The Scientific World Journal

The Scientific World Journal / 2002 / Article

Short Communication | Open Access

Volume 2 |Article ID 629784 | https://doi.org/10.1100/tsw.2002.855

Ulrich Arnold, Matthew P. Hinderaker, Ronald T. Raines, "Semisynthesis of Ribonuclease A using Intein-Mediated Protein Ligation", The Scientific World Journal, vol. 2, Article ID 629784, 5 pages, 2002. https://doi.org/10.1100/tsw.2002.855

Semisynthesis of Ribonuclease A using Intein-Mediated Protein Ligation

Received19 Mar 2002
Revised15 May 2002
Accepted16 May 2002

Abstract

The introduction of non-natural amino acid residues or modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding behavior, or biological function. We exploited intein-mediated protein ligation to produce a semisynthetic ribonuclease A. Of the 124 residues of RNase A, residues 1–94 were linked to an intein. After expression of the fusion protein and thiol-induced cleavage, the RNase A(1–94) fragment possessed a C-terminal thioester. A peptide identical to the C-terminal residues 95–124 of RNase A (with residue 95 being cysteine) was successfully ligated to that thioester thereby reconstituting full-length wild-type RNase A. In mass spectrometry, this semisynthetic RNase A proved to be undistinguishable from the control protein, namely recombinant wild-type RNase A. Recombinant wild-type RNase A was obtained by expression of RNase A(1–124)–intein fusion protein followed by thiol-induced cleavage and hydrolysis of the thioester. Both proteins showed thermal stabilities (Tm ) and catalytic activities comparable to the wild-type enzyme, indicating that both proteins folded properly. These results might serve as basis for the semisynthesis of RNase A variants containing non-natural modules in the aforementioned peptide.


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