Sang-Chul Lee^a,1, Keunwan Park^b,1, Jieun Han^a,1, Joong-jae Lee^a,1, Hyun Jung Kim^c,d, Seungpyo Hong^b, Woosung Heu^a, Yu Jung Kim^e, Jae-Seok Ha^e, Seung-Goo Lee^e, Hae-Kap Cheong^c, Young Ho Jeon^c, Dongsup Kim^b,2, and Hak-Sung Kim^a,2

^aDepartment of Biological Sciences, and
^bDepartment of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 305-701, Korea;
^cDivision of Magnetic Resonance Research, Korea Basic Science Institute, Cheongwon, Chungbuk, 363-883, Korea;
^dCollege of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, 361-763, Korea; and
^eIndustrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea

Edited by Max D. Cooper, Emory University, Atlanta, GA, and approved December 28, 2011 (received for review August 12, 2011)

Abstract
Repeat proteins have recently been of great interest as potential alternatives to immunoglobulin antibodies due to their unique structural and biophysical features. We here present the development of a binding scaffold based on variable lymphocyte receptors, which are nonimmunoglobulin antibodies composed of Leucine-rich repeat modules in jawless vertebrates, by module engineering. A template scaffold was first constructed by joining consensus repeat modules between the N- and C-capping motifs of variable lymphocyte receptors. The N-terminal domain of the template scaffold was redesigned based on the internalin-B cap by analyzing the modular similarity between the respective repeat units using a computational approach. The newly designed scaffold, termed “Repebody,” showed a high level of soluble expression in bacteria, displaying high thermodynamic and pH stabilities. Ease of molecular engineering was shown by designing repebodies specific for myeloid differentiation protein-2 and hen egg lysozyme, respectively, by a rational approach. The crystal structures of designed repebodies were determined to elucidate the structural features and interaction interfaces. We demonstrate general applicability of the scaffold by selecting repebodies with different binding affinities for interleukin-6 using phage display.

non-antibody scaffold, repeat protein, modular architecture, molecular binder

Footnotes
¹ S.-C.L., K.P., J.H., and J.-j.L. contributed equally to this work.

²To whom correspondence may be addressed.

Author contributions: S.-C.L., K.P., J.H., J.-j.L., D.K., and H.-S.K. designed research; S.-C.L., K.P., J.H., J.-j.L., H.J.K., S.H., W.H., Y.J.K., and J.-S.H. performed research; S.-C.L., K.P., J.H., J.-S.H., S.-G.L., H.-K.C., Y.H.J., D.K., and H.-S.K. analyzed data; and S.-C.L., K.P., J.H., S.-G.L., H.-K.C., and H.-S.K. wrote the paper.