Here, we enhanced the popular yeast display method by multiple rounds of DNA and protein engineering. We introduced surface exposure-tailored reporters, eUnaG2 and DnbALFA, creating a new platform of C and N terminal fusion vectors. The optimization of eUnaG2 resulted in five times brighter fluorescence and 10 °C increased thermostability than UnaG. The optimized DnbALFA has 10-fold the level of expression of the starting protein. Following this, different plasmids were developed to create a complex platform allowing a broad range of protein expression organizations and labeling strategies. Our platform showed up to five times better separation between nonexpressing and expressing cells compared with traditional pCTcon2 and c-myc labeling, allowing for fewer rounds of selection and achieving higher binding affinities. Testing 16 different proteins, the enhanced system showed consistently stronger expression signals over c-myc labeling. In addition to gains in simplicity, speed, and cost-effectiveness, new applications were introduced to monitor protein surface exposure and protein retention in the secretion pathway that enabled successful protein engineering of hard-to-express proteins. As an example, we show how we optimized the WD40 domain of the ATG16L1 protein for yeast surface and soluble bacterial expression, starting from a nonexpressing protein. As a second example, we show how using the here-presented enhanced yeast display method we rapidly selected high-affinity binders toward two protein targets, demonstrating the simplicity of generating new protein-protein interactions. While the methodological changes are incremental, it results in a qualitative enhancement in the applicability of yeast display for many applications.

Institut Pasteur Unité de Biologie cellulaire de l'infection microbienne 25 rue du Dr Roux Paris 75015 France

Institute of Biotechnology CAS v v i Prumyslova 595 Vestec 252 50 Prague region Czech Republic

Weizmann Institute of Science Herzl St 234 Rehovot 7610001 Israel

Zobrazit více v PubMed

Schreuder M. P.; Brekelmans S.; Van Den Ende H.; Klis F. M. Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae. Yeast 1993, 9, 399–409. 10.1002/yea.320090410. PubMed DOI

Hetrick K. J.; Walker M. C.; van der Donk W. A. Development and Application of Yeast and Phage Display of Diverse Lanthipeptides. ACS Cent. Sci. 2018, 4, 458–467. 10.1021/acscentsci.7b00581. PubMed DOI PMC

Cohen-Khait R.; Dym O.; Hamer-Rogotner S.; Schreiber G. (2017) Promiscuous Protein Binding as a Function of Protein Stability. Structure London England 1993, 25, 1867–1874. PubMed

Uchański T.; Zögg T.; Yin J.; Yuan D.; Wohlkönig A.; Fischer B.; Rosenbaum D. M.; Kobilka B. K.; Pardon E.; Steyaert J. An improved yeast surface display platform for the screening of nanobody immune libraries. Sci. Rep. 2019, 9, 382.10.1038/s41598-018-37212-3. PubMed DOI PMC

Zhang K.; Nelson K. M.; Bhuripanyo K.; Grimes K. D.; Zhao B.; Aldrich C. C.; Yin J. Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display. Chem. Biol. 2013, 20, 92–101. 10.1016/j.chembiol.2012.10.020. PubMed DOI PMC

Niyonzima N.; Lambert A. R.; Werther R.; De Silva Feelixge H.; Roychoudhury P.; Greninger A. L.; Stone D.; Stoddard B. L.; Jerome K. R. Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses. Protein Eng. Des. Sel. 2017, 30, 503–522. 10.1093/protein/gzx037. PubMed DOI PMC

Benatuil L.; Perez J. M.; Belk J.; Hsieh C. M. An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng. Des. Sel. 2010, 23, 155–159. 10.1093/protein/gzq002. PubMed DOI

Swers J. S.; Kellogg B. A.; Wittrup K. D. Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display. Nucleic Acids Res. 2004, 32, 36e.10.1093/nar/gnh030. PubMed DOI PMC

Chao G.; Lau W. L.; Hackel B. J.; Sazinsky S. L.; Lippow S. M.; Wittrup K. D. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 2006, 1, 755–768. 10.1038/nprot.2006.94. PubMed DOI

Boder E. T.; Wittrup K. D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 1997, 15, 553–557. 10.1038/nbt0697-553. PubMed DOI

Wang Z.; Mathias A.; Stavrou S.; Neville D. M. Jr. A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities. Protein Eng. Des. Sel. 2005, 18, 337–343. 10.1093/protein/gzi036. PubMed DOI

Gai S. A.; Wittrup K. D. Yeast surface display for protein engineering and characterization. Curr. Opin. Struct. Biol. 2007, 17, 467–473. 10.1016/j.sbi.2007.08.012. PubMed DOI PMC

