The results are presented as mean fluorescence intensity (MFI) values of at least 20,000 lactococcal cells

The results are presented as mean fluorescence intensity (MFI) values of at least 20,000 lactococcal cells. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis. and that produce lactic acid as an end product of carbohydrate metabolism [18]. Several of them are part of human intestinal and vaginal microbiota. Some LAB are used as probiotics that are defined as live microorganisms that when administered in adequate quantity confer health benefit on host [19]. is a model lactic acid bacterium with well-developed tools for genetic engineering. It has been widely used in the food industry as a starter for the production of cheese. Apart from its industrial importance [20], has also been recognized in recent years as a potential probiotic with beneficial effects E7449 in experimental colitis [21,22], which supports its role as a delivery vehicle in IBD treatment. can survive passage through the intestinal tract but does not colonize it [23]. Intrinsic probiotic efficacy of can be further strengthened by genetic engineering, and several proof-of-principle applications have already been developed [24,25,26]. Delivery of anti-TNF Nanobody [27], anti-TNF Affibody [28], trefoil factors [29] and elafin [30], by recombinant BL21(DE3), purified from inclusion bodies and refolded from 8 M urea extracts. Final product of calculated molecular weight 40 kDa is shown as a stained band after SDS polyacrylamide gel electrophoresis. Right: 96-well Polysorp ELISA plate was coated with the ILP317 protein variant in the form of a fusion with TolA-Avitag protein. p19-TRX was used as an analyte, detected by anti-IL-23 (p19) polyclonal antibody and anti-mouse IgG-HRP conjugate. The result represents three individual measurements and the error bars indicate standard deviations. 2.2. Expression of ILP-Fusion Proteins in L. lactis To verify whether IL-23-specific ILP protein binders can be produced in host cells, DNA sequences coding for ILP proteins (ILP030, ILP317 and ILP323 variants) E7449 were genetically fused to Usp45 secretion signal and C-terminal domain of AcmA protein (cA) containing LysM repeats (LysM) for peptidoglycan anchoring. Fusion genes were under the control of inducible nisin promoter. To simplify detection of particular ILP proteins, FLAG-tag sequence consensus was added between secretion signal and ILP coding sequences. Previously reported construct for the display of IL-23-binding Adnectin variant ADN23 [38] was also modified by inserting FLAG-tag. All three ILP fusion proteins, as well as the Adnectin fusion protein, were detected in bacterial cell lysates after nisin-stimulated induction using anti-FLAG antibodies (Figure 2A). No signal could be detected in empty plasmid pNZ8148-containing control cells. Also, the fusion proteins could not be observed in Coomassie Brilliant Blue stained gel (Figure 2B). However, visualization of protein lysates demonstrates their uniform concentration. The acquired data documents that all ILP proteins are expressed. Among them, ILP317-containing fusion protein was expressed at the lowest level. Open in a separate window Figure 2 Analysis of protein expression by Western blot (A) and Coomassie Brilliant Blue-stained SDS PAGE gel (B) of lysates of cells expressing ILP binding proteins and ADN23. All proteins are in fusion with Usp45 secretion signal, FLAG tag and LysM-containing cA surface anchor. 2.3. Display of ILP-Fusion E7449 Proteins on the Surface of L. lactis To investigate a secretion efficacy of the particular ILP variants produced CXCL12 in lactococcal cells, cell-surface E7449 display of AcmA-anchored ILP proteins and ADN23 was used. The bound E7449 proteins were detected with anti-FLAG antibodies using flow cytometry. The data confirm that all ILP proteins as well as ADN23 Adnectin were effectively secreted and displayed in comparison to the used negative control (Figure 3A,B). However, they were displayed in different amounts. The results document that all three ILP binding proteins were displayed in higher amounts than ADN23. The best cell-surface display was achieved with ILP317 variant, followed by ILP030 and ILP323. Open in a separate window Figure 3 Flow cytometry of cells displaying ILP proteins, or ADN23, detected with Anti-FLAG tag antibodies demonstrating mean fluorescence intensity (MFI; A) or a shift in fluorescence intensity (B). Vertical bars denote standard error. Significant differences ( 0.05) are marked with an asterisk. 2.4. Binding of Recombinant p19 by ILP-Displaying L. lactis The ability.