Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit.
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.
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia.|The Chair of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000, Ljubljana, Slovenia.
Petra
P
Zadravec
Laboratory of Ligand Engineering, Institute of Biotechnology CAS, v. v. i., BIOCEV Research Center, Průmyslová 595, 252 50, Vestec, Czech Republic.
Lucie
L
Marečková
Laboratory of Ligand Engineering, Institute of Biotechnology CAS, v. v. i., BIOCEV Research Center, Průmyslová 595, 252 50, Vestec, Czech Republic.
Hana
H
Petroková
Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia.|National Institute of Chemistry, Hajdrihova 19, 1000, Ljubljana, Slovenia.
Vesna
V
Hodnik
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia.
Milica
M
Perišić Nanut
Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia.|National Institute of Chemistry, Hajdrihova 19, 1000, Ljubljana, Slovenia.
Gregor
G
Anderluh
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia.|The Chair of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000, Ljubljana, Slovenia.
Borut
B
Štrukelj
Laboratory of Ligand Engineering, Institute of Biotechnology CAS, v. v. i., BIOCEV Research Center, Průmyslová 595, 252 50, Vestec, Czech Republic.
Petr
P
Malý
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia.
Aleš
A
Berlec
D000418:Albumins / Q000378:metabolism*; D061505:Cell Surface Display Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D013294:Lactococcus lactis / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D021122:Protein Subunits / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D022622:Shiga Toxin 1 / Q000737:chemistry* / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D000418:Albumins; D055767:Immobilized Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D022622:Shiga Toxin 1