Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: In situ cleavage of genomic target site by transferrin-ZFN1/transferrin-ZFN2 pair. ( A ) Schematic diagram of the U2OS 2-6-3 transgene array, modified from . Approximately 200 copies of this array are integrated at a single genomic site. Each repeat in the tandem array contains 256 copies of the lac operator recognition site and a single ZFN-cleavable CFP cDNA sequence. The transgene is not induced in these experiments, and several other elements not used here, and relevant only to gene expression are depicted in gray: 96 copies of a tetracycline response element, a minimal CMV promoter, a peroxisomal targeting signal (SKL), 24 copies of the MS2 translational operator, a rabbit beta-globin intron/exon module and a polyadenylation signal . ( B ) Merged images of fluorescent lac repressor (LacI-ECFP) and the DSB marker 53BP1 in untreated control cells (top) or in two representative fields of transferrin-ZFN treated cells (two lower rows). Insets show colocalization of Laci-ECFP and anti-53BP1 staining in treated cells. ( C ) Tabulation of co-localization in the indicated numbers of untreated and transferrin-ZFN treated U2OS 2-6-3 cells.

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: In Situ, Modification, Sequencing, Expressing, Marker, Staining

Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: Receptor mediated delivery concept. ( A ) Conjugation scheme. Human holo-transferrin was activated by incubation with SPDP, and conjugates were isolated and incubated with purified ZFN as described in ‘Materials and Methods’ section. A scissile disulfide bond joins the transferrin and the ZFN. ( B ) Cellular delivery. 1. The transferrin-nuclease complex binds to its cognate receptor, which induces uptake of the cargo to the cell interior in the early recycling endosome. 2. Under the reducing conditions of the endosomal milieu, the ZFN protein is released from the ligand-receptor complex by ‘self-immolation’ of the disulfide bond. 3. ZFN protein escapes the endosome. 4. ZFN protein translocates to the cell nucleus where the two ZFN subunits bind to opposing DNA strands and cleave the target sequence. Pm, plasma membrane; End, endosome; Nuc, nucleus.

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: Conjugation Assay, Incubation, Isolation, Purification, Sequencing

Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: Production and cleavage activity of transferrin-ZFN conjugates. ( A ) Purification. S-75 gel filtration chromatography of transferrin and ZFN containing reaction mix. Elution positions of conjugates and reactants are indicated. ( B ) SDS–PAGE analysis of fractions from panel (A) as indicated. Sample buffer was either non-reducing (top) or reducing (bottom). Reduction of transferrin-ZFN2 (tf-ZFN2) yields free ZFN2, free transferrin and an additional band migrating just ahead of free transferrin, the identity of which is not known. ( C ) ZFN and tf-ZFN DNA cleavage activity. Figure shows titration of ZFN2 or tf-ZFN2 with ZFN1 held constant. Titrations of ZFN1 and tf-ZFN1 were similar ( Supplementary Figure S2 ). Position of substrate and products are indicated. ( D ) Quantification of data from panel (C) showing substrate and products as a percentage of total DNA in each lane. ( E ) Cleavage activity of tf-ZFN1 and tf-ZFN2 in combination.

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: Activity Assay, Purification, Filtration, Chromatography, SDS Page, Titration

Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: Cellular delivery of transferrin-conjugated ZFN. ( A ) ZFN protein levels in HEK293/A658 cells visualized by indirect immunofluorescence using ant-FLAG primary antibody. Cells were transfected with ZFN1/2 cDNA. Alternatively, they were incubated with 100 nM transferrin-ZFN1, or 100 nM non-conjugated ZFN1, as indicated, for 1 h at 37°C. Competition experiments were performed with a 10-fold molar excess of free holo-transferrin. Z-stacks were collected with a Deltavision microscope and deconvolved. Each panel corresponds to a single Z-section. ( B ) Quantification of nuclear uptake of tf-ZFN1 protein based on anti-FLAG fluorescence intensity. HEK293/A658 cells were incubated for 60 min with various concentration of tf-ZFN1 as indicated, then fixed and stained. Measurements were based on one representative Z-section from each of 25 cells total (from five different fields) for each experimental group. Graph shows mean and standard deviation. ( C ) Quantification of nuclear uptake of tf-ZFN1 as in panel (B), except that tf-ZFN1 concentration was fixed at 100 nM, and continuous incubation was performed for the indicated times. ( D ) ZFN protein levels in U2OS 2-6-3 human osteosarcoma cells. Cells were transfected with ZFN1/2 cDNA expression plasmid or incubated with tf-ZFN1 (100 nM, 60 min) as indicated. ( E ) Same as panel (D) but with murine adult fibroblasts. ( F ) Same as panel (D) but with primary human HSPCs (CD34 + ). ( G ) Same as panel (D) but with primary mouse HSPCs (LSK). Panels compare ZFN distribution with labeled free transferrin, note difference in localization. Scale bars, 10 μm for panels (A–E), 5 μm for panels (F) and (G).

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: Immunofluorescence, Transfection, Incubation, Microscopy, Fluorescence, Concentration Assay, Staining, Standard Deviation, Expressing, Plasmid Preparation, Labeling

Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: Transferrin-ZFN mediated gene correction in HEK293/A658 cells. ( A ) Schematic diagram of the integrated GFP transgene in HEK293/A658 cells. A frameshift mutation has been introduced near the ZFN recognition site, leading to premature termination of translation (‘shown as STOP’). Treatment of the mutant cells with a GFP donor template that lacks the first 12 nt of the wild-type cDNA sequence, together with a GFP-targeting ZFN pair, induces homology-directed repair of the mutant locus and functional expression of GFP. ( B ) Representative examples of green fluorescent, GFP gene corrected HEK293/A658 cells after treatment with donor DNA and ZFN expression plasmid, middle column or tf-ZFN1/2, right column. ( C ) Representative flow cytometry plots demonstrating quantification of gene corrected, GFP-positive HEK293/A658 3 days after treatment with the transferrin-ZFN pair (left panel) or donor DNA alone (right panel). Treatment with donor DNA alone results in rare homologous recombination events at the transgene locus. ( D ) Results of independent experiments comparing the gene correction efficiency of ZFN cDNA and transferrin-ZFN-treated HEK293/A658 cells (n = 5 for transferrin-ZFN, ZFN cDNA, and no donor/no nuclease controls; n = 2 for donor/no nuclease control). Prior work has shown that there is no gene correction with nuclease in the absence of donor template .

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: Mutagenesis, Sequencing, Functional Assay, Expressing, Plasmid Preparation, Flow Cytometry, Homologous Recombination

Journal: Tissue Engineering. Part A

Article Title: Insulin, Ascorbate, and Glucose Have a Much Greater Influence Than Transferrin and Selenous Acid on the In Vitro Growth of Engineered Cartilage in Chondrogenic Media

doi: 10.1089/ten.tea.2012.0596

Figure Lengend Snippet: Representative curve fits for constructs with high E-Y (1× ITS) and with low E-Y (30× AAP) during stress-relaxation test of ramp to 10% strain, followed by 1500-s relaxation. ITS, insulin-transferrin-selenium; AAP, ascorbic acid 2-phosphate.

Article Snippet: ITS+ Premix, human holo transferrin, and bovine serum albumin/linoleic acid complex were obtained from BD Biosciences (San Jose, CA).

Techniques: Construct

Journal: Tissue Engineering. Part A

Article Title: Insulin, Ascorbate, and Glucose Have a Much Greater Influence Than Transferrin and Selenous Acid on the In Vitro Growth of Engineered Cartilage in Chondrogenic Media

doi: 10.1089/ten.tea.2012.0596

Figure Lengend Snippet: Compressive Young's moduli (E-Y) of constructs at day 42. Asterisks (*) denote p<0.05 versus corresponding 1× control; daggers (†) denote p<0.05 versus corresponding 0× group; hash marks (#) denote other statistical differences (p<0.05). INS, insulin; TR, transferrin; SA, selenous acid; GLU, glucose.