Simeon R.; Chen Z. In vitro-engineered non-antibody protein therapeutics. Protein & cell 2018, 9, 3–14. 10.1007/s13238-017-0386-6. PubMed DOI PMC

Mata-Fink J.; Kriegsman B.; Yu H. X.; Zhu H.; Hanson M. C.; Irvine D. J.; Wittrup K. D. Rapid conformational epitope mapping of anti-gp120 antibodies with a designed mutant panel displayed on yeast. J. Mol. Biol. 2013, 425, 444–456. 10.1016/j.jmb.2012.11.010. PubMed DOI PMC

Cohen-Khait R.; Schreiber G. Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders. Proc. Natl. Acad. Sci. U. 2016, 113, 14982–14987. 10.1073/pnas.1613122113. PubMed DOI PMC

Traxlmayr M. W.; Obinger C. Directed evolution of proteins for increased stability and expression using yeast display. Arch. Biochem. Biophys. 2012, 526, 174–180. 10.1016/j.abb.2012.04.022. PubMed DOI

Mei M.; Zhou Y.; Peng W.; Yu C.; Ma L.; Zhang G.; Yi L. Application of modified yeast surface display technologies for non-Antibody protein engineering. Microbiol. Res. 2017, 196, 118–128. 10.1016/j.micres.2016.12.002. PubMed DOI

Szczupak A.; Alfonta L.. The Use of Yeast Surface Display in Biofuel Cells. In Yeast Surface Display: Methods, Protocols, and Applications; (Liu B., Ed.), Springer: New York, New York, NY, 2015, 261–268. PubMed

Colby D. W.; Kellogg B. A.; Graff C. P.; Yeung Y. A.; Swers J. S.; Wittrup K. D. Engineering antibody affinity by yeast surface display. Meth. Enzymol. 2004, 388, 348–358. 10.1016/S0076-6879(04)88027-3. PubMed DOI

van den Ent F.; Löwe J. RF cloning: A restriction-free method for inserting target genes into plasmids. J. Biochem. Biophys. Methods 2006, 67, 67–74. 10.1016/j.jbbm.2005.12.008. PubMed DOI

Wyatt R. G.; Okamoto G. A.; Feigin R. D. Stability of Antibiotics in parental solutions. Pediatrics 1972, 49, 22–29. PubMed

Peleg Y.; Unger T. Application of the Restriction-Free (RF) cloning for multicomponents assembly. Methods Mol. Biol. 2014, 1116, 73–87. 10.1007/978-1-62703-764-8_6. PubMed DOI

Davey H. M.; Hexley P. Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide. Environ. Microbiol. 2011, 13, 163–171. 10.1111/j.1462-2920.2010.02317.x. PubMed DOI

Shaner N. C.; Lambert G. G.; Chammas A.; Ni Y.; Cranfill P. J.; Baird M. A.; Sell B. R.; Allen J. R.; Day R. N.; Israelsson M.; Davidson M. W.; Wang J. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 2013, 10, 407–409. 10.1038/nmeth.2413. PubMed DOI PMC

Huang D.; Shusta E. V. Secretion and Surface Display of Green Fluorescent Protein Using the Yeast Saccharomyces cerevisiae. Biotechnol. Prog. 2005, 21, 349–357. PubMed

Kumagai A.; Ando R.; Miyatake H.; Greimel P.; Kobayashi T.; Hirabayashi Y.; Shimogori T.; Miyawaki A. A Bilirubin-Inducible Fluorescent Protein from Eel Muscle. Cell 2013, 153, 1602–1611. 10.1016/j.cell.2013.05.038. PubMed DOI

Chapman S.; Faulkner C.; Kaiserli E.; Garcia-Mata C.; Savenkov E. I.; Roberts A. G.; Oparka K. J.; Christie J. M. The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection. Proc. Natl. Acad. Sci. U. 2008, 105, 20038–20043. 10.1073/pnas.0807551105. PubMed DOI PMC

Sheehan M. M.; Magaraci M. S.; Kuznetsov I. A.; Mancini J. A.; Kodali G.; Moser C. C.; Dutton P. L.; Chow B. Y. Rational Construction of Compact de Novo-Designed Biliverdin-Binding Proteins. Biochemistry 2018, 57, 6752–6756. 10.1021/acs.biochem.8b01076. PubMed DOI PMC

