Abstract
We have identified a 101-amino-acid polypeptide derived from the sequence of the IIA binding site of human albumin. The polypeptide contains residues that make contact with IIA ligands in the parent protein, and eight cysteine residues to form disulfide bridges, that stabilize the polypeptide structure. Seventy-four amino acids are located in six α-helical regions, while the remaining thirty-seven amino acids form six connecting coil/loop regions. A soluble GST fusion protein was expressed in E. coli in yields as high as 4 mg/l. This protein retains the IIA fragment’s capacity to bind typical ligands such as warfarin and efavirenz and other albumin’s functional properties such as aldolase activity and the ability to direct the stereochemical outcome of a diketone reduction. This newly cloned polypeptide thus represents a valuable starting point for the construction of libraries of binders and catalysts with improved proficiency.
Highlights
In recent years, a number of different binding proteins have been proposed as alternatives to conventional antibody-based technologies
In this report we describe the identification, cloning as a soluble glutathione S-transferase (GST) fusion protein and characterization of a 101-amino-acid polypeptide derived from the sequence of the IIA binding site of human albumin
Based on structural and functional analyses, we have identified a peptide corresponding to a 101-residue stretch of the human serum albumin sequence (A194 to E294) named HSA100 (Fig. 1a)
Summary
A number of different binding proteins have been proposed as alternatives to conventional antibody-based technologies. The engineering of novel protein scaffolds for binding small molecules has several drawbacks common to antibody-based technologies. Small haptens are chemically modified at a suitable functional group to introduce a reactive linker for conjugation to a carrier protein or immobilization on a solid surface, often via biotinylation. This modification reduces the number of available interactions and constrains the orientation of the small ligand with respect to potential receptors. A detailed example has been reported involving a testosterone-binding peptide derived from neocarzinostatin [9]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
