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High-throughput analysis and protein engineering using microcapillary arrays

Nature Chemical Biology volume 12, pages 76–81 (2016)Cite this article

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Abstract

We describe a multipurpose technology platform, termed μSCALE (microcapillary single-cell analysis and laser extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. μSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding-affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With μSCALE, we discovered a new antibody against a clinical cancer target, evolved a fluorescent protein biosensor and engineered an enzyme to reduce its sensitivity to its inhibitor. These protein analysis and engineering applications each have unique assay requirements and different host organisms, highlighting the flexibility and technical capabilities of the μSCALE platform.

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Figure 1: μSCALE overview.
Figure 2: High-throughput screening of binding proteins using μSCALE.
Figure 3: Engineering an orange-hued fluorescent protein with μSCALE.
Figure 4: High-throughput kinetic measurements and enzyme library screening with μSCALE.

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References

  1. Lander, E.S. Initial impact of the sequencing of the human genome. Nature 470, 187–197 (2011).

    Article  CAS  Google Scholar 

  2. Rahman, N. Realizing the promise of cancer predisposition genes. Nature 505, 302–308 (2014).

    Article  CAS  Google Scholar 

  3. Rhodes, D.R. & Chinnaiyan, A.M. Integrative analysis of the cancer transcriptome. Nat. Genet. 37 (suppl.) S31–S37 (2005).

    Article  CAS  Google Scholar 

  4. Zhang, Y. et al. Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules. Mol. Cell. Proteomics 4, 1240–1250 (2005).

    Article  CAS  Google Scholar 

  5. Romero, P.A. & Arnold, F.H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).

    Article  CAS  Google Scholar 

  6. Kariolis, M.S. et al. An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis. Nat. Chem. Biol. 10, 977–983 (2014).

    Article  CAS  Google Scholar 

  7. Wu, I. & Arnold, F.H. Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures. Biotechnol. Bioeng. 110, 1874–1883 (2013).

    Article  CAS  Google Scholar 

  8. Alford, S.C., Ding, Y., Simmen, T. & Campbell, R.E. Dimerization-dependent green and yellow fluorescent proteins. ACS Synth. Biol. 1, 569–575 (2012).

    Article  CAS  Google Scholar 

  9. Chen, I., Dorr, B.M. & Liu, D.R. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc. Natl. Acad. Sci. USA 108, 11399–11404 (2011).

    Article  CAS  Google Scholar 

  10. Gai, S.A. & Wittrup, K.D. Yeast surface display for protein engineering and characterization. Curr. Opin. Struct. Biol. 17, 467–473 (2007).

    Article  CAS  Google Scholar 

  11. Lee, C.V. et al. High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J. Mol. Biol. 340, 1073–1093 (2004).

    Article  CAS  Google Scholar 

  12. Martis, E. High-throughput screening: the hits and leads of drug discovery. J. Appl. Pharm. Sci. 01, 02–10 (2011).

    Google Scholar 

  13. Povolotskaya, I.S. & Kondrashov, F.A. Sequence space and the ongoing expansion of the protein universe. Nature 465, 922–926 (2010).

    Article  CAS  Google Scholar 

  14. Agresti, J.J. et al. Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc. Natl. Acad. Sci. USA 107, 4004–4009 (2010).

    Article  CAS  Google Scholar 

  15. Ostafe, R., Prodanovic, R., Nazor, J. & Fischer, R. Ultra-high-throughput screening method for the directed evolution of glucose oxidase. Chem. Biol. 21, 414–421 (2014).

    Article  CAS  Google Scholar 

  16. Zinchenko, A. et al. One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution. Anal. Chem. 86, 2526–2533 (2014).

    Article  CAS  Google Scholar 

  17. Fischlechner, M. et al. Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nat. Chem. 6, 791–796 (2014).

    Article  CAS  Google Scholar 

  18. Romero, P.A., Tran, T.M. & Abate, A.R. Dissecting enzyme function with microfluidic-based deep mutational scanning. Proc. Natl. Acad. Sci. USA 112, 7159–7164 (2015).

    Article  CAS  Google Scholar 

  19. Lafferty, M. & Dycaico, M.J. GigaMatrix: an ultra high-throughput tool for accessing biodiversity. J. Lab. Autom. 9, 200–208 (2004).

    Article  CAS  Google Scholar 

  20. Fukuda, T., Shiraga, S., Kato, M. & Yamamura, S. Construction of novel single-cell screening system using a yeast cell chip for nano-sized modified-protein-displaying libraries. NanoBiotechnology 1, 105–111 (2005).

    Article  CAS  Google Scholar 

  21. Gorris, H.H. & Walt, D.R. Mechanistic aspects of horseradish peroxidase elucidated through single-molecule studies. J. Am. Chem. Soc. 131, 6277–6282 (2009).

    Article  CAS  Google Scholar 

  22. Liebherr, R.B., Renner, M. & Gorris, H.H. A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase. J. Am. Chem. Soc. 136, 5949–5955 (2014).

    Article  CAS  Google Scholar 

  23. Rissin, D.M. et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat. Biotechnol. 28, 595–599 (2010).

    Article  CAS  Google Scholar 

  24. Love, J.C., Ronan, J.L., Grotenbreg, G.M., van der Veen, A.G. & Ploegh, H.L. A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat. Biotechnol. 24, 703–707 (2006).

    Article  CAS  Google Scholar 

  25. Fitzgerald, V. et al. Exploiting highly ordered subnanoliter volume microcapillaries as microtools for the analysis of antibody producing cells. Anal. Chem. 87, 997–103 (2015).

    Article  CAS  Google Scholar 

  26. Kovac, J.R. & Voldman, J. Intuitive, image-based cell sorting using optofluidic cell sorting. Anal. Chem. 79, 9321–9330 (2007).

    Article  CAS  Google Scholar 

  27. Gach, P.C., Attayek, P.J., Whittlesey, R.L., Yeh, J.J. & Allbritton, N.L. Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma. Biosens. Bioelectron. 54, 476–483 (2014).

    Article  CAS  Google Scholar 

  28. Waters, J.C. Accuracy and precision in quantitative fluorescence microscopy. J. Cell Biol. 185, 1135–1148 (2009).

    Article  CAS  Google Scholar 

  29. Lemke, G. & Rothlin, C.V. Immunobiology of the TAM receptors. Nat. Rev. Immunol. 8, 327–336 (2008).

    Article  CAS  Google Scholar 

  30. Graham, D.K., DeRyckere, D., Davies, K.D. & Earp, H.S. The TAM family: phosphatidylserine-sensing receptor tyrosine kinases gone awry in cancer. Nat. Rev. Cancer 14, 769–785 (2014).

    Article  CAS  Google Scholar 

  31. Sidhu, S.S. et al. Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J. Mol. Biol. 338, 299–310 (2004).

    Article  CAS  Google Scholar 

  32. Ai, H.-W., Baird, M.A., Shen, Y., Davidson, M.W. & Campbell, R.E. Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications. Nat. Protoc. 9, 910–928 (2014).

    Article  CAS  Google Scholar 

  33. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  34. Alford, S.C., Abdelfattah, A.S., Ding, Y. & Campbell, R.E. A fluorogenic red fluorescent protein heterodimer. Chem. Biol. 19, 353–360 (2012).

    Article  CAS  Google Scholar 

  35. Ding, Y. et al. Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange. Nat. Methods 12, 195–198 (2015).

    Article  CAS  Google Scholar 

  36. Coleman, J. Structure and mechanism of alkaline phosphatase. Annu. Rev. Biophys. Biomol. Struct. 21, 441–483 (1992).

    Article  CAS  Google Scholar 

  37. O'Brien, P.J. & Herschlag, D. Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase. Biochemistry 40, 5691–5699 (2001).

    Article  CAS  Google Scholar 

  38. Alberstein, M., Eisenstein, M. & Abeliovich, H. Removing allosteric feedback inhibition of tomato 4-coumarate:CoA ligase by directed evolution. Plant J. 69, 57–69 (2012).

    Article  CAS  Google Scholar 

  39. Yang, J.-S., Seo, S.W., Jang, S., Jung, G.Y. & Kim, S. Rational engineering of enzyme allosteric regulation through sequence evolution analysis. PLoS Comput. Biol. 8, e1002612 (2012).

    Article  CAS  Google Scholar 

  40. Hu, X., Robin, S., O'Connell, S., Walsh, G. & Wall, J.G. Engineering of a fungal β-galactosidase to remove product inhibition by galactose. Appl. Microbiol. Biotechnol. 87, 1773–1782 (2010).

    Article  CAS  Google Scholar 

  41. Coelho, P.S., Brustad, E.M., Kannan, A. & Arnold, F.H. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339, 307–310 (2013).

