Design of a binding scaffold based on variable lymphocyte receptors of jawless vertebrates by module engineering.
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.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 305-701, Korea.
Sang-Chul
SC
Lee
Keunwan
K
Park
Jieun
J
Han
Joong-jae
JJ
Lee
Hyun Jung
HJ
Kim
Seungpyo
S
Hong
Woosung
W
Heu
Yu Jung
YJ
Kim
Jae-Seok
JS
Ha
Seung-Goo
SG
Lee
Hae-Kap
HK
Cheong
Young Ho
YH
Jeon
Dongsup
D
Kim
Hak-Sung
HS
Kim
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D016384:Consensus Sequence; D018360:Crystallography, X-Ray; D006196:Hagfishes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007798:Lampreys / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D011971:Receptors, Immunologic / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D010446:Peptide Fragments; D019151:Peptide Library; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins