Journal Article

Engineering of a femtomolar affinity binding protein to human serum albumin

Andreas Jonsson, Jakob Dogan, Nina Herne, Lars Abrahmsén and Per-Åke Nygren

in Protein Engineering, Design and Selection

Volume 21, issue 8, pages 515-527
Published in print August 2008 | ISSN: 1741-0126
Published online May 2008 | e-ISSN: 1741-0134 | DOI: https://dx.doi.org/10.1093/protein/gzn028
Engineering of a femtomolar affinity binding protein to human serum albumin

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We describe the development of a novel serum albumin binding protein showing an extremely high affinity (KD) 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.

Keywords: affinity; combinatorial protein engineering; femtomolar; human serum albumin; phage display

Journal Article.  9087 words.  Illustrated.

Subjects: Proteins

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