Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A CD95L can exist either in a membrane-bound or in a soluble form, the latter resulting from protease cleavage. Key domains include the CD95 binding domain (CD95 Bind), the TNF homology domain (THD) for its homotrimerization, and the proline-rich domain (PRD). B Workflow of the CD95L-IZ construct design, secretory expression, affinity purification, and modification. The recombinant protein consists of a signaling peptide (Sig), an affinity tag (Tag), a cleavage site (Clv), a Cysteine (Cys), an isoleucine zipper domain (IZ) fused via a linker to the extracellular CD95L domain. In method 1 transient CD95-IZ expression was used. Here, a His-tag was used as Tag and a TEV protease sequence as Clv. Affinity purification was done by anti-His-tag antibody coupled agarose beads. In method 2 a cell line stably expressing CD95-IZ was used. Here, a Twin-Strep-Tag was used as Tag and a PreScission cleavage site as Clv. Affinity purification was done by Strep-TactinXT4Flow beads. C Upper panel: examination of cell morphology before and up to three days after transfection. Lower panel: photos of cell culture and transfection using a 10-layer cell factory in a cell culture hood (left) or incubator (right). A T175 cell culture flask was seeded with the same cell density as in a 10-layer cell factory to check the cell condition and confluency. D CD95L-IZ protein expression and secretion was verified by a Western blot of the cell culture medium using anti-CD95L antibody. The image contrast was adjusted for better visibility.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Membrane, Binding Assay, Construct, Expressing, Affinity Purification, Modification, Recombinant, Sequencing, Stable Transfection, Strep-tag, Transfection, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A ELISA analysis of CD95L-IZ-biotin and CD95L-Enzo binding to CD95. B Comparison of representative bright field images showing morphological changes of cells treated with CD95L-IZ-X or soluble CD95L (Enzo Life Science) after 2, 6, and 15 h. Red asterisks indicate the position of apoptotic cell. Scale bars are 50 µm. C Apoptosis dynamics analysis of CD95L-IZ apoptosis induction at different stages: post-purification, post-His-tag cleavage, post-biotinylation, and comparison with CD95L-Enzo. D The efficiency of CD95L-IZ-His apoptosis induction is increased by anti-His-tag antibody crosslinking. E The presence of CD95-His (in 10x, 50x, 100x, and 200x molar excess in comparison to CD95L-IZ-biotin) as competitive binders to the natural CD95 on the cell membrane, successively inhibits CD95L-IZ-biotin apoptosis induction. In panels C, D, and E data points are marked with dots. Hill equation fit curves are represented by solid lines, and standard deviations are indicated with shaded regions.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Comparison, Purification, Membrane

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A CD95L can exist either in a membrane-bound or in a soluble form, the latter resulting from protease cleavage. Key domains include the CD95 binding domain (CD95 Bind), the TNF homology domain (THD) for its homotrimerization, and the proline-rich domain (PRD). B Workflow of the CD95L-IZ construct design, secretory expression, affinity purification, and modification. The recombinant protein consists of a signaling peptide (Sig), an affinity tag (Tag), a cleavage site (Clv), a Cysteine (Cys), an isoleucine zipper domain (IZ) fused via a linker to the extracellular CD95L domain. In method 1 transient CD95-IZ expression was used. Here, a His-tag was used as Tag and a TEV protease sequence as Clv. Affinity purification was done by anti-His-tag antibody coupled agarose beads. In method 2 a cell line stably expressing CD95-IZ was used. Here, a Twin-Strep-Tag was used as Tag and a PreScission cleavage site as Clv. Affinity purification was done by Strep-TactinXT4Flow beads. C Upper panel: examination of cell morphology before and up to three days after transfection. Lower panel: photos of cell culture and transfection using a 10-layer cell factory in a cell culture hood (left) or incubator (right). A T175 cell culture flask was seeded with the same cell density as in a 10-layer cell factory to check the cell condition and confluency. D CD95L-IZ protein expression and secretion was verified by a Western blot of the cell culture medium using anti-CD95L antibody. The image contrast was adjusted for better visibility.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Membrane, Binding Assay, Construct, Expressing, Affinity Purification, Modification, Recombinant, Sequencing, Stable Transfection, Strep-tag, Transfection, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A CD95L-IZ purification and functionalization. Lane 1 Affinity purification of CD95L-IZ-His using anti-His-tag antibody coupled agarose beads followed by 10 % SDS-PAGE analysis of the purified protein. Lane 2-3 Western blot analysis using anti-His-tag antibody: 100 ng CD95L-IZ-His before (lane 2) and 100 ng CD95L-IZ after His-tag cleavage (lane 3). Lane 4-5 Western blot analysis of site-specific biotinylation using the cysteine maleimide reaction: 300 ng CD95L-IZ before (lane 4) and after (lane 5) biotinylation were loaded, probed with AF488-streptavidin. B Size exclusion chromatography (SEC) analysis of CD95L-IZ-His after His-tag affinity purification compared with a protein standard mix run under identical conditions. The SEC data exhibits peaks corresponding to the trimeric, the dimeric, and the monomeric form of CD95L-IZ-His. C From the SEC data, a standard curve of the protein standard mix is calculated and peak positions of the CD95L-IZ-His data are plotted to derive the corresponding molecular weight. D Droplet assay to verify biotinylation. Top: a sketch of the assay. Bottom: Fluorescence colocalization images of the 488 nm and the 594 nm channel. CD95L-IZ-biotin was non-specifically labeled with ATTO594 NHS-ester and incubated with AF488-streptavidin on a BSA passivated glass coverslip. E DNA origami functionalized with CD95L-IZ-ssDNA. The DNA handle on the CD95L-IZ hybridizes to a complementary anti-handle on the DNA origami, attaching the ligand site-specifically to the DNA origami. The TEM micrograph on the lower right is duplicated and origami outlines and proteins are indicated for better interpretability.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Purification, Affinity Purification, SDS Page, Western Blot, Size-exclusion Chromatography, Molecular Weight, Fluorescence, Labeling, Incubation

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A ELISA analysis of CD95L-IZ-biotin and CD95L-Enzo binding to CD95. B Comparison of representative bright field images showing morphological changes of cells treated with CD95L-IZ-X or soluble CD95L (Enzo Life Science) after 2, 6, and 15 h. Red asterisks indicate the position of apoptotic cell. Scale bars are 50 µm. C Apoptosis dynamics analysis of CD95L-IZ apoptosis induction at different stages: post-purification, post-His-tag cleavage, post-biotinylation, and comparison with CD95L-Enzo. D The efficiency of CD95L-IZ-His apoptosis induction is increased by anti-His-tag antibody crosslinking. E The presence of CD95-His (in 10x, 50x, 100x, and 200x molar excess in comparison to CD95L-IZ-biotin) as competitive binders to the natural CD95 on the cell membrane, successively inhibits CD95L-IZ-biotin apoptosis induction. In panels C, D, and E data points are marked with dots. Hill equation fit curves are represented by solid lines, and standard deviations are indicated with shaded regions.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Comparison, Purification, Membrane