PMID:8453373

Citation	No author listed (1993) Ribonuclease S-peptide as a carrier in fusion proteins. Protein Sci. 2:348-56
Abstract	S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).
Links	PubMed PMC2142386 Online version:10.1002/pro.5560020307
Keywords	Amino Acid Sequence; Base Sequence; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; DNA, Recombinant/genetics; Escherichia coli/genetics; Escherichia coli/metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments/chemistry; Peptide Fragments/genetics; Peptide Fragments/metabolism; Plasmids; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Ribonucleases/chemistry; Ribonucleases/genetics; Ribonucleases/metabolism
edit table

Significance

You can help EcoliWiki by summarizing why this paper is useful

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.

Annotations

Gene product	Qualifier	GO ID	GO term name	Evidence Code	with/from	Aspect	Notes	Status
edit table

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

References

See Help:References for how to manage references in EcoliWiki.