X-ray Data collection and refinement statistics.

a<p>The values in parentheses refer to statistics in the highest shell.</p>b<p>Rmerge = |Ii−<i>|/|Ii| where Ii is the intensity of the ith measurement, and <i>is the mean intensity for that reflection.</i></i></p><i><i>c<p>Rwork = Σh|Fo(h)−Fc(h)|/ΣhFo(h), where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.</p>d<p>Rfree was calculated with 10% of the reflections in the test set.</p>e<p>Categories were defined by MolProbity.</p></i></i

Comparison of three structures of Cbx3 chromodomain binding to methylated histone H3, H1 and G9a peptides.

<p>(A) Superposition of human Cbx3 chromodomain in complex with methylated histone H1 peptide (yellow), histone H3 peptide (orange), G9a peptide (cyan), Cbx3 chromodomains are colored as magenta, gray and green, respectively. (B) Superposition of histone H1 peptide (yellow), histone H3 peptide (orange). (C) Structure of Cbx3-H3K9me3 complex (magenta) was superposed to one protomer of the tetramer of Cbx3-G9aK185me3 complex (green) formed in one asymmetric unit. (D) The α helix (residues 70 to 79) of the chromodomain in the structure of Cbx3-G9aK185me3 complex (green) shifts 4.9 Å away from its counterpart in the structures of Cbx3-H3K9me3 (magenta).</p

Human Cbx3 chromodomain binds to methylated histone H1K26 and G9aK185.

<p>ITC data for Cbx3 chromodomain binding to (A) H1K26 peptides (residues 18–29) and (B) G9aK185 peptides (residues 179–190). Lower panel show fit to a one-site binding model to the binding isotherms.</p

Structure basis for Cbx3 binding to methylated histone H1K26 and G9aK185 peptide.

<p>(A and C) Electrostatic surface depiction of human Cbx3-histone H1K26me2, and Cbx3-G9aK185me3 complex. Peptide substrates are shown in a stick mode. Surfaces with positive electrostatic potential are blue, and negative potential are red. The side chain of residue H1A24 (G9aA183) inserts into the small hydrophobic pocket formed by Phe48 and Leu49 of human Cbx3. The size of the pocket is only sufficient to accommodate a methyl group but not other residue side chains. (B and D) Binding of histone H1 peptide and G9a peptide in the binding groove of Cbx3 chromodomain, respectively. Hydrogen-bonds are shown as dashed lines. Yellow: histone H1 peptide; Gray: Cbx3 chromodomain in Cbx3-histone H1K26me2 complex. Cyan: G9a peptide; Green: Cbx3 chromodomain in Cbx3-G9aK185me3 complex.</p

Peptide binding specificity of human Cbx3 chromodomain.

*<p>N/B: No binding was detected.</p

DS_DISC766278 – Supplemental material for Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain

<p>Supplemental material, DS_DISC766278 for Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain by Guillermo Senisterra, Hugh Y. Zhu, Xiao Luo, Hailong Zhang, Guoliang Xun, Chunliang Lu, Wen Xiao, Taraneh Hajian, Peter Loppnau, Irene Chau, Fengling Li, Abdellah Allali-Hassani, Peter Atadja, Counde Oyang, En Li, Peter J. Brown, Cheryl H. Arrowsmith, Kehao Zhao, Zhengtian Yu, and Masoud Vedadi in SLAS Discovery</p

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

<div><p>We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that <i>in vivo</i> biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 <i>in vivo</i> biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce <i>in vivo</i> biotinylated Fabs in <i>E</i>. <i>coli</i>. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.</p></div

Fab and IgG production.

<p>(A) Comparison of purification yields between different expression vectors using an anti-MBP Fab as an example. The large-scale purification method on the ÄKTA Xpress including a heat denaturation step was used. (B) SDS-PAGE gel showing the anti-MBP Fab produced with various expression vectors and purified in triplicate. (C) IgG production yields with and without the dilution strategy.</p

Overview of targets and their success rates.

<p>Overview of the 265 antigen domains included in the study and what protein families they belong to. Family success rate was calculated as percentage of targets that were successful in selections and which also yielded at least one antibody that passed cell-based validation.</p><p>Overview of targets and their success rates.</p

Performance consistency among Fabs and IgGs generated against the same target.

<p>Multiple Fabs and IgGs against several targets were used to immunoprecipitate their corresponding FLAG-tagged antigens. Western blot was performed and the presence of the FLAG-tagged immunoprecipitated protein was detected with an antibody against the tag. A) CBX3. B) L3MBTl2, C) SFMBT2, D) TDRD3. FLAG-tagged GFP was used as control (data not shown). Fab batches are labeled with a trailer “-Bxxx” and IgG batches are labeled with a trailer “-IBxxx”. Fabs against CBX3 and SFMBT2 have been produced twice (CBX3 (B002, B004); SFMBT2 (B002, B004)) while Fabs against L3MBTL2 and TDRD3 have been produced only once (L3MBTL2 (B001); TDRD3 (B001)). Multiple IgGs have been produced with corresponding IB numbers. Fabs/IgGs derived from the same phagemid clone have similar efficiencies and show a high lot-to-lot consistency.</p