The void volume (V 0) and elution positions for bovine serum albumin (BSA 67 kDa) and ribonuclease A (RNaseA 13.7 kDa) are indicated with the arrows. The labels show the identities of analyzed samples. The chromatographs are shown with vertical offsets for clarity. ( A) Size-exclusion chromatograms of monobodies. Oligomerization state and stability of monobodies. The errors indicated are the standard deviation of the global fits from at least triplicate data sets for the kinetic experiments, or the standard deviation from global curve fitting of duplicate data sets for the equilibrium experiments. The insets show dose-dependence analysis of the sensorgrams (black) and the best fit of the 1:1 binding model (gray). The thin lines show the best global fit of the 1:1 binding model.

( D) SPR sensorgrams for target binding of representative monobodies. A portion of data for point mutants of As15 and As27 is shown for clarity. The errors indicated are the standard deviations from curve fitting of the 1:1 binding model. The mean fluorescence intensities of yeast cells displaying a monobody are plotted as a function of the concentration of the target as indicated in panel A. ( C) Binding measurements by yeast surface display of representative monobodies. “X” denotes a mixture of 30% Tyr, 15% Ser, 10% Gly, 5% Phe, 5% Trp and 2.5% each of all the other amino acids except for Cys “B”, a mixture of Gly, Ser and Tyr “J”, a mixture of Ser and Tyr “O”, a mixture of Asn, Asp, His, Ile, Leu, Phe, Tyr and Val “U”, a mixture of His, Leu, Phe and Tyr “Z”, a mixture of Ala, Glu, Lys and Thr. Amino acid sequences of monobodies generated from the new “side and loop” library ( A) and the “loop only” library ( B). Monobody library designs and generated clones. Together, this work illustrates the utilization of different surfaces of a single immunoglobulin-like scaffold to generate binding proteins with distinct characteristics.Ĭopyright © 2011 Elsevier Ltd. This mode of interaction differs from a common binding mode for single-domain antibodies and antibody mimics in which recognition loops recognize clefts in targets. The crystal structure of a monobody generated from the new library in complex with its target, the Abl SH2 domain, revealed that a concave surface of the monobody, as intended in our design, bound to a convex surface of the target with the interface area being among the largest of published structures of monobody-target complexes. The new library produced binding proteins (termed 'monobodies') to multiple target proteins, generally with similar efficacy as the original, loop-focused library. We diversified positions on a β-sheet and surface loops that together form a concave surface. Here, we report an FN3 library that utilizes alternative positions for presenting amino acid diversity. Because of its structural homology to the immunoglobulin domain, combinatorial libraries of FN3 designed to date have primarily focused on introducing amino acid diversity into three loops that are equivalent to antibody complementarity-determining regions. The fibronectin type III domain (FN3) has become one of the most widely used non-antibody scaffolds for generating new binding proteins.

John bailey protein scaffold