The Albumin-Binding Domain as a Scaffold for Protein Engineering

Abstract

The albumin-binding domain is a small, three-helical protein domain found in various surface proteins expressed by gram-positive bacteria. Albumin binding is important in bacterial pathogenesis and several homologous domains have been identified. Such albumin-binding regions have been used for protein purification or immobilization. Moreover, improvement of the pharmacokinetics, through the non-covalent association to albumin, by fusing such domains to therapeutic proteins has been shown to be successful. Domains derived from streptococcal protein G and protein PAB from Finegoldia magna, which share a common origin and therefore represent an interesting evolutionary system, have been thoroughly studied structurally and functionally. Their albumin-binding sites have been mapped and these domains form the basis for a wide range of protein engineering approaches. By substitution-mutagenesis they have been engineered to achieve a broader specificity, an increased stability or an improved binding affinity, respectively. Furthermore, novel binding sites have been incorporated either by replacing the original albumin-binding surface, or by complementing it with a novel interaction interface. Combinatorial protein libraries, where several residues have been randomized simultaneously, have generated a large number of new variants with desired binding characteristics. The albumin-binding domain has also been utilized to explore the relationship between three-dimensional structure and amino acid sequence. Proteins with latent structural information built into their sequence, where a single amino acid substitution shifts the equilibrium in favor of a different fold with a new function, have been designed. Altogether, these examples illustrate the versatility of the albumin-binding domain as a scaffold for protein engineering.

Conjugate of Doxorubicin to Albumin‐Binding Peptide Outperforms Aldoxorubicin

“…In T-cell proliferation assays, this variant showed a very weak immunogenic response (Affibody AB, unpublished data). [81] In conclusion, the system demonstrated here allows site-specific conjugation of chemotherapeutics to an ABD that is readily overexpressed in E. coli and purified with high yield and purity without the need for chromatography. This system should also enable other functional elements-such as targeting protein domains-to be easily incorporated at the gene level to yield a modular and flexible system for delivery of small-molecule drugs to tumors for cancer therapy.…”

“…Nevertheless, to address any potential immunogenicity concerns, a deimmunized ABD variant (ABD094) has been generated by mutagenesis of the ABD sequence by substituting the T‐cell epitope residues located within the ABD. In T‐cell proliferation assays, this variant showed a very weak immunogenic response (Affibody AB, unpublished data) …”

Antibody-Targeted Chemotherapy for the Treatment of Melanoma

“…2A). The binding affinity of bevacizumab and Abraxane is within the range of dissociation constants observed between albumin and natural or engineered albumin-binding domains of some bacterial proteins (14). We tested whether binding affinity was pH-and/or temperature-dependent by suspending the Abraxane in bevacizumab and diluting the mixture with saline at pH 3, 5, 7, or 9 before incubation at various temperatures (room temperature, 37 C, and 58 C) to allow particle formation.…”

The future of protein scaffolds as affinity reagents for purification

“…ABD was affinity maturated by phage display to increase affinity to Human Serum Albumin (HSA) from a nanomolar to femtomolar range ( Fig. One of the variants with improved properties was applied in HSA purification (Jonsson et al, 2008), and also in half-life extension of therapeutic proteins by generation of bivalent domains that bind HSA and Tumor Necrosis Factor-a (TNF-a) (Nilvebrant and Hober, 2013). The protein was engineered in 15 out of 46 amino acids, located in the contact region between the ABD and HSA.…”