  
M A Brown ◽  
A J Barrett
Keyword(s):  
Affinity Chromatography ◽  
Cathepsin B ◽  
Single Step ◽  
Starting Material ◽  
High Yield ◽  
Cysteine Proteinases ◽  
New Form ◽  
Human Cathepsin ◽  
Sepharose 4B

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2′-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.

scholarly journals Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

2020 ◽  
Vol 175 (2) ◽  
pp. 251-265 ◽  
Author(s):  
Xilin Li ◽  
Si Chen ◽  
Xiaoqing Guo ◽  
Qiangen Wu ◽  
Ji-Eun Seo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Expression System ◽  
Cytochrome P450s ◽  
Mass Spectrometry Analysis ◽  
Established Cell Line ◽  
Micronucleus Formation ◽  
Tk6 Cells ◽  
Genotoxicity Testing

Abstract Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress 1 of 8 cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using 2 prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 cells with CYP1A1, 1B1, 2B6, and 2C19 expression as compared with EV controls. B[a]P significantly increased cytotoxicity, DNA damage, and chromosomal damage in TK6 cells overexpressing CYP1A1 and 1B1 when compared with EV controls. B[a]P also induced micronucleus formation in TK6 cells expressing CYP1A2. These results suggest that our CYP-expressing TK6 cell system can be used to detect the genotoxicity of compounds requiring metabolic transformation.

scholarly journals Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro

2020 ◽  
Vol 11 ◽  
Author(s):  
Balamurugan Shanmugaraj ◽  
Kaewta Rattanapisit ◽  
Suwimon Manopwisedjaroen ◽  
Arunee Thitithanyanont ◽  
Waranyoo Phoolcharoen
Keyword(s):  
Monoclonal Antibodies ◽  
Nicotiana Benthamiana ◽  
Specific Binding ◽  
Expression System ◽  
Protein A ◽  
Single Step ◽  
Affinity Column ◽  
Plant Expression ◽  
Plant Expression System

The ongoing coronavirus disease 2019 (COVID-19) outbreak caused by novel zoonotic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially reported in Wuhan city, Hubei Province of China, in late December 2019. The rapid global spread of the virus calls for the urgent development of vaccines or therapeutics for human applications to combat the coronavirus infection. Monoclonal antibodies (mAbs) have been utilized as effective therapeutics for treating various infectious diseases. In the present study, we evaluated the feasibility of plant expression system for the rapid production of recently identified therapeutically suitable human anti-SARS-CoV-2 mAbs B38 and H4. Transient co-expression of heavy-chain and light-chain sequences of both the antibodies by using plant expression geminiviral vector resulted in rapid accumulation of assembled mAbs in Nicotiana benthamiana leaves within 4 days post-infiltration. Furthermore, both the mAbs were purified from the plant crude extracts with single-step protein A affinity column chromatography. The expression level of mAb B38 and H4 was estimated to be 4 and 35 μg/g leaf fresh weight, respectively. Both plant-produced mAbs demonstrated specific binding to receptor binding domain (RBD) of SARS-CoV-2 and exhibited efficient virus neutralization activity in vitro. To the best of our knowledge, this is the first report of functional anti-SARS-CoV-2 mAbs produced in plants, which demonstrates the ability of using a plant expression system as a suitable platform for the production of effective, safe, and affordable SARS-CoV-2 mAbs to fight against the spread of this highly infectious pathogen.

scholarly journals Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

2006 ◽  
Vol 80 (16) ◽  
pp. 7832-7843 ◽  
Author(s):  
Ying-Chuan Lin ◽  
Ashraf Brik ◽  
Aymeric de Parseval ◽  
Karen Tam ◽  
Bruce E. Torbett ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Feline Immunodeficiency Virus ◽  
Ex Vivo ◽  
Expression System ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
Virus Mutant ◽  
Substrate Inhibitor ◽  
Hiv 1

ABSTRACT We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations.

scholarly journals An improved in vitro and in vivo Sindbis virus expression system through host and virus engineering

2009 ◽  
Vol 141 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Toey Nivitchanyong ◽  
Yien Che Tsai ◽  
Michael J. Betenbaugh ◽  
George A. Oyler
Keyword(s):  
Sindbis Virus ◽  
Expression System ◽  
Virus Expression

scholarly journals Generation of dual specific bivalent BiTEs (dbBIspecific T-cell Engaging antibodies) for cellular immunotherapy

2019 ◽  
Author(s):  
Maciej Kujawski ◽  
Lin Li ◽  
Supriyo Bhattacharya ◽  
Patty Wong ◽  
Wen-Hui Lee ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Antigen ◽  
Structural Model ◽  
Molecular Size ◽  
High Yield ◽  
Cellular Immunotherapy ◽  
Target Cells ◽  
Antigen Antibody

AbstractBispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. Here we show that an intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (db BiTES, 300 kDa). The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of the 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES maintain high in vivo tumor targeting as demonstrated by PET imaging, and redirect dbBiTE coated T-cells (1 microgram/10 million cells) to kill CEA+ target cells both in vitro, and in vivo in CEA transgenic mice.

scholarly journals Role of NH2- and COOH-Terminal Domains of the P Protein of Human Parainfluenza Virus Type 3 in Transcription and Replication

2000 ◽  
Vol 74 (13) ◽  
pp. 5886-5895 ◽  
Author(s):  
Bishnu P. De ◽  
Michael A. Hoffman ◽  
Suresh Choudhary ◽  
Clayton C. Huntley ◽  
Amiya K. Banerjee
Keyword(s):  
Expression System ◽  
Recombinant Vaccinia Virus ◽  
Luciferase Expression ◽  
Terminal Domain ◽  
Amino Acid Region ◽  
P Protein ◽  
Virus Expression ◽  
Transcription And Replication

ABSTRACT The phosphoproteins (P proteins) of paramyxoviruses play a central role in transcription and replication of the viruses by forming the RNA polymerase complex L-P and encapsidation complex (N-P) with nucleocapsid protein (N) and binding to N protein-encapsidated genome RNA template (N-RNA template). We have analyzed the human parainfluenza virus type 3 (HPIV3) P protein and deletion mutants thereof in an in vitro transcription and in vivo replication system. The in vitro system utilizes purified N-RNA template and cell extract containing L and P proteins coexpressed via plasmids using a recombinant vaccinia virus expression system. The in vivo system takes advantage of minigenome replication, which measures luciferase reporter gene expression from HPIV3 minigenomes by viral proteins in a recombinant vaccinia virus expression system. These studies revealed that the C-terminal 20-amino-acid region of P is absolutely required for transcription in vitro and luciferase expression in vivo, suggesting its critical role in viral RNA synthesis. The N-terminal 40-amino-acid region, on the other hand, is essential for luciferase expression but dispensable for transcription in vitro. Consistent with these findings, the C-terminal domain is required for binding of P protein to the N-RNA template involved in both transcription and replication, whereas the N-terminal domain is required for the formation of soluble N-P complex involved in encapsidation of nascent RNA chains during replication. Coimmunoprecipitation analysis showed that the P protein forms a stable homooligomer (perhaps a trimer) that is present in L-P and N-P complexes in the higher oligomeric forms (at least a pentamer). Interestingly, coexpression of a large excess of N- or C-terminally deleted P with wild-type P had no effect on minigenome replication in vivo, notwithstanding the formation of heterooligomeric complexes. These data indicate that P protein with a deleted terminal domain can function normally within the P heterooligomeric complex to carry out transcription and replication in vivo.

scholarly journals Preparation of a recombinant chimaera of insulin-like growth factor II and interleukin 3 with high proliferative potency for haemopoietic cells

1997 ◽  
Vol 326 (2) ◽  
pp. 407-413 ◽  
Author(s):  
Marcos R. DIFALCO ◽  
L. Fernando CONGOTE
Keyword(s):  
Growth Factor ◽  
Insect Cells ◽  
Human Peripheral Blood ◽  
Biological Activities ◽  
Expression System ◽  
High Yield ◽  
Insulin Like Growth Factor ◽  
Interleukin 3 ◽  
Factor Ii

