Advertisement

Rapid Antigen and Antibody-Like Molecule Discovery by Staphylococcal Surface Display

  • Marco CavallariEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)

Abstract

Ever since the discovery of antibodies, they have been generated by complicated multi-step procedures. Typically, these involve sequencing, cloning, and screening after expression of the antibodies in a suitable organism and format. Here, a staphylococcal nanobody display is described that omits many the abovementioned intermediate steps and allows for simultaneous screening of multiple targets without prior knowledge nor expression of the binders. This paper reports a detailed, general step-by-step protocol to achieve nanobodies of high affinity. Apart from its focus on radioactive and fluorescent targets, it gives options for various other target formats and additional applications for the staphylococcal library; including flow cytometry and immunoprecipitation. This provides a system for antibody engineers that can be easily adopted to their specific needs.

Key words

Nanobody (VHH) Bacterial surface display Target identification Staphylococcal sortase A (SrtA) Immunoprecipitation 

Abbreviations

BSA

Bovine serum albumin

Cm

Chloramphenicol

DTT

Dithiothreitol

EDTA

Ethylenediaminetetraacetic acid

GFP

Green fluorescent protein

MES

2-(N-morpholino)ethanesulfonic acid

MSG

Monosodium glutamate

OD

Optical density

PBS

Phosphate-buffered saline

RT

Room temperature

SDS

Sodium dodecyl sulfate

Tris

Tris(hydroxymethyl)aminomethane

TSA

Tryptic soy agar

TSB

Tryptic soy broth

References

  1. 1.
    Hamers-Casterman C, Atarhouch T, Muyldermans S et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448.  https://doi.org/10.1038/363446a0CrossRefPubMedGoogle Scholar
  2. 2.
    Conrath KE, Wernery U, Muyldermans S, Nguyen VK (2003) Emergence and evolution of functional heavy-chain antibodies in Camelidae. Dev Comp Immunol 27:87–103CrossRefGoogle Scholar
  3. 3.
    Ingram JR, Schmidt FI, Ploegh HL (2018) Exploiting nanobodies’ singular traits. Annu Rev Immunol 36:695–715.  https://doi.org/10.1146/annurev-immunol-042617-053327CrossRefPubMedGoogle Scholar
  4. 4.
    Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797.  https://doi.org/10.1146/annurev-biochem-063011-092449CrossRefPubMedGoogle Scholar
  5. 5.
    Muyldermans S, Cambillau C, Wyns L (2001) Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci 26:230–235CrossRefGoogle Scholar
  6. 6.
    Finlay WJJ, Almagro JC (2012) Natural and man-made V-gene repertoires for antibody discovery. Front Immunol 3:342.  https://doi.org/10.3389/fimmu.2012.00342CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Govaert J, Pellis M, Deschacht N et al (2012) Dual beneficial effect of interloop disulfide bond for single domain antibody fragments. J Biol Chem 287:1970–1979.  https://doi.org/10.1074/jbc.M111.242818CrossRefPubMedGoogle Scholar
  8. 8.
    Marraffini LA, Dedent AC, Schneewind O (2006) Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol Mol Biol Rev 70:192–221.  https://doi.org/10.1128/MMBR.70.1.192-221.2006CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Mazmanian SK, Liu G, Ton-That H, Schneewind O (1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285:760–763.  https://doi.org/10.1126/science.285.5428.760CrossRefPubMedGoogle Scholar
  10. 10.
    Ton-That H, Liu G, Mazmanian SK et al (1999) Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc Natl Acad Sci U S A 96:12424–12429CrossRefGoogle Scholar
  11. 11.
    Chen I, Dorr BM, Liu DR (2011) A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci U S A 108:11399–11404.  https://doi.org/10.1073/pnas.1101046108CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hirakawa H, Ishikawa S, Nagamune T (2012) Design of Ca2+−independent Staphylococcus aureus sortase A mutants. Biotechnol Bioeng 109:2955–2961.  https://doi.org/10.1002/bit.24585CrossRefPubMedGoogle Scholar
  13. 13.
    Wu Q, Ploegh HL, Truttmann MC (2017) Hepta-mutant Staphylococcus aureus Sortase A (SrtA7m) as a tool for in vivo protein labeling in Caenorhabditis elegans. ACS Chem Biol 12(3):664–673.  https://doi.org/10.1021/acschembio.6b00998CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Dorr BM, Ham HO, An C et al (2014) Reprogramming the specificity of sortase enzymes. Proc Natl Acad Sci U S A 111:13343–13348.  https://doi.org/10.1073/pnas.1411179111CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Cavallari M (2017) Rapid and direct VHH and target identification by staphylococcal surface display libraries. Int J Mol Sci 18:1507.  https://doi.org/10.3390/ijms18071507CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Nair D, Memmi G, Hernandez D et al (2011) Whole-genome sequencing of Staphylococcus aureus strain RN4220, a key laboratory strain used in virulence research, identifies mutations that affect not only virulence factors but also the fitness of the strain. J Bacteriol 193:2332–2335.  https://doi.org/10.1128/JB.00027-11CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Monk IR, Shah IM, Xu M et al (2012) Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. MBio 3:e00277.  https://doi.org/10.1128/mBio.00277-11CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Moutel S, Bery N, Bernard V et al (2016) NaLi-H1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife 5:e16228.  https://doi.org/10.7554/eLife.16228CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    McMahon C, Baier AS, Pascolutti R et al (2018) Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol 25:289–296.  https://doi.org/10.1038/s41594-018-0028-6CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making antibodies by phage display technology. Annu Rev Immunol 12:433–455.  https://doi.org/10.1146/annurev.iy.12.040194.002245CrossRefPubMedGoogle Scholar
  21. 21.
    Benhar I (2007) Design of synthetic antibody libraries. Expert Opin Biol Ther 7:763–779.  https://doi.org/10.1517/14712598.7.5.763CrossRefPubMedGoogle Scholar
  22. 22.
    Chan CEZ, Lim APC, MacAry PA, Hanson BJ (2014) The role of phage display in therapeutic antibody discovery. Int Immunol 26:649–657.  https://doi.org/10.1093/intimm/dxu082CrossRefPubMedGoogle Scholar
  23. 23.
    Pardon E, Laeremans T, Triest S et al (2014) A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9:674–693.  https://doi.org/10.1038/nprot.2014.039CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Deschaght P, Vintém AP, Logghe M et al (2017) Large diversity of functional Nanobodies from a Camelid immune library revealed by an alternative analysis of next-generation sequencing data. Front Immunol 8:420.  https://doi.org/10.3389/fimmu.2017.00420CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Daugherty PS (2007) Protein engineering with bacterial display. Curr Opin Struct Biol 17:474–480.  https://doi.org/10.1016/j.sbi.2007.07.004CrossRefPubMedGoogle Scholar
  26. 26.
    Löfblom J (2011) Bacterial display in combinatorial protein engineering. Biotechnol J 6:1115–1129.  https://doi.org/10.1002/biot.201100129CrossRefPubMedGoogle Scholar
  27. 27.
    van Bloois E, Winter RT, Kolmar H, Fraaije MW (2011) Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol 29:79–86.  https://doi.org/10.1016/j.tibtech.2010.11.003CrossRefPubMedGoogle Scholar
  28. 28.
    Gautam S, Gniadek TJ, Kim T, Spiegel DA (2013) Exterior design: strategies for redecorating the bacterial surface with small molecules. Trends Biotechnol 31:258–267.  https://doi.org/10.1016/j.tibtech.2013.01.012CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Salema V, Marín E, Martínez-Arteaga R et al (2013) Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies. PLoS One 8:e75126.  https://doi.org/10.1371/journal.pone.0075126CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Nicolay T, Vanderleyden J, Spaepen S (2015) Autotransporter-based cell surface display in Gram-negative bacteria. Crit Rev Microbiol 41:109–123.  https://doi.org/10.3109/1040841X.2013.804032CrossRefPubMedGoogle Scholar
  31. 31.
    Salema V, Fernández LÁ (2017) Escherichia coli surface display for the selection of nanobodies. Microb Biotechnol 10:1468–1484.  https://doi.org/10.1111/1751-7915.12819CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Fleetwood F, Devoogdt N, Pellis M et al (2013) Surface display of a single-domain antibody library on Gram-positive bacteria. Cell Mol Life Sci 70:1081–1093.  https://doi.org/10.1007/s00018-012-1179-yCrossRefPubMedGoogle Scholar
  33. 33.
    Kronqvist N, Löfblom J, Jonsson A et al (2008) A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry. Protein Eng Des Sel 21:247–255.  https://doi.org/10.1093/protein/gzm090CrossRefPubMedGoogle Scholar
  34. 34.
    Nelson JW, Chamessian AG, McEnaney PJ et al (2010) A biosynthetic strategy for re-engineering the Staphylococcus aureus cell wall with non-native small molecules. ACS Chem Biol 5:1147–1155.  https://doi.org/10.1021/cb100195dCrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Schneewind O, Mihaylova-Petkov D, Model P (1993) Cell wall sorting signals in surface proteins of Gram-positive bacteria. EMBO J 12:4803–4811CrossRefGoogle Scholar
  36. 36.
    Schneewind O, Model P, Fischetti VA (1992) Sorting of protein a to the staphylococcal cell wall. Cell 70:267–281.  https://doi.org/10.1016/0092-8674(92)90101-HCrossRefPubMedGoogle Scholar
  37. 37.
    Klock HE, Koesema EJ, Knuth MW, Lesley SA (2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71:982–994.  https://doi.org/10.1002/prot.21786CrossRefPubMedGoogle Scholar
  38. 38.
    Klock HE, Lesley SA (2009) The polymerase incomplete primer extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis. Methods Mol Biol 498:91–103.  https://doi.org/10.1007/978-1-59745-196-3_6CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.BIOSS Centre for Biological Signalling StudiesUniversity of FreiburgFreiburgGermany

Personalised recommendations