scholarly journals Activation Process of Dipteran-Specific Insecticidal Protein Produced by Bacillus thuringiensissubsp. israelensis

1999 ◽  
Vol 65 (8) ◽  
pp. 3464-3469 ◽  
Author(s):  
Masashi Yamagiwa ◽  
Motoyuki Esaki ◽  
Kanao Otake ◽  
Manabu Inagaki ◽  
Tohru Komano ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Culex Pipiens ◽  
Insecticidal Protein ◽  
Activation Process ◽  
Mosquito Larvae ◽  
In Vitro Processing ◽  
Significant Toxicity

ABSTRACT Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp.israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathioneS-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST–20-kDa fusion protein (GST-20) nor the GST–45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST–60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.

Preclinical Research on Idiotype Vaccine Against B Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4744-4744
Author(s):  
Fuxu Wang ◽  
Bing Zhao ◽  
Ling Pan ◽  
Xuejun Zhang ◽  
Jianmin Luo ◽  
...  
Keyword(s):  
Fusion Protein ◽  
B Cell ◽  
Fusion Proteins ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Host Cells ◽  
Fusion Genes ◽  
Preclinical Research

Abstract The idiotype (Id) of immunoglobulin expressed by B cell lymphoma can serve as the only widely accepted tumor associated antigen. But the Id vaccines have failed to elicit anti-tumor immunity for its weak immunogenic. Monocyte chemoattractant protein-3 (MCP3) can recruit various subsets of immune cells, such as DCs, which would uptake and properly process and present Id, activating both arms of the immune system, humoral and cellular. So the Id-MCP3 fusion proteins are potential vaccines for immunotherapy of B cell lymphoma. In this study, two vaccine candidates were constructed by fusing allogeneous MCP3 to the amino-(MCP3-scFv) or carboxyl-(scFv-MCP3) terminus of the A20 (BABL/c murine B-lymphocyte) Id scFv with a flexible polypeptide spacer encoding NDAQAPKS to prevent dissociation and keep their respective natural construction and function. And VH and VL domains were linked with a current linker encoding (Gly4Ser)3. Firstly, the cDNAs of Ig VH and Ig VL were amplified by RT-PCR from A20 mRNA, and then assembled into scFv by recombinant PCR method. Secondly the fusion genes of scFv/MCP3 were formed using the same method. After sequencing, MCP3/scFv fusion genes were cloned into pET-39b vector. Lastly MCP3/scFv fusion proteins were expressed in E.coli BL21. And the fusion protein is about 62 kD. We found that, under the same condition, MCP3-scFv fusion protein was expressed successfully and accounted for 40% of the total protein of the bacteria but not scFv-MCP3. Our result indicated that fusing MCP3 to carboxyl-terminus of scFv protein may have cytotoxicity to the host cells or maybe not stable inside the host cells. Next we will determine the activity of the fusion protein MCP3-scFv with cell-chemotatic-experiment in vitro and bearing-tumor mice experiment in vivo. Once the results would suggest that there may be an anti-tumor effect, we can make individual vaccines to lead to a better survival.

Blockade Of CD47 Using SIRPαFc: Role Of The Fc Region In Anti-Leukemic Activity and Tolerability

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3935-3935 ◽  
Author(s):  
Robert A. Uger ◽  
Xinli Pang ◽  
Mark Wong ◽  
Violetta House ◽  
Karen Dodge ◽  
...  
Keyword(s):  
Stem Cell ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Research Funding ◽  
Cell Therapeutics ◽  
Human Igg1 ◽  
Effector Activity ◽  
Stem Cell Therapeutics

