title: Semisynthesis of ribonuclease A using intein-mediated protein ligation.
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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA. arnold@biochemtech.uni-halle.de
authors.forename: Ulrich
authors.initials: U
authors.lastname: Arnold
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Hinderaker
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Raines
mesh_terms: D008958:Models, Molecular; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D012259:Ribonuclease, Pancreatic