Engineering of a femtomolar affinity binding protein to human serum albumin

Jonsson, Andreas,1974-KTH,Molekylär Bioteknologi,Biotherapy and Bacterial Display (author)

QC 20100722
We describe the development of a novel serum albumin binding protein showing an extremely high affinity (K(D)) for HSA in the femtomolar range. Using a naturally occurring 46-residue three-helix bundle albumin binding domain (ABD) of nanomolar affinity for HSA as template, 15 residues were targeted for a combinatorial protein engineering strategy to identify variants showing improved HSA affinities. Sequencing of 55 unique phage display-selected clones showed a strong bias for wild-type residues at nine positions, whereas various changes were observed at other positions, including charge shifts. Additionally, a few non-designed substitutions appeared. On the basis of the sequences of 12 variants showing high overall binding affinities and slow dissociation rate kinetics, a set of seven 'second generation' variants were constructed. One variant denoted ABD035 displaying wild-type-like secondary structure content and excellent thermal denaturation/renaturation properties showed an apparent affinity for HSA in the range of 50-500 fM, corresponding to several orders of magnitude improvement compared with the wild-type domain. The ABD035 variant also showed an improved affinity toward serum albumin from a number of other species, and a capture experiment involving human serum indicated that the selectivity for serum albumin had not been compromised from the affinity engineering.

In:Protein Engineering Design & Selection: Oxford University Press (OUP)21:8, s. 515-5271741-01261741-0134

By the author/editor: Jonsson, Andreas ...; Dogan, Jakob; Harne, Nina; Abrahmsén, Lars; Nygren, Per-Åke

About the subject

NATURAL SCIENCES: NATURAL SCIENCES; and Biological Scien ...; and Biochemistry and ...

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