Novel high‐affinity binders of human interferon gamma derived from albumin‐binding domain of protein G

Ahmad, Jawid Nazir; Li, Jingjing; Biedermannová, Lada; Kuchař, Milan; Šípová, Hana; Semerádtová, Alena; Černý, Jiří; Petroková, Hana; Mikulecký, Pavel; Polínek, Jiří; Staněk, Ondřej; Vondrášek, Jiřı́; Homola, Jiřı́; Malý, Jan; Osička, Radim; Šebo, Peter; Malý, Petr

doi:10.1002/prot.23234

Cited by 33 publications

(44 citation statements)

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“…The identified S1B binders were produced in E . coli as fusion proteins with TolA spacer containing AviTag on the C-terminus and a H6 tag on the N-terminus (H6-S1B-TolA-Avi), as reported previously [11–13]. The expression level of the corresponding fusion protein was tested by SDS PAGE and the binding capacity verified by ELISA (Fig 2B).…”

Section: Resultsmentioning

confidence: 93%

“…To obtain novel protein binders of Shiga toxin, a highly complex combinatorial library derived from streptococcal albumin-binding domain scaffold was therefore applied. This has been used successfully for selecting high-affinity binders of IFN-γ [11], for development of IL-23 receptor antagonists [12] and for generation of unique binders of human prostate cancer oncomarker [13]. …”

Section: Resultsmentioning

confidence: 99%

“…To select unique binders of Stx1B we used the highly-complex combinatorial ABD library [11], [12], [13] and ribosome display. Ribosome display enables the selection of binding proteins by associating ribosome-translated proteins with their parent mRNA in a complex.…”

Section: Resultsmentioning

confidence: 99%

“…The combinatorial DNA library of ABD protein was generated as described [11, 12], in vitro transcribed and the resulting mRNA was translated using E . coli extract (EasyXpress Protein Synthesis Mini Kit, QIAGEN, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Compared to antibodies, engineered non-Ig scaffolds are stable, robust and soluble monomeric proteins, that lack disulfide bonds, are easily and inexpensively produced in large amounts in bacteria, exhibit effective tissue penetration, rapid distribution and elimination, can be easily modified by conjugation or gene fusion technology and can exert multivalency or multispecificity [9, 10]. Among them, a highly complex combinatorial library, derived from the three-helix bundle of the albumin-binding domain (ABD) scaffold of streptococcal protein G has been used successfully for the selection of human interferon gamma binders [11], interleukin (IL)-23 receptor antagonist [12] and binders of human prostate secretory protein 94 (PSP94) [13]. In this work we applied the ABD scaffold library for the selection of unique binders of Stx1B.…”

Section: Introductionmentioning

confidence: 99%

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Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

et al. 2016

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Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine.

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Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

et al. 2016

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Biocompatible Size‐Defined Dendrimer–Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications

Malý

Staněk

Frolík

et al. 2016

Macromolecular Bioscience

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For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures.

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Human interleukin‐23 receptor antagonists derived from an albumin‐binding domain scaffold inhibit IL‐23‐dependent ex vivo expansion of IL‐17‐producing T‐cells

et al. 2013

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Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin-23 receptor (IL-23R), which is a key element of proinflammatory IL-23-mediated signaling. A library of variants derived from the three-helix bundle scaffold of the albumin-binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high-affinity binders of recombinant extracellular IL-23R. A collection of 34 IL-23R-binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL-23, or the biologically active human IL-23 cytokine, to the recombinant IL-23R or soluble IL-23R-IgG chimera. The strongest competitors for IL-23R binding in ELISA were confirmed to recognize human IL-23R-IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub- to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K-562, THP-1 and Jurkat, and this binding correlated with IL-23R cell-surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4+ T-cells. Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals. Proteins 2014; 82:975–989.

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Novel high‐affinity binders of human interferon gamma derived from albumin‐binding domain of protein G

Cited by 33 publications

References 50 publications

Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

Biocompatible Size‐Defined Dendrimer–Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications

Human interleukin‐23 receptor antagonists derived from an albumin‐binding domain scaffold inhibit IL‐23‐dependent ex vivo expansion of IL‐17‐producing T‐cells

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