Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: Enzymatic release of tANCHOR-displayed proteins (A) Schematic of TEVp-mediated release of a protein of interest using the tANCHOR display system. Fusion tANCHORed proteins enter the endoplasmic reticulum for N-linked glycosylation and quality control and are then trafficked via the secretory pathway in membrane-bound carriers to the Golgi/TGN for further glycan processing and maturation before reaching the plasma membrane (PM, left side). At the cell surface, the tANCHOR construct presents the protein of interest through a four-helix multipass anchor with an N-terminal FLAG tag and a C-terminal mCherry reporter (right side). The displayed protein is linked to the anchor by TEVp cleavage sites. Addition of TEVp cleaves these sites and releases the protein, while the membrane-anchored domains remain at the cell surface. (B) Schematic of the expression construct for protein release in case of the receptor-binding domain (RBD) of the SARS-CoV-2 delta variant. V5-His-tag, GKPIPNPLLGLDST fused to 6× histidine; FLAG tag: DYKDDDDK; mCherry, red fluorescent protein used as C-terminal reporter tag; POI, protein of interest; CMV: major immediate-early enhancer and promoter of cytomegalovirus; TM, transmembrane domains of the tANCHOR tetraspanin core. (C) HEK293T cells expressing the tANCHORed RBD-V5-His protein were analyzed for protein localization by confocal laser scanning microscopy (CLSM). Scale bars, 10 μm, in the cutout, 5 μm. (D) Cell-based binding assay ( n = 4) for confirming extracellular HeLa cell accessibility of the CD82 tANCHORed RBD (amino acids 319–543) fused to V5-His-tag (dark blue dots). V5-His, cells transfected with ptANCHOR-CD82-V5-His-mCherry (pale blue dots); control, without transfection (gray dots). The dotted line represents the cut-off value. Statistical analysis was performed using an unpaired two-tailed Welch’s t test. Significance levels: non-significant (ns), p > 0.05. (E) Cartoon of the workflow for the isolation and analysis of the released protein. (F) HEK293T cells expressing the tANCHORed SARS-CoV-2 RBD delta variant (amino acids 319–543) -V5-His with flanked TEVp cleavage sites were incubated for 24 h post-transfection with different concentrations of recombinant TEVp. Supernatant and cell lysates were analyzed by western blotting, using the anti-V5 epitope for the detection of released and tANCHORed RBD-V5-His. ACTB, beta-actin loading control. (G) Removal of glycans attached to asparagine of the released SARS-CoV-2 delta variant RBD-V5-His protein bound to Dynabeads with peptide N-glycosidase F (PNGase F). The eluted protein was subsequently examined by western blotting.

Article Snippet: Transfection complexes were added to the cells and incubated for 24 h. The medium was then replaced with pre-warmed (37°C) medium containing 2% FBS and recombinant TEV protease (New England Biolabs, Frankfurt, Germany) at the indicated dilution.

Techniques: Glycoproteomics, Control, Membrane, Clinical Proteomics, Construct, FLAG-tag, Expressing, Binding Assay, Variant Assay, Confocal Laser Scanning Microscopy, Cell Binding Assay, Transfection, Two Tailed Test, Isolation, Incubation, Recombinant, Western Blot

Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: Quantification of released RBD-V5-His and monitoring of cytotoxic effects during TEVp treatment (A) RBD-V5-His, released by added TEVp from transfected HEK293T cells, was isolated from the medium by a V5-specific nanobody trap, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4%–15% gradient gel, stained after separation with Coomassie Brilliant Blue R-250 (top), and subsequently blotted onto a polyvinylidene difluoride (PVDF) membrane and probed for V5-tag (bottom). BSA, bovine serum albumin. (B) Estimation of protein amounts by densitometry, using profiles of the bands and calibrated to BSA protein standard. a.u., arbitrary units. (C–E) Expression analysis in HEK293T cells of tANCHORed RBD-V5-His flanked by TEVp cleavage sites, in the presence or absence of recombinant TEVp. (C) Fluorescence microscopy images of cells transfected with the plasmid ptANCHOR-CD82- TEVp RBD Delta -V5-His- TEVp -mCherry showing TEVp-dependent decrease in the fluorescence intensity of mCherry. Scale bars, 100 μm. (D) Quantification of the expressed mCherry-fused tANCHORed RBD-V5-His within a 6-well plate by the fluorescence signals emitted by the mCherry reporter protein. At 24 h, TEVp was added. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed using an ordinary two-way ANOVA (time × TEVp concentration), followed by Dunnett’s multiple comparison test to compare each TEVp concentration to 800 U/mL TEVp. Time, TEVp concentration, and their interaction were significant ( ∗∗∗∗ p < 0.0001). (E) Western blot analysis indicating the release of the fused RBD-V5-His protein from cells due to the reduction of the mCherry-tagged full-length protein within the cell membrane upon treatment with TEVp (top). Following treatment with TEVp, cell pellets exhibited a white coloration, contrasting with the pink hue observed in untreated pellets (bottom). (F) Monitoring of LDH release from the cells ( n = 3) transfected with the plasmid ptANCHOR-CD82- TEVp -RBD Delta -V5-His- TEVp -mCherry for the identification of cytotoxic effects with the rising recombinant TEVp concentrations; treatment with 1% IGEPAL CA-630 was used as a cell lytic positive control. (G) Extended incubation time ( n = 4) at a TEVp concentration of 800 U/mL. Statistical significance was assessed using a one-way ANOVA followed by Tukey’s post hoc test (F) and groups were compared for significance by an unpaired two-tailed Welch’s t test (G); non-significant (ns), >0.05; ∗ p < 0.05.

Article Snippet: Transfection complexes were added to the cells and incubated for 24 h. The medium was then replaced with pre-warmed (37°C) medium containing 2% FBS and recombinant TEV protease (New England Biolabs, Frankfurt, Germany) at the indicated dilution.

Techniques: Transfection, Isolation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Membrane, Expressing, Recombinant, Fluorescence, Microscopy, Plasmid Preparation, Concentration Assay, Comparison, Western Blot, Positive Control, Incubation, Two Tailed Test

Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: Engineering, secretion, and activity of secreted TEVp- c -Myc-His variants (A) Schematic for the surface model of TEVp, illustrating the relative positions and functional classes of mutations introduced to enable activity during passage through the secretory pathway. The catalytic triad (H46, D81, and C151) and the approximate location of the active-site cleft are shown for orientation only. The construct includes residues 1–236, excluding the autoinhibitory ELVYSQ tail, and carries a C-terminal c -Myc-6×His-tag for detection and purification. (B) Schematic of the expression constructs (not to scale). The mutation sites were introduced in the wild-type TEVp sequence. SP1, signal peptide derived from the Gaussia princeps luciferase gene; c -Myc-His-tag, EQKLISEEDL fused to 6× histidine; TEVp, tobacco etch virus protease; CMV, major immediate-early enhancer and promoter of cytomegalovirus; WT, wildtype; aa, amino acid. The numbers inside the green boxes refer to the positions of mutation sites in the TEVp sequence. (C) Western blot analysis of collected supernatants and cell lysates derived from HEK293T cells transfected with constructs for TEVp- c -Myc-His variants probed by anti- c -Myc-tag antibodies. ACTB, beta-actin control protein probed by using anti-ACTB antibodies. (D) TEVp activity was monitored at 34°C to evaluate the cleavage performance of expressed TEVp- c -Myc-His variants. This assessment utilized supernatants collected from the experiment. Control, no transfection; RLUs, relative light units. (E) Slopes were determined by linear regression. Statistical differences in slopes were evaluated by comparing 95% confidence intervals. Groups with non-overlapping intervals were considered significantly different.

Article Snippet: Transfection complexes were added to the cells and incubated for 24 h. The medium was then replaced with pre-warmed (37°C) medium containing 2% FBS and recombinant TEV protease (New England Biolabs, Frankfurt, Germany) at the indicated dilution.