Rumyantsev K. A.; Shcherbakova D. M.; Zakharova N. I.; Emelyanov A. V.; Turoverov K. K.; Verkhusha V. V. Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence. Sci. Rep. 2016, 5, 18348.10.1038/srep18348. PubMed DOI PMC

Rodriguez E. A.; Tran G. N.; Gross L. A.; Crisp J. L.; Shu X.; Lin J. Y.; Tsien R. Y. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat. Methods 2016, 13, 763–769. 10.1038/nmeth.3935. PubMed DOI PMC

Fuenzalida-Werner J. P.; Janowski R.; Mishra K.; Weidenfeld I.; Niessing D.; Ntziachristos V.; Stiel A. C. Crystal structure of a biliverdin-bound phycobiliprotein: Interdependence of oligomerization and chromophorylation. J. Struct. Biol. 2018, 204, 519–522. 10.1016/j.jsb.2018.09.013. PubMed DOI

Shcherbakova D. M.; Verkhusha V. V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat. Methods 2013, 10, 751.10.1038/nmeth.2521. PubMed DOI PMC

Oliinyk O. S.; Shemetov A. A.; Pletnev S.; Shcherbakova D. M.; Verkhusha V. V. Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing. Nat. Commun. 2019, 10, 279.10.1038/s41467-018-08050-8. PubMed DOI PMC

Braun M. B.; Traenkle B.; Koch P. A.; Emele F.; Weiss F.; Poetz O.; Stehle T.; Rothbauer U. Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy. Sci. Rep. 2016, 6, 19211.10.1038/srep19211. PubMed DOI PMC

Götzke H.; Kilisch M.; Martínez-Carranza M.; Sograte-Idrissi S.; Rajavel A.; Schlichthaerle T.; Engels N.; Jungmann R.; Stenmark P.; Opazo F.; Frey S. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat. Commun. 2019, 10, 4403.10.1038/s41467-019-12301-7. PubMed DOI PMC

Kaishima M.; Ishii J.; Matsuno T.; Fukuda N.; Kondo A. Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity. Sci. Rep. 2016, 6, 35932–35932. 10.1038/srep35932. PubMed DOI PMC

Drew D.; Newstead S.; Sonoda Y.; Kim H.; von Heijne G.; Iwata S. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat. Protoc. 2008, 3, 784–798. 10.1038/nprot.2008.44. PubMed DOI PMC

Yeh J. T. H.; Nam K.; Yeh J. T. H.; Perrimon N. eUnaG: a new ligand-inducible fluorescent reporter to detect drug transporter activity in live cells. Sci.Rep. 2017, 7, 41619–41619. 10.1038/srep41619. PubMed DOI PMC

Goldenzweig A.; Goldsmith M.; Hill S. E.; Gertman O.; Laurino P.; Ashani Y.; Dym O.; Unger T.; Albeck S.; Prilusky J.; Lieberman R. L.; Aharoni A.; Silman I.; Sussman J. L.; Tawfik D. S.; Fleishman S. J. Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability. Mol. Cell 2016, 63, 337–346. 10.1016/j.molcel.2016.06.012. PubMed DOI PMC

Zahradnik J.; Kolarova L.; Peleg Y.; Kolenko P.; Svidenska S.; Charnavets T.; Unger T.; Sussman J. L.; Schneider B. Flexible regions govern promiscuous binding of IL-24 to receptors IL-20R1 and IL-22R1. FEBS J. 2019, 286, 3858–3873. 10.1111/febs.14945. PubMed DOI

Erijman A.; Dantes A.; Bernheim R.; Shifman J. M.; Peleg Y. Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J. Struct. Biol. 2011, 175, 171–177. 10.1016/j.jsb.2011.04.005. PubMed DOI

Magliery T. J. Protein stability: computation, sequence statistics, and new experimental methods. Curr. Opin. Struct. Biol. 2015, 33, 161–168. 10.1016/j.sbi.2015.09.002. PubMed DOI PMC

Gupta R.; Brunak S. Prediction of glycosylation across the human proteome and the correlation to protein function. Pacific Symp. Biocomp. 2002, 310–322. PubMed

Baran D.; Pszolla M. G.; Lapidoth G. D.; Norn C.; Dym O.; Unger T.; Albeck S.; Tyka M. D.; Fleishman S. J. Principles for computational design of binding antibodies. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 10900–10905. 10.1073/pnas.1707171114. PubMed DOI PMC

Rakestraw J. A.; Sazinsky S. L.; Piatesi A.; Antipov E.; Wittrup K. D. Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in Saccharomyces cerevisiae. Biotechnol. Bioeng. 2009, 103, 1192–1201. 10.1002/bit.22338. PubMed DOI PMC

