Sequence similarity analysis (A) and binding affinity (B) of 17 S1B binders selected after five rounds of ribosome display.

<p>A: The sequence of the parental ABD wild-type domain was used as a root of the tree and is highlighted as a square, while S1B variants selected for more detailed analysis are highlighted as circles. B: S1B binders-containing cell lysates were incubated with immobilized Stx1B (grey bars) or BSA (white bars) and detected with HRP-conjugated streptavidin. Error bars denote standard deviations.</p

Influence of S1B binders on Stx1B transport into HeLa cells.

<p>A, B: Flow cytometric analysis of HeLa cells demonstrating shift in fluorescence intensity (A) and mean fluorescence intensity (MFI; B) upon 1h incubation of HeLa cells with mixtures of S1B22, S1B26 or ABDwt (all in fusion with TolA-Avi and H6) and Alexa Fluor 488-labeled Stx1B. Cont: unstained HeLa cells. Stx1B: HeLa cells incubated with Stx1B-Alexa Fluor 488 alone. C, D: Fluorescence microscopy images of HeLa cells incubated with Alexa Fluor 488 labelled Stx1B (green) with or without pre-incubation with S1B22 and S1B26. DAPI staining (blue) was used to label nuclei. Cells were stained either with PKH26 membrane labeling dye (red; C) and Golgi apparatus was detected with mouse monoclonal Golgin-97 antibody and secondary polyclonal goat anti-mouse antibody conjugated with Alexa Fluor 633 (purple; D). Bars = 10 μm.</p

Development of Recombinant <i>Lactococcus lactis</i> Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit - Fig 7

<p>(A) SDS PAGE analysis of lysates of <i>L</i>. <i>lacti</i>s cells expressing S1B22, S1B26, ABDwt and H6-ABDwt (ABDwtH), all in fusion with Usp45 secretion signal and the LysM-containing cA domain, and stained with Coomassie brilliant blue. ABD fusion proteins are high-lighted with arrows. (B) Flow cytometric analysis of ABD surface display, detection with FITC-conjugated human serum albumin. The MFI value of ABDwt was compared with that of the control using Student’s t test. *** p<0.001. Cont.: control containing empty plasmid pNZ8148.</p

Strains, plasmids, gene and primers used in this study.

<p>Strains, plasmids, gene and primers used in this study.</p

Flow cytometric (A) and whole-cell ELISA (B) analyses of binding of recombinant Stx1B by <i>L</i>. <i>lactis</i> cells displaying S1B variants or ABDwt on their surface.

<p>(A) Alexa488-conjugated Stx1B was used for detection. MFI: Mean fluorescence intensity. (B) Mouse antiStx1B antibody and HRP-conjugated anti mouse antibody were used for detection of Stx1B. A₄₅₀: Absorbance at 450 nm. Vertical bars denote standard deviation. MFI or A₄₅₀ values of S1B binders were compared to those of the ABDwt control using Student’s t test. *: p<0.05, ** p<0.01, *** p<0.001.</p

SPR analysis of binding of S1B22, S1B26 and ABDwt in fusion with TolA-Avi and H6 at 1 μM concentration to immobilized recombinant Stx1B.

<p>SPR analysis of binding of S1B22, S1B26 and ABDwt in fusion with TolA-Avi and H6 at 1 μM concentration to immobilized recombinant Stx1B.</p