Article Snippet: ITS+ Premix, human holo transferrin, and bovine serum albumin/linoleic acid complex were obtained from BD Biosciences (San Jose, CA).

Techniques: Construct

Journal: bioRxiv

Article Title: Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

doi: 10.1101/2021.07.13.452223

Figure Lengend Snippet: A . The proposed role of Lbp system in iron acquisition from lactoferrin and protection from lactoferricin (Biorender). B . Summary of LbpB constructs used in this study. C . Solid phase binding assay of holo-lactoferrin (Lf) binding to Nm LbpB (anti-Lf) and Ng LbpB (Lf-HRP). D . Formation of the Nm LbpB-Lf complex over SEC from purified components. A leftward shift is observed for the complex compared to the individual components indicating the formation of the complex. E . SDS-PAGE analysis of the Nm LbpB-Lf complex formed from panel C, indicating the formation of the complex at a 1:1 ratio (lane 4). Similarly, the Ng LbpB-Lf complex was formed by SEC from purified components which also formed a 1:1 complex as shown by SDS-PAGE analysis.

Article Snippet: For complex formation, purified Nm/Ng LbpB was incubated with 2-fold molar excess of human holo-lactoferrin (Lf) (Raybiotech, Inc.) at 4°C for one hour.

Techniques: Construct, Binding Assay, Purification, SDS Page

Journal: bioRxiv

Article Title: Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

doi: 10.1101/2021.07.13.452223

Figure Lengend Snippet: SEC-SAXS analysis for both Nm LbpB (green) and Ng LbpB (orange) produced scattering profiles (top), Guinier plots (middle, offset for clarity), and P(r) plots (bottom) with closely matching Rg and D max values alone ( A ), in complex with lactoferrin (Lf) ( B ), and in complex with lactoferricin (Lfcn) ( C ). D . A superposition of scattering profiles for Nm LbpB alone (green), in complex with Lf (olive), and in complex with Lfcn (gray). The bottom panel shows a zoomed view at lower q values to highlight the large change for the Lf complex and the small, but reproducible change observed for the Lfcn complex (green vs gray). E . A zoomed view at lower q values comparing the Nm LbpB-Lf complex in the absence (olive) and presence (blue) of Lfcn. Again, a reproducible small change is observed in the presence of Lfcn. F . P(r) plots for Nm LbpB in the absence (green) and presence of Lfcn (gray). G . P(r) plots for Nm LbpB-Lf in the absence (olive) and presence of Lfcn (blue). H . A Crysol plot of the calculated scattering curve for the Nm LbpB structure (red line) with the experimental scattering profile (green). I . A Crysol plot of the calculated scattering curve for the Nm LbpB-Lf complex structure (red line) with the experimental scattering profile (olive).

Article Snippet: For complex formation, purified Nm/Ng LbpB was incubated with 2-fold molar excess of human holo-lactoferrin (Lf) (Raybiotech, Inc.) at 4°C for one hour.

Techniques: Produced

Journal: bioRxiv

Article Title: Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

doi: 10.1101/2021.07.13.452223

Figure Lengend Snippet: A . Zoomed view at the interface between Nm LbpB and lactoferrin (Lf) depicting the quality of the electron density shown as a gray isosurface (2F O -F C , 1.0 σ). B . Orthogonal views of the complex with Nm LbpB in green, Lf in violet, and the iron atoms as red spheres. The N-lobe of Nm LbpB interacts with only the C-lobe of Lf along an extended interface. C . The C-lobe of Nm LbpB has high B-factors with the large loops of this lobe not observed in our structure; the black arrow indicates the putative location of these loops. D . An alignment of Lf from the complex with the structure of uncomplexed Lf (PDB ID 2BJJ) shows very little conformational changes upon binding Nm LbpB (RMSD of 1.3 Å). E . A zoomed view of the binding interface shows extensive interactions along an elongated surface covering both the C1 and C2 domains of Lf (buried surface area 1760.8 Å 2 ). F . Solid phase binding assays show Lf binds both full length and N-lobe Nm LbpB, but not C-lobe only, supporting the observations in the complex structure. G . Much like what has been proposed for the role of TbpB in the Tbp system, here we propose that LbpB also serves to bind and lock Lf in an iron-bound state for delivery to LbpA for iron import.