Townsley F. M.; Frigerio G.; Pelham H. R. Retrieval of HDEL proteins is required for growth of yeast cells. J. Cell Biol. 1994, 127, 21–28. 10.1083/jcb.127.1.21. PubMed DOI PMC

Frey S.; Görlich D. A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. J. Chromatogr. A 2014, 1337, 95–105. 10.1016/j.chroma.2014.02.029. PubMed DOI

Boyko K. M.; Gorbacheva M. A.; Korzhenevskiy D. A.; Alekseeva M. G.; Mavletova D. A.; Zakharevich N. V.; Elizarov S. M.; Rudakova N. N.; Danilenko V. N.; Popov V. O. Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation. Biochem. Biophys. Res. Commun. 2016, 477, 595–601. 10.1016/j.bbrc.2016.06.097. PubMed DOI

Tron C. M.; McNae I. W.; Nutley M.; Clarke D. J.; Cooper A.; Walkinshaw M. D.; Baxter R. L.; Campopiano D. J. Structural and functional studies of the biotin protein ligase from Aquifex aeolicus reveal a critical role for a conserved residue in target specificity. J. Mol. Biol. 2009, 387, 129–146. 10.1016/j.jmb.2008.12.086. PubMed DOI

Lim S.; Glasgow J. E.; Filsinger Interrante M.; Storm E. M.; Cochran J. R. Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions. Biotechnol. J. 2017, 12, 160069610.1002/biot.201600696. PubMed DOI PMC

Bajagic M.; Archna A.; Büsing P.; Scrima A. Structure of the WD40-domain of human ATG16L1. Protein Sci. 2017, 26, 1828–1837. 10.1002/pro.3222. PubMed DOI PMC

Hamaoui D.; Cossé M. M.; Mohan J.; Lystad A. H.; Wollert T.; Subtil A. The Chlamydia effector CT622/TaiP targets a nonautophagy related function of ATG16L1. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 26784–26794. 10.1073/pnas.2005389117. PubMed DOI PMC

Schymkowitz J.; Borg J.; Stricher F.; Nys R.; Rousseau F.; Serrano L. The FoldX web server: an online force field. Nucleic Acids Res. 2005, 33, W382–W388. 10.1093/nar/gki387. PubMed DOI PMC

Gera N.; Hussain M.; Wright R. C.; Rao B. M. Highly stable binding proteins derived from the hyperthermophilic Sso7d scaffold. J. Mol. Biol. 2011, 409, 601–616. 10.1016/j.jmb.2011.04.020. PubMed DOI

Hosse R. J.; Rothe A.; Power B. E. A new generation of protein display scaffolds for molecular recognition. Prot. Sci. 2006, 15, 14–27. 10.1110/ps.051817606. PubMed DOI PMC

Kruziki M. A.; Bhatnagar S.; Woldring D. R.; Duong V. T.; Hackel B. J. A 45-Amino-Acid Scaffold Mined from the PDB for High-Affinity Ligand Engineering. Chem. Biol. 2015, 22, 946–956. 10.1016/j.chembiol.2015.06.012. PubMed DOI PMC

Ye K.; Shibasaki S.; Ueda M.; Murai T.; Kamasawa N.; Osumi M.; Shimizu K.; Tanaka A. Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface. Appl. Microbiol. Biotechnol. 2000, 54, 90–96. 10.1007/s002539900307. PubMed DOI

Kunze I.; Hensel G.; Adler K.; Bernard J.; Neubohn B.; Nilsson C.; Stoltenburg R.; Kohlwein S. D.; Kunze G. The green fluorescent protein targets secretory proteins to the yeast vacuole. Biochim. Biophys. Acta, Bioenerg. 1999, 1410, 287–298. 10.1016/S0005-2728(99)00006-7. PubMed DOI

Li J.; Xu H.; Bentley W. E.; Rao G. Impediments to secretion of green fluorescent protein and its fusion from Saccharomyces cerevisiae. Biotechnol. Prog. 2002, 18, 831–838. 10.1021/bp020066t. PubMed DOI

Eiden-Plach A.; Zagorc T.; Heintel T.; Carius Y.; Breinig F.; Schmitt M. J. Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Appl. Environ. Microbiol. 2004, 70, 961–966. 10.1128/AEM.70.2.961-966.2004. PubMed DOI PMC

Roh J. Y.; Koo B. C.; Kwon M. S.; Kim M.; Kim N.-H.; Kim T. Modification of enhanced green fluorescent protein for secretion out of cells. Biotechnol. Bioprocess Eng. 2013, 18, 1135–1141. 10.1007/s12257-013-0333-1. DOI