Abstract Introduction CD47 binds to SIRPα on the surface of macrophages and delivers a “do not eat” signal that suppresses phagocytosis. There is increasing evidence that acute myeloid leukemia (AML) stem cells exploit the CD47-SIRPα pathway to escape macrophage-mediated destruction. Blockade of CD47 using a soluble SIRPα-Fc fusion protein (SIRPαFc) has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of AML cells. However, little information is available regarding the optimal structure of SIRPαFc. In particular, the influence of the Fc region, which can mediate antibody-dependent cellular cytotoxicity and complement activation, on anti-leukemic activity and toxicity has not been explored. Results We have generated three unique human SIRPαFc fusion proteins that vary in their Fc regions: SIRPα-G1, which contains the Fc region from human IgG1 with full effector activity; SIRPα-G4, bearing the Fc region from human IgG4, which has low effector activity; and SIRPα-G4m, which possesses a mutated human IgG4 Fc region that is devoid of any effector activity. These three fusion proteins were tested for their ability to promote macrophage-mediated phagocytosis of patient-derived AML cells in vitro. Although all three proteins were able to stimulate tumor cell destruction, SIRPα-G4m was clearly the least potent, while SIRPα-G1 and SIRPα-G4 showed similar activity. Next, the anti-leukemic activity of the fusion proteins was assessed in an AML xenograft model in NOD.SCID mice. SIRPα-G1 induced a profound anti-leukemic effect and was superior to both SIRPα-G4 and SIRPα-G4m, particularly with respect to eradicating tumor cells within the transplanted femur. Thus, while only a low level of Fc activity was required for maximal pro-phagocytic activity in vitro, full effector activity (human IgG1) provided superior anti-leukemic activity in vivo. The strong anti-tumor activity of this fusion protein presumably results from the simultaneous delivery of a positive macrophage activating signal (through Fc receptors) and blockade of the negative “do not eat” signal from CD47. Increased Fc effector activity could also carry the risk of increased toxicity. Since human SIRPα has no measurable binding to mouse CD47, to assess tolerability in mice we generated a surrogate fusion protein consisting of NOD mouse SIRPα linked to a mouse IgG2a Fc region with full effector function (mSIRPα-G2a). Repeat administration of high dose mSIRPα-G2a to mice (50 mg/kg IP twice per week for 8 weeks) produced no adverse clinical effects. No abnormalities were observed in hematological parameters, (including erythrocyte, platelet and leukocyte counts) or bone marrow CD150+CD48- LSK hematopoietic stem cells, nor were gross or microscopic changes noted in any tissue. Furthermore, taking advantage of a fortuitous cross-reactivity between NOD SIRPα and human CD47, we conducted a xenograft study with patient-derived AML cells using the mSIRPα-G2a fusion protein. Compared to control Fc, mSIRPα-G2a profoundly reduced leukemic burden in both the injected femur and non-injected bone marrow at doses significantly below the 50 mg/kg used in the tolerability studies. Thus, a mouse surrogate fusion that can bind both human CD47 on xenograft AML cells and endogenous CD47 on host tissue is both safe and effective. A pilot repeat-dose toxicity study using various human SIRPαFc proteins is currently underway in non-human primates. Conclusions These results demonstrate that SIRPαFc fusion proteins that combine Fc activity with CD47 blockade lead to effective AML destruction in vitro and in vivo, and are well tolerated in mice. Thus the therapeutic window in a homologous model system appears to be sufficiently wide to proceed with formal IND-enabling studies. On the basis of these findings we are moving forward with the development of a SIRPαFc therapeutic for the treatment of AML. Disclosures: Uger: Trillium Therapeutics/Stem Cell Therapeutics: Employment. Pang:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Wong:Trillium Therapeutics/Stem Cell Therapeutics: Employment. House:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Dodge:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Viau:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Vigo:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Tam:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Truong:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Jin:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Malko:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Ho:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Prasolava:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Danska:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Wang:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Petrova:Trillium Therapeutics/Stem Cell Therapeutics: Employment.

Erythropoietin Fusion Proteins Comprised of Identical Head-to-Tail Repeats with Enhanced Biological Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1162-1162
Author(s):  
Jee-Yeong Jeong ◽  
Changmin Chen ◽  
Kerry L. Davis ◽  
Andreas Breidbach ◽  
Don H. Catlin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Cho Cells ◽  
Half Life ◽  
Fusion Proteins ◽  
Biological Activities ◽  
Cmv Promoter ◽  
Equivalent Unit