    Article  CAS  Google Scholar 

  42. Pitsillides, C.M., Joe, E.K., Wei, X., Anderson, R.R. & Lin, C.P. Selective cell targeting with light-absorbing microparticles and nanoparticles. Biophys. J. 84, 4023–4032 (2003).

    Article  CAS  Google Scholar 

  43. Wu, Y.-C. et al. Massively parallel delivery of large cargo into mammalian cells with light pulses. Nat. Methods 12, 439–444 (2015).

    Article  CAS  Google Scholar 

  44. Otsu, N. A threshold selection method from gray-level histograms. IEEE SMC 9, 62–66 (1979).

    Google Scholar 

  45. Van Deventer, J.A. & Wittrup, K.D. Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation. Methods Mol. Biol. 1131, 151–181 (2014).

    Article  CAS  Google Scholar 

  46. Chao, G. et al. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1, 755–768 (2006).

    Article  CAS  Google Scholar 

  47. Benton, D. & Krishnamoorthy, K. Performance of the parametric bootstrap method in small sample interval estimates. Adv. Appl. Stat. 2, 269–285 (2002).

    Google Scholar 

  48. Andrews, L.D., Zalatan, J.G. & Herschlag, D. Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases. Biochemistry 53, 6811–6819 (2014).

    Article  CAS  Google Scholar 

  49. Brune, M., Hunter, J.L., Corrie, J.E.T. & Webb, M.R. Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33, 8262–8271 (1994).

    Article  CAS  Google Scholar 

  50. Zalatan, J.G., Fenn, T.D. & Herschlag, D. Comparative enzymology in the alkaline phosphatase superfamily to determine the catalytic role of an active-site metal ion. J. Mol. Biol. 384, 1174–1189 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank K.D. Wittrup and J. Van Deventer (MIT) and S. Sidhu (University of Toronto) for providing the yeast-displayed scFv library. This project was funded in part by the Stanford–Wallace H. Coulter Translational Partnership Award Program, the Siebel Stem Cell Institute and the Thomas and Stacey Siebel Foundation (to I.K.D.), the Stanford Photonics Research Center, a Hitachi America Faculty Scholar Award (to J.R.C.) and the US National Institutes of Health (GM49243 to D.H.). We acknowledge support from the National Science Foundation Graduate Fellowship Program (B.C. and A.K.), the Howard Hughes Medical Institute International Student Research Program (S.L.), a Fannie and John Hertz Foundation Graduate Fellowship (A.K.), the Stanford Bio-X Fellowship Program (S.L.), the Stanford Graduate Fellowship Program (B.C.) and the Stanford Dean's Fellowship Program (S.C.A.).

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Author notes

  1. Sungwon Lim, Arvind Kannan and Spencer C Alford: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, California, USA

    Bob Chen, Sungwon Lim, Spencer C Alford & Jennifer R Cochran

  2. Department of Chemical Engineering, Stanford University, Stanford, California, USA

    Arvind Kannan & Jennifer R Cochran

  3. Department of Biochemistry, Stanford University, Stanford, California, USA

    Fanny Sunden & Daniel Herschlag

  4. Department of Chemistry, Stanford University, Stanford, California, USA

    Daniel Herschlag

  5. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA

    Ivan K Dimov

  6. Stanford Photonics Research Center, Stanford University, Stanford, California, USA

    Thomas M Baer

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Contributions

All authors conceived and designed experiments and analyzed data. T.M.B., I.K.D. and B.C. designed and established the μSCALE platform. B.C., S.L. and I.K.D. established the μSCALE workflow. S.L. and B.C. performed protein engineering for binding interactions. S.C.A. and B.C. performed protein engineering for fluorescent proteins. A.K. and B.C. performed enzyme characterization and engineering with guidance from F.S. and D.H. The manuscript was prepared by B.C., S.L., A.K., S.C.A. and J.R.C. with input from all coauthors.

Corresponding authors

Correspondence to Thomas M Baer or Jennifer R Cochran.

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Competing interests

B.C., S.L., A.K., S.C.A, I.K.D, T.M.B. and J.R.C. are listed as inventors on patent applications related to technology described in this work. T.M.B. and I.K.D. are listed as inventors on pending patent applications (PCT/US2013/047792).

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Chen, B., Lim, S., Kannan, A. et al. High-throughput analysis and protein engineering using microcapillary arrays. Nat Chem Biol 12, 76–81 (2016). https://doi.org/10.1038/nchembio.1978

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