Techniques: Activity Assay, Functional Assay, Construct, Purification, Expressing, Mutagenesis, Sequencing, Derivative Assay, Luciferase, Virus, Western Blot, Transfection, Control

Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: Efficient TEVp-mediated protein release is achieved by co-expression of TEVp in tANCHORed RBD-expressing cells (A) Cartoon showing co-cultivation and co-transfection experimental setup for protein release using optimized TEVp- c -Myc-His. (B) Images of fluorescence emitted by HEK293T cells expressing tANCHORed RBD-V5-His fused with mCherry (red); additionally, TEVp- c -Myc-His-transfected cells (top) or all cells (bottom) were stained with the live cell-tracking fluorescence dye 5-chloromethylfluorescein diacetate (CMFDA, green). Cells expressing only the tANCHORed TEV RBD-V5-His TEV are indicated by a white arrow, while cells co-expressing the tANCHORed TEV RBD-V5-His TEV and TEVp N23Q,C130S,T173G,S219V - c -Myc-His are marked with a yellow arrow. Scale bars, 10 μm. (C) Western blot analysis of RBD-V5-His protein released from HEK293T cells transfected with ptANCHOR- TEV RBD Delta -V5-His TEV -mCherry, using co-cultivation with HEK293T cells transfected with the indicated pTEVp- c -Myc-His plasmids (top) or co-transfection approach (bottom). Released RBD-V5-His and the uncleaved tANCHORed RBD-V5-His were probed using anti-V5 antibodies (topmost and third lanes), and TEVp- c -Myc-His expression was detected by anti- c -Myc antibodies (second lane). ACTB, beta-actin control protein probed by anti-ACTB antibodies. (D) Protein structure predictions of the mutated TEVp N23Q,C130S,T173G,S219V - c -Myc-His (left) and the tANCHORed RBD-V5-His (right side). The colors used to represent the various components of the protein are as follows: 6×His-tag (yellow), c -Myc-tag (magenta), V5-tag (cyan), and FLAG tag (blue). The active center of TEVp, which contains the catalytic triad (H46, D81, and C151), is shown in red, along with the TEVp cleavage sites of tANCHORed RBD-V5-His. The scissile site is rendered in stick representation. The light blue shading indicates the membrane, with gray dots representing the lipid carbonyl groups of the membrane bilayer.

Article Snippet: Transfection complexes were added to the cells and incubated for 24 h. The medium was then replaced with pre-warmed (37°C) medium containing 2% FBS and recombinant TEV protease (New England Biolabs, Frankfurt, Germany) at the indicated dilution.

Techniques: Expressing, Cotransfection, Fluorescence, Transfection, Staining, Cell Tracking Assay, Western Blot, Control, FLAG-tag, Membrane

Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: Analysis of de novo RBD expression, secretion, and intracellular TEVp-mediated cleavage in co-transfected HEK293T cells (A) Overview of the experimental set-up to analyze the total RBD-V5-His release and TEVp- c -Myc-His secretion (left side) and de novo RBD-V5-His release (right side) from co-transfected HEK293T cells. (B) Western blot analysis of the samples derived from experiment (A) by detection of V5 or c -Myc tag. (C) Western blot analysis of the secreted and residual intracellular RBD-V5-His (top) and TEVp N23Q,C130S,T173G,S219V - c -Myc-His (bottom) isolated using His-tag from 1 mL of supernatant (left lanes) or lysed co-transfected HEK293T cells from one well of a 6-well plate in 1 mL (right lanes). Cells were lysed with 1% of IGEPAL CA-630, which was also added to the supernatant. (D) Coomassie Brilliant blue-stained polyacrylamide gel quantified by comparison with a bovine serum albumin standard curve (left) and subsequent western blot probed by anti-V5 antibodies (right) of the protein released from the supernatant of co-transfected HEK293T cells obtained by co-expression of tANCHORed RBD-V5-His (top) or CD63LEL-V5-His (bottom) with or without TEVp N23Q,C130S,T173G,S219V - c -Myc-His. Highly glycosylated CD63LEL-V5-His was treated with PNGase F (peptide N-glycosidase F). (E) Lectin blot of the released RBD-V5-His protein, probed with biotinylated Sambucus nigra agglutinin (SNA) and detected using streptavidin-HRP. (F and G) Western blot analysis of the cell lysates for the expression of CD63-mCherry (F) or CD82-tANCHOR-CD63LEL-mCherry (G), with or without modified TEVp expression. Control contains untransfected cells, and TEVp was captured from the supernatant.