Abstract Recombinant human erythropoietin (EPO, epoetin) is used widely for treatment of chronic anemia due to renal failure, cancer, and other causes. However, considerably high and frequent doses of EPO are required to maintain therapeutic effectiveness, since it has a relatively short in vivo half-life. Thus, alternatives with higher efficacy and/or longer half-life are being developed. We have shown previously that EPO-dimers, either produced by chemical cross-linking of monomeric EPO or expressed as a recombinant fusion protein from COS cells, exhibit enhanced biological properties in vitro and in vivo (Sytkowski, et.al. Proc. Natl. Acad. Sci. USA 95, 1184; Sytkowski, et.al. J. Biol. Chem. 274, 24773). We now report increased activities of EPO-dimer fusion protein and EPO-trimer fusion protein comprised of identical head-to-tail repeats and a 15 or 20-amino acid linker (for dimer), or 17-amino acid linkers (for trimer) produced from stably transfected CHO cells. EPO-fusion proteins were expressed under a CMV promoter with a signal peptide present on the first monomer coding sequence. The EPO-dimer fusion protein was connected with either three or four repeats of Gly-Gly-Gly-Gly-Ser as a 15 or 20-amino acid linker sequence, respectively. The expression levels of EPO-dimer fusion protein from cloned CHO cells to supernatant of protein-free medium ranged from 4 to 40 mg/L determined by EPO-ELISA, and from 2.0×105 to 4.5×106 IU/L determined by in vitro bioassay. We selected clones producing EPO-dimer fusion protein with the greatest extent of glycosylation, as indicated by SDS-PAGE and isoelectric focusing. Subcutaneous injection of mice with three doses of EPO-dimer fusion protein resulted in percent increases in mean hematocrit of 32.6% (300 IU/kg) or 18.2% (100 IU/kg), while equivalent unit doses of EPO-monomer increased mean hematocrit by 12.5% (300 IU/kg) or 6.4% (100 IU/kg). Moreover, a single dose of EPO-dimer fusion protein (100 IU/kg) increased their mean hematocrit by 4.3% within 7 days, while an equivalent unit dose of EPO-monomer had no effect. Importantly, three doses of EPO-trimer fusion protein increased their mean hematocrit by 8.83% per IU injected, which was much greater than that observed with EPO-monomer (0.69%) or EPO-dimer fusion protein (1.81%). The results show that EPO-fusion proteins exhibit biological activities superior to those of EPO-monomer, suggesting important therapeutic advantages.

scholarly journals The therapeutic effect of a novel anti-TNF-α/IL-6R triple-specific fusion protein under experimental septic condition

2021 ◽  
Author(s):  
Xiaole Chen ◽  
Shuangyu Tan ◽  
Mengru Yan ◽  
Kaimei Nie ◽  
Qingmei Zheng ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Expression System ◽  
Cecal Ligation ◽  
Mouse Fibroblast ◽  
Potential Candidate ◽  
Tnf Α ◽  
Prokaryotic Expression System

Abstract A novel anti-TNF-α/IL-6R triple-specific fusion protein, by linking 3 single domain chains, was designed and constructed in our lab. The high purity fusion proteins were obtained by our developed prokaryotic expression system process with high binding affinity with TNF-α (94.75 pM), Human Serum Albumin (1.83 nM) and IL-6R (2.29 nM). In this study, the anti-TNF-α/IL-6R triple-specific fusion protein protected the mouse fibroblast fibrosarcoma cell line (L929) from the apoptosis effects induced by TNF-α, establishing that the expressed fusion proteins can selectively combine with TNF-α in vitro. In vivo, the survival rate of cecal ligation and puncture (CLP) was notably increased in the group with anti-TNF-α/IL-6R triple-specific fusion protein treatment, and meaningfully higher compared with the single-targeted IL-6R and TNF-α fusion protein at the same dose. After the treatment with anti-TNF-α/IL-6R triple-specific fusion protein, the level of serum TNF-α, IL-1β and IL-6 were significantly decreased, and sepsis-induced pathological injuries in the kidney were remarkably attenuated. The anti-TNF-α/IL-6R triple-specific fusion protein can be the potential candidate for the development of new drug design against sepsis.

scholarly journals Expression of Pokeweed Antiviral Protein Isoform S1 (PAP-S1) and of Ricin-A-Chain/PAP-S1 novel fusion protein (RTA/PAP-S1) in Escherichia coli and their comparative inhibition of protein synthesis in vitro

2017 ◽  
Author(s):  
Yasser Hassan ◽  
Sherry Ogg
Keyword(s):  
Protein Synthesis ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Antiviral Protein ◽  
Developmental Potential ◽  
E Coli ◽  
Protein Isoform ◽  
A Chain

