Engineering of a femtomolar affinity binding protein to human serum albumin
2008 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 8, 515-527 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2008. Vol. 21, no 8, 515-527 p.
affinity/combinatorial protein engineering
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-10016DOI: 10.1093/protein/gzn028ISI: 000257789400005PubMedID: 18499681ScopusID: 2-s2.0-47649128420OAI: oai:DiVA.org:kth-10016DiVA: diva2:201348
QC 201007222009-03-042009-03-042011-11-08Bibliographically approved