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Engineering Proteins Containing Noncanonical Amino Acids on the Yeast Surface

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Yeast Surface Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2491))

Abstract

Yeast display has been used to advance many critical research areas, including the discovery of unique protein binders and biological therapeutics. In parallel, noncanonical amino acids (ncAAs) have been used to tailor antibody-drug conjugates and enable discovery of therapeutic leads. Together, these two technologies have allowed for generation of synthetic antibody libraries, where the introduction of ncAAs in yeast-displayed proteins allows for library screening for therapeutically relevant targets. The combination of yeast display with genetically encoded ncAAs increases the available chemistry in proteins and advances applications that require high-throughput strategies. In this chapter, we discuss methods for displaying proteins containing ncAAs on the yeast surface, generating and screening libraries of proteins containing ncAAs, preparing bioconjugates on the yeast surface in large scale, generating and screening libraries of aminoacyl-tRNA synthetases (aaRSs) for encoding ncAAs by using reporter constructs, and characterizing ncAA-containing proteins secreted from yeast. The experimental designs laid out in this chapter are generalizable for discovery of protein binders to a variety of targets and aaRS evolution to continue expanding the genetic code beyond what is currently available in yeast.

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Abbreviations

aaRS:

Aminoacyl-tRNA synthetase

ADC:

Antibody–drug conjugate

cAA:

Canonical amino acid

CuAAC:

Copper-catalyzed azide-alkyne cycloaddition

DIYD:

Drop-in yeast display

DPS:

Dual plasmid system

EcLeuRS:

E. coli leucyl-tRNA synthetase

EcTyrRS:

E. coli tyrosyl-tRNA synthetase

epPCR:

Error-prone PCR

FACS:

Fluorescence-activated cell sorting

Fc:

Fragment crystallizable region

MMF:

Maximum misincorporation efficiency

MS:

Mass spectrometry

ncAA:

Noncanonical amino acid

OTS:

Orthogonal translation system

PCR:

Polymerase chain reaction

PDB:

Protein data bank

POI:

Protein of interest

PylRS:

Pyrrolysyl-tRNA synthetase

RRE:

Relative readthrough efficiency

SPS:

Single plasmid system

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Authors and Affiliations

  1. Chemical and Biological Engineering Department, Tufts University, Medford, MA, USA

    Rebecca L. Hershman, Arlinda Rezhdo, Jessica T. Stieglitz & James A. Van Deventer

  2. Biomedical Engineering Department, Tufts University, Medford, MA, USA

    James A. Van Deventer

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  1. Rebecca L. Hershman
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  2. Arlinda Rezhdo
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  3. Jessica T. Stieglitz
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  4. James A. Van Deventer
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Correspondence to James A. Van Deventer .

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Editors and Affiliations

  1. Department of Chemistry, Institute of Biochemistry, University of Natural Resources and Life Sciences, Vienna, Austria

    Michael W. Traxlmayr

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Hershman, R.L., Rezhdo, A., Stieglitz, J.T., Van Deventer, J.A. (2022). Engineering Proteins Containing Noncanonical Amino Acids on the Yeast Surface. In: Traxlmayr, M.W. (eds) Yeast Surface Display. Methods in Molecular Biology, vol 2491. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2285-8_23

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  • DOI: https://doi.org/10.1007/978-1-0716-2285-8_23

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