Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria.

Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase-tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists.

On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

A genetically encoded aza-Michael acceptor for covalent cross-linking of protein-receptor complexes.

Selective covalent bond formation at a protein-protein interface potentially can be achieved by genetically introducing into a protein an appropriately "tuned" electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs that genetically encode three aza-Michael acceptor amino acids, N(ε)-acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 in vitro or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.

Evolution of iron(II)-finger peptides by using a bipyridyl amino acid.

We report the engineering of zinc-finger-like motifs containing the unnatural amino acid (2,2'-bipyridin-5-yl)alanine (Bpy-Ala). A phage-display library was constructed in which five residues in the N-terminal finger of zif268 were randomized to include both canonical amino acids and Bpy-Ala. Panning of this library against a nine-base-pair DNA binding site identified several Bpy-Ala-containing functional Zif268 mutants. These mutants bind the Zif268 recognition site with affinities comparable to that of the wild-type protein. Further characterization indicated that the mutant fingers bind low-spin Fe(II) rather than Zn(II) . This work demonstrates that an expanded genetic code can lead to new metal ion binding motifs that can serve as structural, catalytic, or regulatory elements in proteins.

Loss of CD4 T-cell-dependent tolerance to proteins with modified amino acids.

The site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO(2)Phe) into autologous proteins overcomes self-tolerance and induces a long-lasting polyclonal IgG antibody response. To determine the molecular mechanism by which such simple modifications to amino acids are able to induce autoantibodies, we incorporated pNO(2)Phe, sulfotyrosine (SO(3)Tyr), and 3-nitrotyrosine (3NO(2)Tyr) at specific sites in murine TNF-α and EGF. A subset of TNF-α and EGF mutants with these nitrated or sulfated residues is highly immunogenic and induces antibodies against the unaltered native protein. Analysis of the immune response to the TNF-α mutants in different strains of mice that are congenic for the H-2 locus indicates that CD4 T-cell recognition is necessary for autoantibody production. IFN-γ ELISPOT analysis of CD4 T cells isolated from vaccinated mice demonstrates that peptides with mutated residues, but not the wild-type residues, are recognized. Immunization of these peptides revealed that a CD4 repertoire exists for the mutated peptides but is lacking for the wild-type peptides and that the mutated residues are processed, loaded, and presented on the I-A(b) molecule. Overall, our results illustrate that, although autoantibodies are generated against the endogenous protein, CD4 cells are activated through a neo-epitope recognition mechanism. Therefore, tolerance is maintained at a CD4 level but is broken at the level of antibody production. Finally, these results suggest that naturally occurring posttranslational modifications such as nitration may play a role in antibody-mediated autoimmune disorders.

Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids.

For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269-277]. However, the decreased ability of the immune system to mount a robust immune response to self-antigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO(2)Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-alpha (mTNF-alpha) independently to pNO(2)Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-alpha IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-alpha and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO(2)Phe(43) mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.

Site-specific incorporation of ε-N-crotonyllysine into histones.

A novel post-translationally modified amino acid, crotonyllysine (Kcr), was genetically incorporated into proteins in bacterial and mammalian cells using an evolved pyrrolysyl-tRNA/synthetase-tRNA pair. The ability to produce histones with homogenous, site-specific Kcr modifications will be valuable in elucidating the biological role of this recently identified post-translational modification.

Combination of large volume sample stacking and reversed pH junction in capillary electrophoresis for online preconcentration of glycoforms of recombinant human erythropoietin.

Resolution of glycoforms of a glycoprotein is usually a challenge for an analytical chemist, which is often associated with the difficulty of detecting a large number of glycoforms with sufficient sensitivity. CE is the main workhorse for the analysis of glycoproteins; however, the current methods are only applicable to high concentration samples. Therefore, the development of online preconcentration approaches is important for the CE analysis of glycoproteins. In this study, we present a combined strategy for online preconcentrating glycoforms of glycoproteins, which couples two individual online preconcentration techniques, i.e., large volume sample stacking (LVSS) and reversed pH junction (RPHJ). LVSS allows for compressing a large injected sample volume into a narrow sample zone, contributing to the main sensitivity improvement, while RPHJ coordinates the preconcentration process to improve the resolution. This strategy was verified with recombinant human erythropoietin (rhEPO), a typical glycoprotein, as the test analyte, and the effects of experimental conditions were investigated. It was found that there is a compromise between sensitivity enhancement and resolution for online preconcentration of glycoprotein glycoforms. By using this strategy, the detection sensitivity can be improved by 50-100 times for rhEPO.

Harnessing the power of an expanded genetic code toward next-generation biopharmaceuticals.

Synthetic biology has been revolutionizing the biopharmaceutical industry from drug discovery, clinical development to the manufacturing of biopharmaceuticals. As one of its most promising areas, genetic incorporation of noncanonical amino acids (ncAA) into proteins via an expanded genetic code emerged with great promise in the pharmaceutical industry recently with multiple therapeutic candidates tested in human clinical trials and one approved veterinary drug. Expanded building blocks enable proteins to have new or modified functions, providing ample opportunities for innovative or improved medicines. Here we review the current progress in the technology platforms for manufacturing ncAA-containing therapeutics and the pharmaceutical applications of an expanded genetic code.

On-line preconcentration of sodium dodecyl sulfate-protein complexes using electrokinetic supercharging method with a prefilled water plug in capillary sieving electrophoresis.

An electrokinetic supercharging (EKS) method with a prefilled water plug at the head column of capillary was developed for on-line preconcentration of sodium dodecyl sulfate (SDS)-protein complexes in capillary sieving electrophoresis (CSE). Conventional EKS is a combination of electrokinetic injection with transient isotachophoresis (tr-ITP). The capillary is first filled with background electrolyte, then an appropriate amount of a leading electrolyte is filled and electro-injection is carried out for certain duration. After that, terminating electrolyte is filled, and tr-ITP is subsequently initiated, followed by capillary electrophoresis (CE) separation. In this work, the performance of EKS was evaluated by integrating multiple sub-methods step by step, and a water plug containing polymer was introduced before electrokinetic injection in order to further improve the concentration effect. The positive effects of the sub-methods were verified, including molecular sieving effect of polymer, field enhanced sample injection (FESI) with and without a water plug, and transient isotachophoretic electrophoresis-based FESI. It was observed that analyte discrimination usually encountered in conventional electrokinetic injection was eliminated due to the similar charge to mass ratios of SDS-protein complexes. Based on these results, a hybrid on-line preconcentration method, EKS with injecting a water plug containing polymer before sample electrokinetic injection, was proposed and used to indiscriminately preconcentrate SDS-protein complexes, which provided a sensitivity enhancement factor of more than 1000. It was very suitable for the analysis of low-abundance proteins, providing the information of their molecular mass.