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Subtiligase: a tool for semisynthesis of proteins
T K Chang1, D Y Jackson, J P Burnier
1Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080.
Summary
Subtiligase enables site-specific peptide ligation onto proteins, creating modified human growth hormone and atrial natriuretic peptide. This protein engineering tool enhances semisynthesis and rational protein design.
Area of Science:
- Biochemistry
- Protein Engineering
- Molecular Biology
Background:
- Site-specific modification of proteins is crucial for developing novel therapeutics and research tools.
- Traditional protein modification methods can be inefficient or lack specificity.
Purpose of the Study:
- To introduce a novel enzyme, subtiligase, for efficient and site-specific ligation of peptides to proteins.
- To demonstrate the utility of subtiligase in creating modified proteins with desired functionalities.
Main Methods:
- Utilized a variant of subtilisin BPN' (subtiligase) for peptide ligation.
- Produced biotinylated and heavy-atom derivatives of methionyl-extended human growth hormone (Met-hGH).
- Generated polyethylene glycol (PEG)-modified atrial natriuretic peptide (ANP) and established N-terminal sequence requirements for ligation.
Main Results:
- Achieved high-yield ligation of esterified peptides onto protein N termini in aqueous solution.
- Successfully created modified Met-hGH and ANP with biotin, heavy atoms, or PEG.
- Developed a more stable subtiligase variant effective in 4 M guanidine hydrochloride for challenging substrates.
Conclusions:
- Subtiligase is a versatile tool for protein semisynthesis and rational protein design.
- The enzyme facilitates the creation of complex protein conjugates with diverse applications.
- Subtiligase expands the toolkit for modifying proteins with buried or structured N termini.