scholarly journals Structural insight and stability of TNFR-Fc fusion protein (Etanercept) produced by using transgenic silkworms

Author(s):  
Masato Kiyoshi ◽  
Ken-Ichiro Tatematsu ◽  
Minoru Tada ◽  
Hideki Sezutsu ◽  
Hiroko Shibata ◽  
...  

Abstract Therapeutic proteins expressed using transgenic animals have been of great interest for several years. Especially, transgenic silkworm has been studied intensively because of its ease in handling, low-cost, high-yield and unique glycosylation patterns. However, the physicochemical property of the therapeutic protein expressed in transgenic silkworm remains elusive. Here, we constructed an expression system for the TNFR-Fc fusion protein (Etanercept) using transgenic silkworm. The TNFR-Fc fusion protein was employed to N-glycan analysis, which revealed an increased amount of afucosylated protein. Evidence from surface plasmon resonance analysis showed that the TNFR-Fc fusion protein exhibit increased binding affinity for Fcγ receptor IIIa and FcRn compared to the commercial Etanercept, emphasizing the profit of expression system using transgenic silkworm. We have further discussed the comparison of higher order structure, thermal stability and aggregation of the TNFR-Fc fusion protein.

2011 ◽  
Vol 100 (11) ◽  
pp. 4597-4606 ◽  
Author(s):  
Hao Li ◽  
Shufeng Bai ◽  
Julie Y. Wei ◽  
Steven A. Berkowitz ◽  
Mark L. Brader

2022 ◽  
Vol 44 (1) ◽  
pp. 301-308
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.


2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Xiaoyan Pan ◽  
Jian Shi ◽  
Xue Hu ◽  
Yan Wu ◽  
Liang Zeng ◽  
...  

AbstractThe pandemic of COVID-19 caused by SARS-CoV-2 has raised a new challenges to the scientific and industrious fields after over 1-year spread across different countries. The ultimate approach to end the pandemic is the timely application of vaccines to achieve herd immunity. Here, a novel SARS-CoV-2 receptor-binding domain (RBD) homodimer was developed as a SARS-CoV-2 vaccine candidate. Formulated with aluminum adjuvant, RBD dimer elicited strong immune response in both rodents and non-human primates, and protected mice from SARS-CoV-2 challenge with significantly reducing viral load and alleviating pathological injury in the lung. In the non-human primates, the vaccine could prevent majority of the animals from SARS-CoV-2 infection in the respiratory tract and reduce lung damage. In addition, antibodies elicited by this vaccine candidate showed cross-neutralization activities to SARS-CoV-2 variants. Furthermore, with our expression system, we provided a high-yield RBD homodimer vaccine without additional biosafety or special transport device supports. Thus, it may serve as a safe, effective, and low-cost SARS-CoV-2 vaccine candidate.


1999 ◽  
Vol 342 (3) ◽  
pp. 625-632 ◽  
Author(s):  
Samantha C. WILLIAMS ◽  
Justin HINSHELWOOD ◽  
Stephen J. PERKINS ◽  
Robert B. SIM

Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system. It binds to other complement proteins C3b and properdin, and is activated by the protease factor D. The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A). A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys267 → Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of 125I-labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain. The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains. Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b.


2019 ◽  
Vol 16 (8) ◽  
pp. 676-682
Author(s):  
Ankusab Noorahmadsab Nadaf ◽  
Kalegowda Shivashankar

The polycyclic dihydropyridine nucleus represents the heterocyclic system of invaluable core motifs with wide applications in chemical, biological and physical properties. Although this kind of compounds have been extensively synthesized by other groups, the synthesis of these compounds under CFL light intensity were not explored. The synthesis of polycyclic dihydropyridine derivatives were achieved through the reaction of 4-hydroxycoumarin, aromatic aldehydes and ammonium acetate under CFL light irradiation conditions. A series of polycyclic dihydropyridine derivatives were prepared under CFL light irradiation conditions with high yield, short reaction time, ambient condition and without the use of catalyst. The results displayed an efficient method for the synthesis of polycyclic dihydropyridine derivatives. Clean profile, short reaction time, low cost and use of CFL light intensity instead of catalyst making it a genuinely green protocol.


