High level expression and secretion of Fc-X fusion proteins in mammalian cells

Lo, Kin-Ming; Sudo, Yuki; Chen, Jie; Li, Yue; Lan, Yan; Kong, Spencer; Chen, Lily Y.; An, Qing; Gillies, S. D.

doi:10.1093/protein/11.6.495

Cited by 79 publications

(59 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The E-Fc proteins were secreted as soluble proteins that 230 formed detergent-stable homodimers, presumably via disulfide bonds between Fc domains. These 231 findings are consistent with the oligomeric characteristics of soluble fusion proteins in other studies 232 (Lo et al, 1998, Loureiro et al, 2011, Zaharatos et al, 2011. Importantly, the E-Fc protein had nocross-reactivity with anti-human and anti-mouse IgG antibodies (data not shown), suggesting that the 234 antigen was a good candidate for multi-species serodiagnostic studies.…”

Section: ) 224supporting

confidence: 85%

Development of a tick-borne encephalitis serodiagnostic ELISA using recombinant Fc-antigen fusion proteins

Chidumayo

Yoshii

Saasa

et al. 2014

Diagnostic Microbiology and Infectious Disease

View full text Add to dashboard Cite

Current diagnostic tests for tick-borne encephalitis virus (TBEV) infections require high-biosafety facilities for antigen preparation and can cross-react with other flaviviruses. There is, therefore, a need to develop safe, inexpensive serodiagnostic tools with high specificity and sensitivity. In this study, a recombinant plasmid that expresses the membrane and envelope (E) proteins of TBEV fused to the Fc domain of rabbit IgG was constructed and expressed in mammalian cells. The E-Fc proteins were secreted as soluble homodimers, which retained reactivity with anti-TBEV and anti-rabbit IgG antibodies. The E-Fc proteins were then used to develop an enzyme-linked immunosorbent assay (ELISA) to detect TBEV antibodies in rodent and human sera. Compared with the neutralization test, the ELISA had over 90% sensitivity and specificity. In addition, the assay showed no cross-reactivity with Japanese encephalitis virus antibodies. These findings suggest that the E-Fc ELISA may be a useful tool for TBEV serodiagnosis. (C) 2014 Elsevier Inc. All rights reserved

show abstract

Section: ) 224supporting

confidence: 85%

Development of a tick-borne encephalitis serodiagnostic ELISA using recombinant Fc-antigen fusion proteins

Chidumayo

Yoshii

Saasa

et al. 2014

Diagnostic Microbiology and Infectious Disease

View full text Add to dashboard Cite

show abstract

“…The 5Ј primer fuses a human heavy-chain signal sequence to the Fc␥1 sequence enabling secretion of the Fc protein. The N-terminal Cys-5 in the ␥1 hinge (1EPKSCDKTHTCPPCP15), that normally forms a disulfide bridge with a C-terminal Cys in the constant domain of the light chain, was mutated to Ser as described (28). A silent HindIII site was introduced through the 3Ј primer, replacing the codons of Leu-121, Ser-122, and Leu-123 at the C terminus of Fc␥1 upstream of the natural stop codon without changing the amino acid sequence.…”

Section: Methodsmentioning

confidence: 99%

An engineered selenocysteine defines a unique class of antibody derivatives

Höfer

Thomas

Burke

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

View full text Add to dashboard Cite

Selenocysteine is cotranslationally inserted into proteins by recoding the stop codon UGA from termination to selenocysteine insertion. The nucleophilic selenol group of selenocysteine endows this rare amino acid with unique chemical reactivity that allows regiospecific covalent conjugation in the presence of the other natural amino acids. Using a mammalian expression system, we generated an IgG1-derived Fc fragment with a C-terminal selenocysteine in yields comparable to conventional monoclonal antibodies and conjugated it to an electrophilic derivative of a peptidomimetic that binds with high affinity and specificity to integrin ␣4␤1. Through this conjugation, both the biological and chemical components are endowed with pharmacological advantages. We demonstrate that whereas the Fc protein increases the circulatory half-life from minutes to days and mediates transcytosis through binding to the neonatal Fc receptor, the peptidomimetic introduces cross-species binding to cell surface integrin ␣4␤1 and blocks its interaction with vascular cell adhesion molecule-1. Compared with conventional monoclonal antibodies, our technology benefits economically from combining a generic biological component with a variable chemical component.antibody engineering ͉ Fc fragment ͉ neonatal Fc receptor ͉ small synthetic molecules ͉ integrin ␣4␤1

show abstract

“…This construct was inserted into the pdCs expression vector, 36 where cFcES is under the control of human cytomegalovirus (CMV) enhancer and promoter.…”

Section: Cell Culture and Reagentsmentioning

confidence: 99%

Endostatin therapy reveals a U-shaped curve for antitumor activity

et al. 2006

View full text Add to dashboard Cite

Developing continuous systemic delivery of endostatin has been a goal of many laboratories. We have employed a method of gene therapy utilizing different viral constructs. Here, we report that a new serotype of adeno-associated viruses, which incorporates canine endostatin, provides dose-dependent transgene expression in the circulation after intramuscular injection in mice. Elevated levels of endostatin remained stable in the circulation for at least 4 months. In vitro assays determined that the protein expressed was biologically active. Antitumor activities of the above construct demonstrated a U-shape curve, where the maximum activity was observed within a certain critical concentration range. These data suggest that an optimum dose range may be required to achieve therapeutic efficacy in large animal models.

show abstract

High level expression and secretion of Fc-X fusion proteins in mammalian cells

Cited by 79 publications

References 0 publications

Development of a tick-borne encephalitis serodiagnostic ELISA using recombinant Fc-antigen fusion proteins

Development of a tick-borne encephalitis serodiagnostic ELISA using recombinant Fc-antigen fusion proteins

An engineered selenocysteine defines a unique class of antibody derivatives

Endostatin therapy reveals a U-shaped curve for antitumor activity

Contact Info

Product

Resources

About