Introduction of unnatural amino acids into proteins using expressed protein ligation

Brenda Ayers, Ulrich K. Blaschke, Julio A. Camarero, Graham J. Cotton, Mande Holford, Tom W. Muir

Research output: Contribution to journalArticlepeer-review

67 Scopus citations

Abstract

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2- mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine- containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins. (C) 2000 John Wiley and Sons, Inc.

Original languageEnglish (US)
Pages (from-to)343-354
Number of pages12
JournalBiopolymers - Peptide Science Section
Volume51
Issue number5
DOIs
StatePublished - Dec 1 1999
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

Keywords

  • Expressed protein ligation
  • Protein semisynthesis

Fingerprint Dive into the research topics of 'Introduction of unnatural amino acids into proteins using expressed protein ligation'. Together they form a unique fingerprint.

Cite this