High-throughput sorting of an Anticalin library via EspP-mediated functional display on the Escherichia coli cell surface

Journal of Molecular Biology
Uli BinderArne Skerra

Abstract

We demonstrate that small engineered single-chain binding proteins based on the lipocalin scaffold, so-called Anticalins, can be functionally displayed on the Gram-negative bacterial cell envelope. To this end, the beta-domains of five different bacterial autotransporters (the IgA protease from Neisseria gonorrhoeae, the esterase EstA from Pseudomonas aeruginosa, the YpjA autotransporter from E. coli K12, the AIDA-I adhesin from enteropathogenic E. coli O127:H27 strain 2787 and the protease EspP from enterohemorrhagic E. coli O157:H7 strain EDL933) were compared with respect to display level, functional variance, and bacterial cell viability. Use of the EspP autotransporter led to a system with high genetic stability for the display of fully functional Anticalins in high density on the cell surface of E. coli as shown by quantitative flow cytofluorimetry. This system was applied to engineer an immunostimulatory Anticalin that binds and blocks the extracellular region of human CTLA-4 to achieve a slower dissociation rate. A combinatorial library of the original Anticalin was generated by error-prone PCR, subjected to E. coli cell surface display, and applied to repeated cycles of cell sorting after incubation with the fluorescen...Continue Reading

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Citations

Sep 29, 2012·The Journal of Biological Chemistry·Balakrishnan RameshNavin Varadarajan
Jan 24, 2016·Protein Engineering, Design & Selection : PEDS·Cyril BarinkaArne Skerra
May 10, 2012·Journal of Biotechnology·Joachim JoseMark George Teese
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Dec 1, 2017·Journal of Industrial Microbiology & Biotechnology·Murali Kannan MaruthamuthuSoon Ho Hong
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Apr 13, 2017·Biotechnology and Bioengineering·Jan SchüürmannJoachim Jose
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Oct 20, 2020·Expert Opinion on Biological Therapy·Friedrich-Christian DeuschleArne Skerra
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