We have found that a slightly modified insulin-like growth factor II (IGF II) consisting of a chimaera of bombyxin and human IGF II (BOMIGF) is properly secreted in insect cells by using the baculovirus expression system. Human interleukin 3 (IL-3) was attached to the C-terminal amino acid residue of BOMIGF with peptide linkers containing five or twelve residues. Only the chimaera with the 12-residue linker had biological activities of both IGF II and IL-3. The chimaera had a significantly higher mitogenic activity than IL-3 in cell cultures of the human haemopoietic cell line TF-1 and its effect could be observed even at femtomolar concentrations. It was also able to stimulate thymidine incorporation in IGF II-dependent bovine fetal erythroid cells. The chimaera significantly increased the number of macroscopic haemopoietic colonies in cultures of human peripheral blood in comparison with IL-3 or mixtures of IL-3 and BOMIGF in vitro. Subcutaneous injection of a BOMIGF–mouse IL-3 chimaera in normal C57BL/6 mice resulted in a significant increase of the number of spleen stem cells producing macroscopic haemopoietic colonies. This new system for the biosynthesis of IGF–cytokine fusion proteins in insect cells might prove advantageous for the low-cost and high-yield production of molecules with complementary or synergistic biological activities.

In vitro expression of a mouse tissue specific glutathione-peroxidase-like protein lacking the selenocysteine can protect stably transfected mammalian cells against oxidative damage

1996 ◽  
Vol 74 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Patrick Vernet ◽  
Nicole Rigaudiére ◽  
Norbert Ghyselinck ◽  
Jean Pierre Dufaure ◽  
Joël R. Drevet
Keyword(s):  
Glutathione Peroxidase ◽  
Mammalian Cells ◽  
Expression System ◽  
Complete Sequence ◽  
Protein Domain ◽  
Mouse Tissue ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Mouse Epididymis

The complete sequence of the mouse epididymal protein (MEP24) was cloned. It contains a 663 bp open-reading frame that, after conceptual translation, shows extensive identity with proteins belonging to the glutathione peroxidase (GPX) family. However, a major difference between GPX5 (MEP24) and other known GPXs concerns a protein domain known to be critical for GPX function. To find out what could be the physiological function of such a protein in the mouse epididymis, we have used a mammalian expression system to overexpress the GPX5 protein. Cells constitutively expressing the GPX5 protein were generated and assayed for their ability to metabolize regular substrates of GPX enzymes. Data presented here show that the GPX5-expressing cells can metabolize hydrogen peroxide in a manner that is consistent with a peroxidase activity. However, the substrate preference of the GPX5-expressing cells and their apparent insensitivity to a regular inhibitor of GPX enzymes suggest that the GPX5 protein belongs to a particular class of GPX proteins. Involvement of this protein in the physiology of the mouse epididymis is discussed.Key words: glutathione peroxidase (GPX), mouse epididymis, MEP24, GPX5 cDNA, selenocysteine, GPX5 polyclonal antibody, spermatozoa, CHO-KI cells.

Indipendent and Tandem Expression of a Novel Antimicrobial Peptides Plectasin in Escherichia coli

2011 ◽  
Vol 345 ◽  
pp. 134-138 ◽  
Author(s):  
Li Hui Lv ◽  
Xue Gang Luo ◽  
Meng Ni ◽  
Xiao Lan Jing ◽  
Nan Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Expression System ◽  
High Yield ◽  
Gene Synthesis ◽  
Post Translational Modification ◽  
Iptg Induction ◽  
E Coli ◽  
Conventional Antibiotics

Plectasin, a novel antimicrobial peptide, is isolated from a saprophytic fungus Pseudoplectania nigrella. Plectasin showed potent antibacterial activity in vitro against Gram-positive, especially the Streptococcus pneumoniae and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In our previous study, plectasin had been expressed at a high yield as a thioredoxin (Trx) – fused protein in Escherichia coli. However, it couldn’t exhibit the antimicrobial activity unless the Trx-tag had been cleaved, which made the producing process be complicated. Concerning that plectasin has no complex post-translational modification and toxicity on E. coli, on the basis of the former works, we further establish the independent and tandem expression system of plectasin in E. coli. In the present study, the coding sequence of plectasin was obtained from pET32a-PLEC with four primers to amplify the independent and tandem plectasin fragments by overlapping PCR-based gene synthesis, and then cloned into pET22b (+) vector. The recombinant protein was expressed successfully in E. coli with IPTG induction. These works might throw light on the production or study of plectasin, and contribute to the development of novel anti-infectious drugs in the future.

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