An albumin-derived peptide scaffold capable of binding and catalysis

Elisa Maurizio,Alessandro Tossi,Immacolata Luisi,Riccardo Sgarra,Giampaolo Fontanive,Silvia Pavan,Fabio Benedetti,Adriano Savoini,Federico Berti,Daniele Sblattero

doi:10.1038/npre.2012.6894.1

Elisa Maurizio, Alessandro Tossi + Show 8 more

Open Access

https://doi.org/10.1038/npre.2012.6894.1

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Abstract

AbstractWe have identified a 101-amino-acid polypeptide derived from the sequence surrounding the IIA binding site of human albumin. The polypeptide contains residues that make contact with ligands as warfarin in the parent protein, and eight cysteine residues to form disulfide bridges, which stabilize the polypeptide structure. Seventy-four amino acids are located in six [alpha]-helical regions, with the remaining amino acids forming six connecting coil/loop regions. Codon usage optimization was used to express a GST fusion protein in E. coli in yields as high as 4 mg/l. This fusion protein retains its structural integrity and aldolase activity, the ability to direct the stereochemical outcome of a diketone reduction, and its binding capacity to warfarin and efavirenz. Notably, this newly cloned polypeptide 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

Introduction

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]

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