AbstractFusion protein therapeutics engineering is advancing to meet the need for novel medicine. Herein, we further characterize the development of novel RTA & PAP-S1 antiviral fusion proteins. In brief, RTA/PAP-S1 and PAP-S1/RTA fusion proteins were produced in both cell free and E. coli in vivo expression systems, purified by His-tag affinity chromatography, and protein synthesis inhibitory activity assayed by comparison to the production of a control protein, CalmL3. Results showed that the RTA/PAP-S1 fusion protein is amenable to standardized production and purification and has both increased potency and less toxicity compared to either RTA or PAP-S1 alone. Thus, this research highlights the developmental potential of novel fusion proteins with reduced cytotoxic risk and increased potency.

scholarly journals Incorporation of GP64 into Helicoverpa armigera nucleopolyhedrovirus enhances virus infectivity in vivo and in vitro

2012 ◽  
Vol 93 (12) ◽  
pp. 2705-2711 ◽  
Author(s):  
Shu Shen ◽  
Yinyin Gan ◽  
Manli Wang ◽  
Zhihong Hu ◽  
Hualin Wang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Helicoverpa Armigera ◽  
Fusion Proteins ◽  
Virus Infectivity ◽  
F Protein ◽  
Recombinant Viruses ◽  
Group I ◽  
Helicoverpa Armígera

The envelope fusion proteins of baculoviruses, glycoprotein GP64 from group I nucleopolyhedrovirus (NPV) or the F protein from group II NPV and granulovirus, are essential for baculovirus morphogenesis and infectivity. The F protein is considered the ancestral baculovirus envelope fusion protein, while GP64 is a more recent evolutionary introduction into baculoviruses and exhibits higher fusogenic activity than the F protein. Each of the fusion proteins is required by the respective virus to spread infection within larval tissues. A recombinant Helicoverpa armigera NPV (HearNPV) expressing GP64 from Autographa californica multiple nucleopolyhedrovirus, vHaBac-gp64-egfp, was constructed, which still retained the native F protein, and its infectivity was assayed in vivo and in vitro. Analyses by one-step growth curve to determine viral titre and by quantitative PCR to determine viral DNA copy number showed that vHaBac-gp64-egfp was more infectious in vitro than the control, vHaBac-egfp. The polyhedrin gene (polh) was reintroduced into the recombinant viruses and bioassays showed that vHaBac-gp64-polh accelerated the mortality of infected larvae compared with the vHaBac-egfp-polh control, and the LC50 (median lethal concentration) of vHaBac-gp64-polh was reduced to approximately 20 % of that of vHaBac-egfp-polh. Therefore, incorporation of GP64 into HearNPV budded virions improved virus infectivity both in vivo and in vitro. The construction of this bivalent virus with a more efficient fusion protein could improve the use of baculoviruses in different areas such as gene therapy and biocontrol.

scholarly journals Specific Enzyme-Linked Immunosorbent Assays for Quantitation of Antibody-Cytokine Fusion Proteins

1999 ◽  
Vol 6 (2) ◽  
pp. 236-242 ◽  
Author(s):  
J. Gan ◽  
K. Kendra ◽  
M. Ricci ◽  
J. A. Hank ◽  
S. D. Gillies ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Interleukin 2 ◽  
Intact Protein ◽  
Mouse Serum ◽  
Clinical Testing ◽  
Immunosorbent Assays ◽  
Macrophage Colony

ABSTRACT Preliminary testing has shown in vitro and in vivo that antitumor activity can be obtained with fusion proteins linking tumor-reactive monoclonal antibodies to cytokines, such as granulocyte-macrophage colony-stimulating factor or interleukin 2 (IL-2). Preclinical and clinical testing of these reagents requires their in vitro and in vivo quantitation and pharmacokinetic evaluation. We have focused on the detection of a fusion protein which links one human IL-2 molecule to the carboxy terminus of each heavy chain of the tumor-reactive human-mouse chimeric anti-GD2 antibody, ch14.18. We have developed enzyme-linked immunosorbent assays (ELISAs) to evaluate intact tumor-reactive fusion proteins. By these ELISAs we can reliably measure nanogram quantities of intact ch14.18-IL-2 fusion protein and distinguish the intact protein from its components (ch14.18 and IL-2) in buffer, mouse serum, and human serum with specificity and reproducibility. The measurement of intact ch14.18-IL-2 fusion protein is not confounded by free IL-2 or free ch14.18 when 100 ng or less of total immunoglobulin per ml is used during the assay procedure. Our results indicate that these ELISAs are suitable for preclinical and clinical testing and with slight modifications are applicable to the analysis of a variety of other fusion proteins.

