PEDS Advance Access originally published online on May 22, 2008
Protein Engineering Design and Selection 2008 21(8):515-527; doi:10.1093/protein/gzn028
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Engineering of a femtomolar affinity binding protein to human serum albumin
1 Affibody AB, Voltavägen 13, SE-161 02 Bromma 2 Department of Molecular Biotechnology, Royal Institute of Technology (KTH), School of Biotechnology, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden
3 To whom correspondence should be addressed. E-mail: perake{at}biotech.kth.se (P-Å.N.)
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
Received January 28, 2008; revised April 8, 2008; accepted April 19, 2008.