Journal:

Article Title: Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands

doi: 10.1110/ps.051897806

Figure Lengend Snippet: Fluorescence microscopy analysis of peptide surface localization using CPX display. Bacterial cells coexpressing GFP and CPX1-S1 (A) or bacterial cells coexpressing GFP and CPX (no peptide) (B) were labeled with streptavidin-conjugated quantum dots. Images were acquired using GFP and PE filter sets and then overlaid.

Article Snippet: Reagents and their suppliers were as follows: primers (Integrated DNA Technologies and Operon), restriction enzymes (New England BioLabs), streptavidin-R-phycoerythrin (SAPE) (Molecular Probes), biotinylated anti-T7 tag monoclonal IgG (Novagen); MyOne streptavidin-coated magnetic microbeads (Dynal), B-PER II bacterial protein extraction reagent (Pierce Biotechnology), anti-biotin mAb R-phycoerythrin conjugate (Miltenyi Biotec), Qdot655 streptavidin conjugate (Quantum Dot Corp), and Ni-NTA agarose (Qiagen).

Techniques: Fluorescence, Microscopy, Labeling

Journal:

Article Title: Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands

doi: 10.1110/ps.051897806

Figure Lengend Snippet: Enrichment of streptavidin-binding clones from a CPX library as measured using flow cytometry. Flow cytometric analysis after labeling with 5 nM SA-PE revealed 0.01% SA-PE positive cells prior to selection (A), 1% after two rounds of MACS (B), and 85% after two rounds of MACS and one round of FACS (C). Plots use a four-decade log scale.

Article Snippet: Reagents and their suppliers were as follows: primers (Integrated DNA Technologies and Operon), restriction enzymes (New England BioLabs), streptavidin-R-phycoerythrin (SAPE) (Molecular Probes), biotinylated anti-T7 tag monoclonal IgG (Novagen); MyOne streptavidin-coated magnetic microbeads (Dynal), B-PER II bacterial protein extraction reagent (Pierce Biotechnology), anti-biotin mAb R-phycoerythrin conjugate (Miltenyi Biotec), Qdot655 streptavidin conjugate (Quantum Dot Corp), and Ni-NTA agarose (Qiagen).

Techniques: Binding Assay, Clone Assay, Flow Cytometry, Labeling, Selection

Journal:

Article Title: Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands

doi: 10.1110/ps.051897806

Figure Lengend Snippet: Streptavidin binding peptides identified using OmpX (OX) or CPX (CX) bacterial display libraries

Article Snippet: Reagents and their suppliers were as follows: primers (Integrated DNA Technologies and Operon), restriction enzymes (New England BioLabs), streptavidin-R-phycoerythrin (SAPE) (Molecular Probes), biotinylated anti-T7 tag monoclonal IgG (Novagen); MyOne streptavidin-coated magnetic microbeads (Dynal), B-PER II bacterial protein extraction reagent (Pierce Biotechnology), anti-biotin mAb R-phycoerythrin conjugate (Miltenyi Biotec), Qdot655 streptavidin conjugate (Quantum Dot Corp), and Ni-NTA agarose (Qiagen).

Techniques: Binding Assay

Journal:

Article Title: Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands

doi: 10.1110/ps.051897806

Figure Lengend Snippet: Dissociation rate constants of selected clones measured on the cell surface or for soluble proteins. (A) Measurement of the dissociation rate of several streptavidin binding bacterial clones after labeling with SA-PE and addition of biotin as a competitor. (Diamonds) CX71-S1, (triangles) OX7-S1, (circles) CX72-S2, (asterisks) OX7-S2, (squares) CX72-SA3. (B) Measurement of SA binding peptide–YFP fusions bound to SA-conjugated polymeric beads after addition of biotin as a competitor. (Diamonds) CX71-S1, (circles) CX72-S2, (triangles) OX7-S1b.

Article Snippet: Reagents and their suppliers were as follows: primers (Integrated DNA Technologies and Operon), restriction enzymes (New England BioLabs), streptavidin-R-phycoerythrin (SAPE) (Molecular Probes), biotinylated anti-T7 tag monoclonal IgG (Novagen); MyOne streptavidin-coated magnetic microbeads (Dynal), B-PER II bacterial protein extraction reagent (Pierce Biotechnology), anti-biotin mAb R-phycoerythrin conjugate (Miltenyi Biotec), Qdot655 streptavidin conjugate (Quantum Dot Corp), and Ni-NTA agarose (Qiagen).

Techniques: Clone Assay, Binding Assay, Labeling

Journal:

Article Title: Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands

doi: 10.1110/ps.051897806

Figure Lengend Snippet: Specificity of isolated clones for streptavidin. The extent of peptide binding to unrelated proteins, including anti-T7 tag monoclonal antibody at 200 nM, human serum albumin (HSA) at 500 nM, C-reactive protein (CRP) at 500 nM, and the Mona/Gads SH3 domain at 500 nM, as measured using flow cytometry.

Article Snippet: Reagents and their suppliers were as follows: primers (Integrated DNA Technologies and Operon), restriction enzymes (New England BioLabs), streptavidin-R-phycoerythrin (SAPE) (Molecular Probes), biotinylated anti-T7 tag monoclonal IgG (Novagen); MyOne streptavidin-coated magnetic microbeads (Dynal), B-PER II bacterial protein extraction reagent (Pierce Biotechnology), anti-biotin mAb R-phycoerythrin conjugate (Miltenyi Biotec), Qdot655 streptavidin conjugate (Quantum Dot Corp), and Ni-NTA agarose (Qiagen).

Techniques: Isolation, Clone Assay, Binding Assay, Flow Cytometry

Journal:

Article Title: Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 ? resolution

doi:

Figure Lengend Snippet: Statistics of data collection and structure refinement

Article Snippet: The statistics of the data collection were judged to be fit for use as listed in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Enzyme Wild type F120A F120G F120W Data collection Space group P3 2 21 Unit cell dimension (Å) a = b = 64.05, c = 63.36 a = b = 63.66, c = 63.49 a = b = 63.73, c = 63.49 a = b = 63.90, c = 63.33 (deg) γ = 120 γ = 120 γ = 120 γ = 120 X-ray source SPring-8 BL40B2 SPring-8 BL44B2 SPring-8 BL40B2 SPring-8 BL44B2 Wavelength (Å) 0.968641 0.7 0.7 0.7 Detector Rigaku RAXIS IV++ IP ADSC Quantum 4R CCD Rigaku RAXIS IV ++ IP ADSC Quantium 4D CCD Mount Cryo protectant (300 mg/ml trehalose) Maximum resolution (Å) 1.35 1.00 1.20 1.00 Data collection resolution (Å) 55.5–1.35 16.8–1.00 41.7–1.20 16.8–1.00 Number of total reflections 241,908 1,446,353 404,905 886,320 Number of unique reflections 33,471 80,089 41,727 80,099 Completeness (%) 98.8 99.7 88.8 99.6 Average I/σ 14.3 36.9 12.6 34.9 R merge 0.05 0.07 0.04 0.06 Structure refinement Refinement resolution (Å) 6.00–1.40 5.00–1.40 6.00–1.40 6.00–1.40 R-factor 0.204 0.201 0.210 0.218 R-free 0.242 0.254 0.265 0.272 RMS (bond distances) (Å) 1.2 1.2 1.1 1.2 RMS (bond angle) (deg) 0.006 0.005 0.005 0.005 Open in a separate window Statistics of data collection and structure refinement The structures were refined by rigid-body, positional, slow-cool, and b-refinement in X-PLOR.

Techniques: