Core-directed protein design. I. An experimental method for selecting stable proteins from combinatorial libraries

The design of proteins represents a significant challenge to modern-day str uctural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive the folding and stabilization of the target. Com putational 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 fol lows. Hexahistidine-tagged proteins were displayed on the surface of phage. These protein-phage were immobilized onto Ni-coated surfaces. The bound fu sion-phage were treated with protease to remove unstable or poorly folded p roteins. Stable phage fusions were eluted and infected into Escherichia col i, which allowed amplification for further selection, sequencing, or protei n expression. Two Ni-derivatized supports were tested: Ni-NTA chips for sur face plasmon resonance (SPR) and Ni-NTA agarose beads. SPR had an advantage in that the selection process could be monitored directly. This allowed in dividual clones and experimental conditions to be tested rapidly prior to p reparative panning of the library, which was carried out using Ni-NTA agaro se beads. We demonstrate the method by selecting stable core mutants of ubi quitin, 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 pro teins of de novo design, and in establishing rules that link sequence and s tructure.