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 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 Expression of novel fusion antiviral proteins Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro

2018 ◽  
Author(s):  
Yasser Hassan ◽  
Sherry Ogg ◽  
Hui Ge
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Therapeutic Index ◽  
Antiviral Protein ◽  
E Coli ◽  
Protein Isoform ◽  
Antiviral Proteins ◽  
A Chain

AbstractRicin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with 50% protein synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and protein synthesis inhibition activity assayed. Results showed that the optimized protein RTA mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on protein synthesis inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infection.

scholarly journals A Neutralizing Antibody to the A Chain of Abrin Inhibits Abrin Toxicity both In Vitro and In Vivo

2008 ◽  
Vol 15 (5) ◽  
pp. 737-743 ◽  
Author(s):  
Kalpana Surendranath ◽  
Anjali A. Karande
Keyword(s):  
Monoclonal Antibody ◽  
Protein Synthesis ◽  
Neutralizing Antibody ◽  
Type Ii ◽  
Inhibit Protein Synthesis ◽  
Ribosome Inactivating Proteins ◽  
A Chain ◽  
Lethal Doses

ABSTRACT Plant ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that inhibit protein synthesis in cells. Abrin, a type II RIP, is an AB type toxin, which is one of the most lethal types of toxin known. The B chain facilitates the entry of the molecule into the cell, whereas the A chain exerts the toxic effect. We have generated hybridomas secreting antibodies of the immunoglobulin G class specific to the recombinant A chain of abrin. One monoclonal antibody, namely, D6F10, rescued cells from abrin toxicity. Importantly, the antibody also protected mice from lethal doses of the toxin. The neutralizing effect of the antibody was shown to be due to interference with abrin attachment to the cell surface.

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.

scholarly journals Production and Evaluation of an Avian IgY Immunotoxin against CD133+ for Treatment of Carcinogenic Stem Cells in Malignant Glioma: IgY Immunotoxin for the Treatment of Glioblastoma

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Elda-Georgina Chavez-Cortez ◽  
Gustavo Vargas Felix ◽  
Edgar Rangel López ◽  
Julio Sotelo ◽  
Carlos Martínez-Canseco ◽  
...  
Keyword(s):  
Stem Cells ◽  
Malignant Glioma ◽  
Potential Therapeutic Target ◽  
E Coli ◽  
Novel Approach ◽  
Original Goal ◽  
Design Production ◽  
A Chain

Background. Glioblastoma is the most common malignant tumor of Central Nervous System. Despite the research in therapeutics, the prognosis is dismal. Malignant glioma stem cells (MGSCs) are a major cause of treatment failure and increasing tumor recurrence. In general, cancer stem cells (CSCs) express prominin-1 (CD133), considered as a potential therapeutic target. In this study, we produced an avian immunotoxin directed against the subpopulation of CD133+ CSCs within a malignant glioma. We used the avian IgY because it has various advantages as increased affinity to mammal antigens and inexpensive obtention of large amounts of specific antibodies (approximately 1 mg/per egg). The design, production, purification and use of IgY anti CD133 immunotoxin constitute an original goal of this research.Methods. The immunodominant peptide of CD133 was designed to immunize hens; also, the extracellular domain of CD133 was cloned to probe the IgY antibodies. In parallel, a recombinant abrin A chain was produced inE. coliin order to join it to the Fc domain of the anti-CD133 IgY to conform the immunotoxin. This anti-CD133 IgY anti-tumor immunotoxin was testedin vitroandin vivo. Results. The cytotoxicity of the immunotoxinin vitroshowed that IgY-abrin immunotoxin reduced 55% cell viability. After subcutaneous MGSCs implantation, the animals treated intraperitoneally or intratumorally with the IgY-abrin immunotoxin showed more than 50% decrease of tumor volume.Conclusion. Results showed that the IgY-abrin immunotoxin had cytotoxic activity against CD133+ MGSCs and provides a novel approach for the immunotherapy of glioblastoma.

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 Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions.

1997 ◽  
Vol 41 (10) ◽  
pp. 2132-2136 ◽  
Author(s):  
D L Shinabarger ◽  
K R Marotti ◽  
R W Murray ◽  
A H Lin ◽  
E P Melchior ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Pathogenic Bacteria ◽  
Coli Strain ◽  
In Vitro Translation ◽  
E Coli ◽  
Initiation Phase ◽  
New Class ◽  
Bacterial Translation

The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

scholarly journals Production of a novel heterodimeric two-chain insulin-Fc fusion protein

2020 ◽  
Vol 33 ◽  
Author(s):  
Christine Faust ◽  
Christian Ochs ◽  
Marcus Korn ◽  
Ulrich Werner ◽  
Jennifer Jung ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Physiological Role ◽  
Peptide Hormone ◽  
In Vitro Assays ◽  
Fusion Partner ◽  
Insulin Analogs ◽  
Fc Fusion Protein ◽  
A Chain

Abstract Insulin is a peptide hormone produced by the pancreas. The physiological role of insulin is the regulation of glucose metabolism. Under certain pathological conditions the insulin levels can be reduced leading to the metabolic disorder diabetes mellitus (DM). For type 1 DM and, dependent on the disease progression for type 2 DM, insulin substitution becomes indispensable. To relieve insulin substitution therapy for patients, novel insulin analogs with pharmacokinetic and pharmacodynamic profiles aiming for long-lasting or fast-acting insulins have been developed. The next step in the evolution of novel insulins should be insulin analogs with a time action profile beyond 1–2 days, preferable up to 1 week. Nowadays, insulin is produced in a recombinant manner. This approach facilitates the design and production of further insulin-analogs or insulin-fusion proteins. The usage of the Fc-domain from immunoglobulin as a fusion partner for therapeutic proteins and peptides is widely used to extend their plasma half-life. Insulin consists of two chains, the A- and B-chain, which are connected by two disulfide-bridges. To produce a novel kind of Fc-fusion protein we have fused the A-chain as well as the B-chain to Fc-fragments containing either ‘knob’ or ‘hole’ mutations. The ‘knob-into-hole’ technique is frequently used to force heterodimerization of the Fc-domain. Using this approach, we were able to produce different variants of two-chain-insulin-Fc-protein (tcI-Fc-protein) variants. The tcI-Fc-fusion variants retained activity as shown in in vitro assays. Finally, prolonged blood glucose lowering activity was demonstrated in normoglycemic rats. Overall, we describe here the production of novel insulin-Fc-fusion proteins with prolonged times of action.

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