Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In ...contrast, selective ligation to the other sulfur-containing amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, we report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. We highlight the broad utility of this conjugation method to enable precise addition of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes.
Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study ...proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1–3mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (Tm of 81°C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.
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•High-throughput antibody generation requires improved scaffolds.•Improved phage display for a scFab with long linkers and optimized signal peptide.•Rapid reformatting for high yield, stable scFab from bacterial and mammalian hosts.•New, optimized scFab to advance antibody discovery.
In response to enteric pathogens, the inflamed intestine produces antimicrobial proteins, a process mediated by the cytokines IL-17 and IL-22. Salmonella enterica serotype Typhimurium thrives in the ...inflamed intestinal environment, suggesting that the pathogen is resistant to antimicrobials it encounters in the intestinal lumen. However, the identity of these antimicrobials and corresponding bacterial resistance mechanisms remain unknown. Here, we report that enteric infection of rhesus macaques and mice with S. Typhimurium resulted in marked Il-17- and IL-22-dependent intestinal epithelial induction and luminal accumulation of lipocalin-2, an antimicrobial protein that prevents bacterial iron acquisition. Resistance to lipocalin-2, mediated by the iroBCDE iroN locus, conferred a competitive advantage to the bacterium in colonizing the inflamed intestine of wild-type but not of lipocalin-2-deficient mice. Thus, resistance to lipocalin-2 defines a specific adaptation of S. Typhimurium for growth in the inflamed intestine.
Yiddish was spoken in pre-war Poland by just under 3 million people and thrived as a literary, theatrical, cinematic and political language in addition to being the daily vernacular of the majority ...of Polish Jewry. Today, a few native speakers of the Polish dialect of Yiddish remain in Poland but are more likely to be found in centres of Jewish culture around the world, such as London or New York. The status of Yiddish as an endangered language worldwide is well establishedi and thorough attempts to document its characteristics have already been carried out (e.g. Jacobs 2005). However, the Polish dialect of Yiddish, despite its pre-war numerical superiority, faces even greater endangerment in the face of a drive to revitalize the language based on the notion of ‘standard’ language. Illogically, the Northern form of Yiddish (Latvian and Lithuanian Yiddish) was used for the basis of standard Yiddish by the YIVO Institute for Jewish Research, New York (despite having fewer speakers) and this is the form used in textbooks and summer schools around the world to teach the language. As a result, descendants of holocaust survivors who wish to connect with their Polish inheritance via the Yiddish language are exposed to non-Polish Yiddish phonological and lexical features, which can alienate them in their attempts to reconnect to the language.
Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial ...fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.
Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized ...platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.
Heterochromatin protein 1 (HP1) family proteins are conserved chromatin binding proteins involved in gene silencing, chromosome packaging, and chromosome segregation. These proteins recognize histone ...H3 lysine 9 methylated tails via their chromodomain and recruit additional ligand proteins with diverse activities through their dimerization domain, the chromoshadow domain. Species that have HP1 proteins possess multiple paralogs that perform non-overlapping roles in vivo. How different HP1 proteins, which are highly conserved, perform different functions is not well understood. Here, we use the two Schizosaccharomyces pombe HP1 paralogs, Swi6 and Chp2, as model systems to compare and contrast their biophysical properties. We find that Swi6 and Chp2 have similar dimerization and oligomerization equilibria, and that Swi6 binds slightly (~3-fold) more strongly to nucleosomes than Chp2. Furthermore, while Swi6 binding to the H3K9me3 mark is regulated by a previously described auto-inhibition mechanism, the binding of Chp2 to the H3K9me3 mark is not analogously regulated. In the context of chromoshadow domain interactions, we show using a newly identified peptide sequence from the Clr3 histone deacetylase and a previously identified sequence from the protein Shugoshin that the Swi6 chromoshadow domain binds both ligands more strongly than the Chp2. Overall, our findings uncover quantitative differences in how Swi6 and Chp2 interact with nucleosomal and non-nucleosomal ligands and qualitative differences in how their assembly on nucleosomes is regulated. These findings provide a biochemical framework to explain the varied functions of Chp2 and Swi6 in vivo.
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•Swi6 and Chp2 play different roles in heterochromatin formation and maintenance.•Swi6 binds peptides from Sgo1 and Clr3 more strongly than Chp2.•Chp2 is not regulated by a Swi6-like auto-inhibition-based mechanism.•Chp2 and Swi6 biophysical differences found here help explain in vivo differences.
Discourses which seek to position different speakers/users of Breton through the use of labels such as 'traditional', 'new', 'learner', 'néo-bretonnant', 'brittophone', etc. draw on persistent ...essentialist ideologies of language and create, in the process, contested elites and counter-elites in Breton-speaking networks. These discourses can be counter-productive towards projects which aim at producing multilingual citizens in Brittany at the present time. This article examines how stances of different speakers towards other speakers of Breton can involve jostling for positions of power within the Breton-speaking community and how attempts at creating elites and counter-elites seem to be a defining feature of contemporary revitalisation efforts in Brittany. This characterisation may, however, miss a 'third space' which some social actors may seek to engage with. These discourses are examined in this article through a critical sociolinguistic exploration of how (elite) multilingualism is constructed, maintained and contested by different actors in the Breton language community.
Michael Hornsby-Smith offers an overview of Catholic social thought particularly in recent decades. While drawing on official teaching such as papal encyclicals and the pastoral letters of bishops' ...conferences, he takes seriously the need for dialogue with secular thought. The 2006 book is organized in four stages. Part I outlines the variety of domestic and international injustices and seeks to offer a social analysis of the causes of these injustices. Part II offers a theological reflection on the characteristics of the kingdom of God which Christians are urged to seek. Part III reviews Catholic social thought in six main areas: human rights, the family and bioethical issues, economic life, social exclusion, authentic development, and war and peace. Part IV completes the cycle with a consideration of appropriate social action responses to the injustices which the author has identified and analysed.
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