Article Snippet: Transfection complexes were added to the cells and incubated for 24 h. The medium was then replaced with pre-warmed (37°C) medium containing 2% FBS and recombinant TEV protease (New England Biolabs, Frankfurt, Germany) at the indicated dilution.

Techniques: Expressing, Transfection, Western Blot, Derivative Assay, Isolation, Staining, Comparison, Modification, Control

Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: Analysis of cytotoxicity and cell viability (A) Top: Monitoring of cytotoxic effects ( n = 3) employing lactate dehydrogenase (LDH) release from HEK293T cells co-transfected with pTEVp N23Q,C130S,T173G,S219V - c -Myc-His and ptANCHOR-CD82- TEV RBD Delta -V5-His TEV -mCherry (top). Positive LDH release control is represented by the cells lysed by 1% IGEPAL CA-630, and negative control is represented by untransfected cells. Bottom: Western blot of LDH release samples probed with anti-V5 antibodies for released or tANCHORed RBD Delta -V5-His in supernatant (top) and cell lysate (bottom), with anti- c -Myc antibodies for TEVp- c -Myc-His; control protein beta-actin (ACTB) was detected by anti-ACTB antibodies. (B) Cell viability after 48 h post-transfection was determined using the MTT assay ( n = 7) and is expressed as a percentage relative to cells treated with transfection reagent only. Cells treated with 15 mM sodium azide (NaN 3 ) served as a cytotoxicity control. Data were analyzed for significance by one-way ANOVA, followed by Tukey’s post hoc test. Significance levels: non-significant (ns), p > 0.05.

Article Snippet: Transfection complexes were added to the cells and incubated for 24 h. The medium was then replaced with pre-warmed (37°C) medium containing 2% FBS and recombinant TEV protease (New England Biolabs, Frankfurt, Germany) at the indicated dilution.

Techniques: Transfection, Control, Negative Control, Western Blot, MTT Assay

Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: ENCOREP enables TEVp-mediated release of small extracellular peptides (A) Schematic of canine CD20 (cCD20) topology, highlighting the second extracellular loop (EC2), the C46 peptide derived from HIV-1 gp41, and the EC2 of CXCR4. The indicated regions (red) correspond to extracellular domains that were fused to the tANCHOR backbone. (B) Western blot analysis of released cCD20-EC2-V5-His, gp41-C46-V5-His, and CXCR4-EC2-V5-His peptides in the culture supernatant (left). The arrowhead indicates the band corresponding to the released peptide. Transfected cells collected and centrifuged from the release experiment (right) show reduced pink coloration due to TEVp-mediated peptide release. (C) mCherry fluorescence (red) indicates expression and subcellular localization of CD82 tANCHORed peptides; nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 10 μm. (D) Cell-based binding assay ( n = 4) using anti-V5-HRP antibody on transfected HeLa cells to confirm extracellular accessibility of the tANCHOR-fused peptides. Groups were compared using one-way ANOVA, followed by Dunnett’s multiple comparison test (reference group: V5-His). Significance levels: non-significant (ns), p > 0.05; ∗∗ p < 0.01. Dotted line indicates the cut-off value.

Article Snippet: Transfection complexes were added to the cells and incubated for 24 h. The medium was then replaced with pre-warmed (37°C) medium containing 2% FBS and recombinant TEV protease (New England Biolabs, Frankfurt, Germany) at the indicated dilution.

Techniques: Derivative Assay, Western Blot, Transfection, Fluorescence, Expressing, Cell Binding Assay, Comparison