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Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry
Published on: July 20, 2016
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A Selenium-based Cysteine Surrogate for Protein Chemical Synthesis
Olga Firstova1, Vangelis Agouridas1,2, Vincent Diemer3
1Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France.
Methods in Molecular Biology (Clifton, N.J.)
|June 27, 2022
Summary
N-selenoethyl cysteine (SetCys) is a cysteine surrogate that enables redox-controlled peptide assembly via native chemical ligation (NCL). Its conversion to cysteine is triggered by tris(2-carboxyethyl)phosphine (TCEP), facilitating controlled protein synthesis.
Area of Science:
- Biochemistry
- Organic Chemistry
- Synthetic Biology
Background:
- Native chemical ligation (NCL) is a powerful method for synthesizing peptides and proteins.
- Controlling the reactivity of amino acid residues is crucial for precise molecular assembly.
- Existing cysteine surrogates may lack tunable reactivity for redox-controlled ligation.
Purpose of the Study:
- To introduce N-selenoethyl cysteine (SetCys) as a novel cysteine surrogate for redox-controlled peptide synthesis.
- To detail the synthesis and incorporation of Fmoc-protected SetCys into peptides.
- To demonstrate the application of SetCys in the one-pot, redox-controlled assembly of proteins.
Main Methods:
- Synthesis of Fmoc-protected N-selenoethyl cysteine (SetCys).
- Incorporation of SetCys into peptide sequences using standard solid-phase peptide synthesis.
- Redox-controlled native chemical ligation (NCL) using SetCys and tris(2-carboxyethyl)phosphine (TCEP).
Main Results:
- SetCys functions as a cysteine surrogate with reactivity dependent on the reducing agent.
- SetCys does not interfere with NCL under mild reducing conditions (without TCEP).
- Treatment with TCEP triggers the spontaneous conversion of SetCys to cysteine, enabling controlled ligation.
- Successful one-pot, redox-controlled assembly of three peptide segments into a protein.
Conclusions:
- N-selenoethyl cysteine (SetCys) offers a versatile tool for redox-controlled peptide and protein synthesis via NCL.
- The tunable reactivity of SetCys, modulated by TCEP, allows for precise control over ligation events.
- This protocol facilitates the efficient chemical synthesis of complex proteins through controlled segment assembly.