2019 ◽  
Vol 9 (2) ◽  
pp. 157-160
Author(s):  
Ali Hasani

Background: Laser ablation method has high-yield and pure SWCNHs. On the other hand, arc discharge methods have low-cost production of SWCNHs. However, these techniques have more desirable features, they need special expertness to use high power laser or high current discharge that either of them produces very high temperature. As for the researches, the temperatures of these techniques are higher than 4727°C to vaporize the graphite. So, to become aware of the advantages of SWCNHs, it is necessary to find a new way to synthesize SWCNHs at a lower temperature. In other words, reaction field can be expandable at a moderate temperature. This paper reports a new way to synthesize SWCNHs at an extremely reduced temperature. Methods: According to this study, the role of N2 is the protection of the copper holder supporting the graphite rod by increasing heat transfer from the holder. After the current of 70 A was supplied to the system, the temperature of graphite rod was raised to 1600°C. It is obvious that this temperature is somehow higher than the melting point of palladium, 1555°C, and much lower than graphite melting point, 3497°C. Results: Based on the results, there are transitional precursors simultaneous with the SWCNHs. This composition can be created by distortion of the primary SWCNTs at the higher temperature. Subsequently, each SWCNTs have a tendency to be broken into individual horns. With increasing the concentration of the free horns, bud-like SWCNHs can be produced. Moreover, there are individual horns almost separated from the mass of single wall carbon nanohorns. This structure is not common in SWCNHs synthesized by the usual method such as arc discharge or laser ablation. Through these regular techniques, SWCNHs are synthesized as cumulative particles with diameters about 30-150 nm. Conclusion: A simple heating is needed for SWCNTs transformation to SWCNHs with the presence of palladium as catalyst. The well-thought-out mechanism for this transformation is that SWCNTs were initially changed to highly curled shape, and after that were formed into small independent horns. The other rout to synthesize SWCNHs is the pyrolysis of palm olein at 950°C with the assistance of zinc nitrate and ferrocene. Palm olein was used as a promising, bio-renewable and inexpensive carbon source for the production of carbon nanohorns.


2019 ◽  
Vol 16 (7) ◽  
pp. 1032-1041
Author(s):  
Dening Pei ◽  
Jialiang Hu ◽  
Chunming Rao ◽  
Pengcheng Yu ◽  
Hanmei Xu ◽  
...  

iScience ◽  
2021 ◽  
pp. 102488
Author(s):  
Takayuki Ozawa ◽  
Masato Morikawa ◽  
Yasuyuki Morishita ◽  
Kazuki Ogikubo ◽  
Fumiko Itoh ◽  
...  

2021 ◽  
Vol 22 (3) ◽  
pp. 1124
Author(s):  
Mafalda Giovanna Reccia ◽  
Floriana Volpicelli ◽  
Eirkiur Benedikz ◽  
Åsa Fex Svenningsen ◽  
Luca Colucci-D’Amato

Neural stem cells represent a powerful tool to study molecules involved in pathophysiology of Nervous System and to discover new drugs. Although they can be cultured and expanded in vitro as a primary culture, their use is hampered by their heterogeneity and by the cost and time needed for their preparation. Here we report that mes-c-myc A1 cells (A1), a neural cell line, is endowed with staminal properties. Undifferentiated/proliferating and differentiated/non-proliferating A1 cells are able to generate neurospheres (Ns) in which gene expression parallels the original differentiation status. In fact, Ns derived from undifferentiated A1 cells express higher levels of Nestin, Kruppel-like factor 4 (Klf4) and glial fibrillary protein (GFAP), markers of stemness, while those obtained from differentiated A1 cells show higher levels of the neuronal marker beta III tubulin. Interestingly, Ns differentiation, by Epidermal Growth Factors (EGF) and Fibroblast Growth Factor 2 (bFGF) withdrawal, generates oligodendrocytes at high-yield as shown by the expression of markers, Galactosylceramidase (Gal-C) Neuron-Glial antigen 2 (NG2), Receptor-Interacting Protein (RIP) and Myelin Basic Protein (MBP). Finally, upon co-culture, Ns-A1-derived oligodendrocytes cause a redistribution of contactin-associated protein (Caspr/paranodin) protein on neuronal cells, as primary oligodendrocytes cultures, suggesting that they are able to form compact myelin. Thus, Ns-A1-derived oligodendrocytes may represent a time-saving and low-cost tool to study the pathophysiology of oligodendrocytes and to test new drugs.


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