title: Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.
abstract: We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2x10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 1 l of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T(m) values ranging from 66 degrees C to well above 85 degrees C. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins.
authors.affiliation: Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: H Kaspar
authors.initials: HK
authors.lastname: Binz
authors.forename: Michael T
authors.initials: MT
authors.lastname: Stumpp
authors.forename: Patrik
authors.initials: P
authors.lastname: Forrer
authors.forename: Patrick
authors.initials: P
authors.lastname: Amstutz
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D001483:Base Sequence; D002942:Circular Dichroism; D016384:Consensus Sequence; D030562:Databases, Protein; D015723:Gene Library; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D013696:Temperature