Yeast surface display for protein engineering and characterization

Gai, S. Annie; Wittrup, K. Dane

doi:10.1016/j.sbi.2007.08.012

Cited by 340 publications

(282 citation statements)

References 66 publications

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“…Larger increases in k cat may require modified selection or screening strategies that explicitly couple survival with multiple turnover kinetics, perhaps by integrating our system with in vitro compartmentalization. Despite the widespread use of yeast display in the evolution of binding interactions (18), to the best of our knowledge, sortase A is only the third enzyme to be evolved using yeast display, in addition to horseradish peroxidase (30,31) and an esterase catalytic antibody (32). Our results highlight the attractive features of yeast display that offer significant advantages for enzyme evolution, including quality control mechanisms within the secretory pathway that ensure display of properly folded proteins and compatibility with FACS (18).…”

Section: Discussionmentioning

confidence: 85%

“…Despite the widespread use of yeast display in the evolution of binding interactions (18), to the best of our knowledge, sortase A is only the third enzyme to be evolved using yeast display, in addition to horseradish peroxidase (30,31) and an esterase catalytic antibody (32). Our results highlight the attractive features of yeast display that offer significant advantages for enzyme evolution, including quality control mechanisms within the secretory pathway that ensure display of properly folded proteins and compatibility with FACS (18). For these reasons, we used yeast as the vehicle for display instead of an M13 phage simultaneously displaying an Sfp peptide substrate and an enzyme library (33).…”

Section: Discussionmentioning

confidence: 99%

“…Cell surface display (17)(18)(19)(20) is an attractive alternative to phage and mRNA display. In contrast with other display methods, the use of bacterial or yeast cells enables up to 100,000 copies of a library member to be linked to one copy of the gene, increasing sensitivity during screening or selection steps.…”

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confidence: 99%

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A general strategy for the evolution of bond-forming enzymes using yeast display

Chen

Dorr

Liu

2011

Proc. Natl. Acad. Sci. U.S.A.

499

584

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The ability to routinely generate efficient protein catalysts of bondforming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A-catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods.

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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A general strategy for the evolution of bond-forming enzymes using yeast display

Chen

Dorr

Liu

2011

Proc. Natl. Acad. Sci. U.S.A.

499

584

View full text Add to dashboard Cite

show abstract

“…In particular, screening of antibody libraries displayed on the surface of yeast cells addresses some of the above mentioned biases prokaryotic systems impose, and has proven to be a very powerful method. 11,12 In order to perform library screening under the most physiological conditions possible, numerous mammalian cell-based antibody expression platforms have been developed, involving simple transient transfection of antibody libraries 13 or their viral transduction using vaccinia, 14 sindbis, 15 or retrovirus vectors. 16 While these approaches are believed to deliver mAbs with favorable biophysical properties that have a higher potential in drug development and therapeutic use, mammalian cell-based screening platforms are currently also associated with a number of limitations, which has led to a slow adoption of these technologies in academia and industry.…”

Section: Introductionmentioning

confidence: 99%

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Waldmeier

Hellmann

Gutknecht

et al. 2016

mAbs

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In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.

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“…In this study, we chose to perform epitope mapping by using the yeast surface display (YSD) technology (10 -12), because this technique facilitates both rapid and quantitative library screening by fluorescence-activated cell sorting (FACS) and the minimization of artifacts resulting from host-expression bias through concurrent expression labeling (12,13). For epitope mapping, in particular, there are two important advantages of YSD as follows: there is no need to produce and purify protein variants (11), and in contrast to phage display, the technique is suitable for eukaryotic proteins, preserving their folds and allowing glycosylation, albeit through slightly different sugar compositions compared with those of humans (15,16).…”

mentioning

confidence: 99%

Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS

Rosenfeld

Shirian

Zur

et al. 2015

Journal of Biological Chemistry

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Background: Identifying residues crucial for M-CSF⅐c-FMS binding remains difficult. Results: Using a combination of experimental and computational methods, we identified mutations on M-CSF that reduce affinity to c-FMS. Conclusion: Affinity-reducing mutations are located both inside and outside of the binding interface. Significance: Knowledge of the critical residues will facilitate a better understanding of the M-CSF mechanism and facilitate drug design.

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Yeast surface display for protein engineering and characterization

Cited by 340 publications

References 66 publications

A general strategy for the evolution of bond-forming enzymes using yeast display

A general strategy for the evolution of bond-forming enzymes using yeast display

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS

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