scholarly journals Expression of Pokeweed Antiviral Protein Isoform S1 (PAP-S1) and of Ricin-A-Chain/PAP-S1 Novel Fusion Protein (RTA/PAP-S1) in Escherichia coli and Their Comparative Inhibition of Protein Synthesis in Vitro

2017 ◽  
Author(s):  
Yasser Hassan ◽  
Sherry Ogg
Keyword(s):  
Protein Synthesis ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Antiviral Protein ◽  
Developmental Potential ◽  
E Coli ◽  
Protein Isoform ◽  
A Chain

Fusion protein therapeutics engineering is advancing to meet the need for novel medicine. Herein, we further characterize the development of novel RTA & PAP-S1 antiviral fusion proteins. In brief, RTA/PAP-S1 and PAP-S1/RTA fusion proteins were produced in both cell free and E. coli in vivo expression systems, purified by His-tag affinity chromatography, and protein synthesis inhibitory activity assayed by comparison to the production of a control protein, CalmL3. Results showed that the RTA/PAP-S1 fusion protein is amenable to standardized production and purification and has both increased potency and less toxicity compared to either RTA or PAP-S1 alone. Thus, this research highlights the developmental potential of novel fusion proteins with reduced cytotoxic risk and increased potency.

scholarly journals MiR-4448 is involved in deltamethrin resistance by targeting CYP4H31 in Culex pipiens pallens

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xixi Li ◽  
Shengli Hu ◽  
Haitao Yin ◽  
Hongbo Zhang ◽  
Dan Zhou ◽  
...  
Keyword(s):  
Culex Pipiens ◽  
Oral Feeding ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Culex Pipiens Pallens ◽  
Deltamethrin Resistance ◽  
Regulatory Functions ◽  
Pipiens Pallens

Abstract Background Culex pipiens (Cx. pipiens) complex, which acts as a vector of viruses and is widespread and abundant worldwide, including West Nile virus, Japanese encephalitis virus, and Sindbis virus, can cause serious vector-borne diseases affecting human health. Unfortunately, mosquitoes have developed deltamethrin resistance because of its long-term overuse, representing a major challenge to mosquito control. Understanding the molecular regulatory mechanisms of resistance is vital to control mosquitoes. MicroRNAs (miRNAs) are short non-coding RNAs that have been demonstrated to be important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. In the present study, we aimed to investigate the regulatory functions of miR-4448 and CYP4H31 in the formation of insecticidal resistance in mosquito Culex pipiens pallens. Methods We used quantitative real-time reverse transcription PCR to measure miR-4448 and CYP4H31 (encoding a cytochrome P450) expression levels. The regulatory functions of miR-4448 and CYP4H31 were assessed using dual-luciferase reporter assays. Then, oral feeding, RNA interference, and the American Centers for Disease Control and Prevention bottle bioassay were used to determine miR-4448’s association with deltamethrin resistance by targeting CYP4H31in vivo. Cell Counting Kit-8 (CCK-8) was also used to detect the viability of pIB/V5-His-CYP4H31-transfected C6/36 cells after deltamethrin treatment in vitro. Results MiR-4448 was downregulated in the deltamethrin-resistant strain (DR strain), whereas CYP4H31 was downregulated in deltamethrin-susceptible strain. CYP4H31 expression was downregulated by miR-4448 recognizing and binding to its 3′ untranslated region. Functional verification experiments showed that miR-4448 overexpression resulted in lower expression of CYP4H31. The mortality of miR-4448 mimic-injected DR strain mosquitoes was higher than that of the controls. CCK-8 assays showed that CYP4H31 decreased cellular resistance to deltamethrin in vitro and the mortality of the DR strain increased when CYP4H31 was knocked down in vivo. Conclusions In mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.

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