Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small molecules of diverse structures to the target proteins with high efficiency and specificity.

Evolution modulates the quantitative characteristics of protein interactions and often uses combinations of weak interactions to achieve a particular specificity. We addressed how quantitative optimization might be used in the design of multidomain proteins, using a chimera containing epidermal growth factor (EGF) as a cell targeting element and interferon-␣-2a (IFN␣-2a) to initiate signal transduction. We first connected EGF and IFN␣-2a via a linker that allows both ligands to bind to their receptors on a cell surface and then incorporated a series of mutations into the IFN␣-2a portion that progressively decrease both the on rate and the dissociation constant of the IFN␣-2a-IFN␣ receptor 2 (IFNAR2) interaction. Using this strategy, we designed chimeric proteins in which the activation of the IFN␣ receptor in HeLa, A431, and engineered Daudi cells depends on the presence of EGF receptor on the same cell. The mutant chimeric proteins also inhibited proliferation of IFN␣-sensitive cells in an EGF receptor-dependent manner. These results provide insights into the quantitative requirements for specific binding to multisubunit receptors and illustrate the value of a quantitative approach in the design of synthetic-biological constructs.Biological recognition events are often mediated by modular protein and nucleic acid segments that can be arbitrarily linked to give functional combinations. In the course of evolution, multidomain proteins have been repeatedly generated and constitute a large fraction of the proteins encoded by metazoan genomes. In the evolutionary improvement of such chimeras after a genetic rearrangement, an important but under studied process is the quantitative optimization of the individual modules.For many years, researchers have also constructed chimeric proteins with properties that derive from the parental modules. For example, one therapeutic approach has sought to use cell surface proteins as addresses to direct the delivery of specific molecules, such as toxins to tumor cells. Pastan's group (1) described a chimeric protein consisting of Pseudomonas exotoxin and interleukin-2, in which the interleukin-2 moiety directed the toxin to cells bearing interleukin-2 receptor; they later described a P. exotoxin-tumor growth factor-␣ chimeric protein that binds to EGFR 3 (2-4), a hallmark of many tumors (5). Similar strategies have been adapted by many groups (6, 7). A universal problem with this kind of approach is that when any targeted agent is administered to a patient, unwanted effects will occur as the drug travels through the body before reaching its target.We therefore sought a different strategy based on quantitative modulation of the signaling part of a targeted molecule. Our strategy builds on the ideas of Adam and Delbrück (8), who proposed that in biological systems, reaction rates are often enhanced by reduction of the dimension of a space in which diffusion occurs. Because a cell surface is effectively two-dimensional, we reasoned that an initial rapid binding reaction to one cell su...

The presence of erythropoietin (Epo) receptors on cells besides red blood cell precursors, such as cancer cells or megakaryocyte precursors, can lead to side effects during Epo therapy including enhanced tumor growth and platelet production. It would be ideal if the action of Epo could be limited to erythroid precursors. To address this issue, we constructed single-chain variable fragment (scFv)-Epo fusion proteins that used the anti-glycophorin 10F7 scFv amino-terminal to Epo analogues that would have minimal activity alone. We introduced the Epo mutations N147A, R150A and R150E, which progressively weakened receptor affinity in the context of Epo alone, as defined by cell proliferation assays using TF-1 or UT-7 cells. Fusion of these mutant proteins to the 10F7 scFv significantly rescued the activity of the mutant proteins, but had a relatively small effect on wild-type Epo. For example, fusion to the 10F7 scFv enhanced the activity of Epo(R150A) by 10- to 27-fold, while a corresponding fusion to wild-type Epo enhanced its activity only up to 2.7-fold. When glycophorin was blocked by antibody competition or reduced by siRNA-mediated inhibition of expression, the activity of 10F7 scFv-Epo(R150A) was correspondingly reduced, while such inhibition had essentially no effect on the activity of 10F7 scFv-Epo(wild-type). In addition, potent stimulation of Epo receptors by 10F7 scFv-Epo(R150A) was observed in long-term proliferation and viability assays. Taken together, these results indicate that a combination of targeting and affinity modulation can be used to engineer forms of Epo with enhanced cell-type specificity.

[reaction: see text] A total solid-phase synthesis of lamellarins U and L has been achieved. The conversion of an aldehyde group into a formate by a Baeyer-Villiger reaction and a intramolecular [3 + 2] cycloaddition of a 3,4-dihydroisoquinolinium salt over a triple bond comprise the key steps of the process. Each transformation has been controlled with the proper spectroscopic and analytical methods.