title: Core-directed protein design. I. An experimental method for selecting stable proteins from combinatorial libraries.
abstract: The design of proteins represents a significant challenge to modern-day structural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive the folding and stabilization of the target. Computational approaches have been used to alleviate this by testing alternate sequences prior to the production and characterization of a few proteins. Here we present the experimental counterpart of this approach. We selected stable variants from a library of ubiquitin hydrophobic-core mutants as follows. Hexahistidine-tagged proteins were displayed on the surface of phage. These protein-phage were immobilized onto Ni-coated surfaces. The bound fusion-phage were treated with protease to remove unstable or poorly folded proteins. Stable phage fusions were eluted and infected into Escherichia coli, which allowed amplification for further selection, sequencing, or protein expression. Two Ni-derivatized supports were tested: Ni-NTA chips for surface plasmon resonance (SPR) and Ni-NTA agarose beads. SPR had an advantage in that the selection process could be monitored directly. This allowed individual clones and experimental conditions to be tested rapidly prior to preparative panning of the library, which was carried out using Ni-NTA agarose beads. We demonstrate the method by selecting stable core mutants of ubiquitin, the characterization of which is described in the following paper [Finucane, M. D., and Woolfson, D. N. (1999) Biochemistry 38, XXXXX-XXXXX]. As our method selects only on the basis of structure and stability, it will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure.
authors.affiliation: Centre for Biomolecular Design, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.
authors.forename: M D
authors.initials: MD
authors.lastname: Finucane
authors.forename: M
authors.initials: M
authors.lastname: Tuna
authors.forename: J H
authors.initials: JH
authors.lastname: Lees
authors.forename: D N
authors.initials: DN
authors.lastname: Woolfson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D019151:Peptide Library; D011506:Proteins / Q000138:chemical synthesis*; D051381:Rats; D020349:Surface Plasmon Resonance
chemical_list: D017931:DNA Primers; D019151:Peptide Library; D011506:Proteins