Article Snippet: For complex formation, purified Nm/Ng LbpB was incubated with 2-fold molar excess of human holo-lactoferrin (Lf) (Raybiotech, Inc.) at 4°C for one hour.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

doi: 10.1101/2021.07.13.452223

Figure Lengend Snippet: A . Zoomed view at the interface between Ng LbpB and lactoferrin (Lf) depicting the quality of the density shown as a gray isosurface. B . Orthogonal views of the full cryoEM map with Ng LbpB in green and Lf in violet. C . Orthogonal view of an alignment of the Ng LbpB-Lf cryoEM structure (green/violet) with the Nm LbpB-Lf crystal structure (gray) (RMSD 1.4 Å along the interacting domains). D . A zoomed view of the binding interface shows extensive interactions along an elongated surface covering both the C1 and C2 domains of Lf (buried surface area 1604 Å 2 ).

Article Snippet: For complex formation, purified Nm/Ng LbpB was incubated with 2-fold molar excess of human holo-lactoferrin (Lf) (Raybiotech, Inc.) at 4°C for one hour.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

doi: 10.1101/2021.07.13.452223

Figure Lengend Snippet: A . A zoomed view of the N-lobe of Nm LbpB along the lactoferrin (Lf) interaction interface, highlighting primary residues involved in binding. B . ELISA assays to test the effects of structure-guided mutations of Nm LbpB on Lf binding along the interaction interface. C . Analysis of the binding parameters using isothermal titration calorimetry analysis of wild type and mutants of Nm LbpB measuring the effects on Lf binding.

Article Snippet: For complex formation, purified Nm/Ng LbpB was incubated with 2-fold molar excess of human holo-lactoferrin (Lf) (Raybiotech, Inc.) at 4°C for one hour.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Isothermal Titration Calorimetry

Journal: bioRxiv

Article Title: Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

doi: 10.1101/2021.07.13.452223

Figure Lengend Snippet: A . Solid phase binding assays of dilutions of Ng LbpB and mutants with HRP-conjugated lactoferrin probe. B . ELISA assays of Ng LbpB and mutants showing normalized absorbance. All experiments were done at least in triplicate.

Article Snippet: For complex formation, purified Nm/Ng LbpB was incubated with 2-fold molar excess of human holo-lactoferrin (Lf) (Raybiotech, Inc.) at 4°C for one hour.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

doi: 10.1101/2021.07.13.452223

Figure Lengend Snippet: A . A zoomed view of the C-lobe of Nm LbpB with the loops indicated in orange. B . An electrostatic surface potential representation along the C-lobe of Nm LbpB depicting the charged surfaces, including a strongly electronegative region (red). C . Analysis of the binding parameters using isothermal titration calorimetry (ITC) analysis of wild type and loop deletion mutants of Nm LbpB measuring the effects on lactoferricin (Lfcn) binding. D . ITC analysis of the Nm LbpB-Lfcn complex titrated with Lf, showing comparable binding to Nm LbpB alone. E . ITC analysis of the Nm LbpB-lactoferrin (Lf) complex titrated with Lfcn, showing comparable binding to Nm LbpB alone. F Model for the dual function of LbpB in mediating Neisserial pathogenesis by serving in both iron piracy and as an antimicrobial peptide (AMP) sink. While not shown, processing by NalP produces a soluble version of LbpB which can diffuse into the host environment to actively locate and neutralize AMP threats.

Article Snippet: For complex formation, purified Nm/Ng LbpB was incubated with 2-fold molar excess of human holo-lactoferrin (Lf) (Raybiotech, Inc.) at 4°C for one hour.

Techniques: Binding Assay, Isothermal Titration Calorimetry