Grzeschik J.; Hinz S. C.; Könning D.; Pirzer T.; Becker S.; Zielonka S.; Kolmar H. A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping. Biotechnol. J. 2017, 12, 160045410.1002/biot.201600454. PubMed DOI

McMahon C.; Baier A. S.; Pascolutti R.; Wegrecki M.; Zheng S.; Ong J. X.; Erlandson S. C.; Hilger D.; Rasmussen S. G. F.; Ring A. M.; Manglik A.; Kruse A. C. Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat. Struct. 2018, 25, 289–296. 10.1038/s41594-018-0028-6. PubMed DOI PMC

Zahradník J.; Marciano S.; Shemesh M.; Zoler E.; Harari D.; Chiaravalli J.; Meyer B.; Rudich Y.; Li C.; Marton I.; Dym O.; Elad N.; Lewis M. G.; Andersen H.; Gagne M.; Seder R. A.; Douek D. C.; Schreiber G. SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution. Nat. Microbiol. 2021, 6, 1188–1198. 10.1038/s41564-021-00954-4. PubMed DOI

Bloom J. D.; Labthavikul S. T.; Otey C. R.; Arnold F. H. Protein stability promotes evolvability. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 5869–5874. 10.1073/pnas.0510098103. PubMed DOI PMC

Tokuriki N.; Tawfik D. S. Stability effects of mutations and protein evolvability. Curr. Opin. Struct. Biol. 2009, 19, 596–604. 10.1016/j.sbi.2009.08.003. PubMed DOI

Wilson D. S.; Keefe A. D.; Szostak J. W. The use of mRNA display to select high-affinity protein-binding peptides. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 3750–3755. 10.1073/pnas.061028198. PubMed DOI PMC

Syedbasha M.; Linnik J.; Santer D.; O’Shea D.; Barakat K.; Joyce M.; Khanna N.; Tyrrell D. L.; Houghton M.; Egli A. An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions. JoVE 2016, e5357510.3791/53575. PubMed DOI PMC

Schreiber G.; Fleishman S. J. Computational design of protein-protein interactions. Curr. Opin. Struct. Biol. 2013, 23, 903–910. 10.1016/j.sbi.2013.08.003. PubMed DOI

Gietz R. D.; Woods R. A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 2002, 350, 87–96. 10.1016/S0076-6879(02)50957-5. PubMed DOI

Zahradník J.; Kolářová L.; Pařízková H.; Kolenko P.; Schneider B. Interferons type II and their receptors R1 and R2 in fish species: Evolution, structure, and function. Fish Shellfish Immunol. 2018, 79, 140–152. 10.1016/j.fsi.2018.05.008. PubMed DOI

Cossé M. M.; Barta M. L.; Fisher D. J.; Oesterlin L. K.; Niragire B.; Perrinet S.; Millot G. A.; Hefty P. S.; Subtil A. The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth. Front. Cell. Infect. Microbiol. 2018, 8, 145.10.3389/fcimb.2018.00145. PubMed DOI PMC

Sofronescu A. G.; Loebs T.; Zhu Y. Effects of temperature and light on the stability of bilirubin in plasma samples. Clin. Chim. Acta 2012, 413, 463–466. 10.1016/j.cca.2011.10.036. PubMed DOI

Starr T. N.; Greaney A. J.; Hilton S. K.; Ellis D.; Crawford K. H. D.; Dingens A. S.; Navarro M. J.; Bowen J. E.; Tortorici M. A.; Walls A. C.; King N. P.; Veesler D.; Bloom J. D. Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. Cell 2020, 182, 1295–1310.e20. 10.1016/j.cell.2020.08.012. PubMed DOI PMC

Pettersen E. F.; Goddard T. D.; Huang C. C.; Couch G. S.; Greenblatt D. M.; Meng E. C.; Ferrin T. E. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612. 10.1002/jcc.20084. PubMed DOI

Kozakov D.; Hall D. R.; Xia B.; Porter K. A.; Padhorny D.; Yueh C.; Beglov D.; Vajda S. The ClusPro web server for protein-protein docking. Nat. Protoc. 2017, 12, 255–278. 10.1038/nprot.2016.169. PubMed DOI PMC

Multiple mutations of SARS-CoV-2 Omicron BA.2 variant orchestrate its virological characteristics

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light

Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants

Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant

Building the SynBio community in the Czech Republic from the bottom up: You get what you give

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant