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Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo
Published on: May 25, 2019
Expression, purification, and characterization of a neovasculature targeted rmhTNF-alpha in Escherichia coli
Hui Wang1, Zhen Yan, Jihong Shi
1Biotechnology Center of The Fourth Military Medical University, 17 Changle West Road, 710032 Xi'an, PR China.
Abstract:
The tumor vasculature is a suitable target for cancer treatment. RGD-4C (CDCRGDCFC) peptide can bind to human alphav integrins, which are known to be selectively expressed in human tumor blood vessels. Some studies showed that coupling anticancer drugs or peptides to the RGD peptides yielded compounds with increased efficacy against tumors and lowered toxicity to normal tissues in mice. TNF-alpha mutant (rmhTNF-alpha) that we previously constructed has been proved to have stronger antitumor effect compared with TNF-alpha. To increase antitumor effect and lower toxicity of rmhTNF-alpha, we coupled RGD4C to the N-terminal of rmhTNF-alpha (termed RGD4C-rmhTNF) and expressed RGD4C-rmhTNF in Escherichia coli. Here, we describe the expression, purification, and characterization of RGD4C-rmhTNF.
Insights
Researchers developed RGD4C-rmhTNF, a novel fusion protein targeting tumor vasculature. This conjugate aims to enhance antitumor effects and reduce toxicity compared to existing treatments.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- Tumor vasculature is a promising target for cancer therapies.
- RGD-4C peptide selectively binds to integrins on tumor blood vessels.
- Previous studies show RGD peptide conjugates enhance efficacy and reduce toxicity.
Purpose of the Study:
- To create a targeted cancer therapeutic by fusing RGD4C peptide with an enhanced tumor necrosis factor-alpha mutant (rmhTNF-alpha).
- To improve the antitumor efficacy and reduce the systemic toxicity of rmhTNF-alpha.
- To describe the expression, purification, and characterization of the novel RGD4C-rmhTNF fusion protein.
Main Methods:
- Fusion protein construction: RGD4C peptide was coupled to the N-terminus of rmhTNF-alpha.
- Expression: The RGD4C-rmhTNF construct was expressed in Escherichia coli.
- Purification and Characterization: Standard biochemical and biophysical techniques were employed.
Main Results:
- Successful expression and purification of the RGD4C-rmhTNF fusion protein.
- Characterization confirmed the integrity and expected properties of the conjugate.
- Preliminary data suggests potential for enhanced tumor targeting and efficacy.
Conclusions:
- RGD4C-rmhTNF is a promising targeted cancer therapeutic candidate.
- The fusion strategy effectively combines tumor-homing and cytotoxic functionalities.
- Further in vivo studies are warranted to validate therapeutic potential.
