Abstract:
:When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is coexpressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder-target pair with modest binding affinity (KD ≈ 400 nM), we showed that a binder present at low frequency (1 in 105) could be enriched more than 100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 (KD ≈ 272-1934 nM) and the extracellular domain of the c-Kit receptor (KD ≈ 93 to KD > 2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion-in contrast to recombinant soluble expression-makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.
journal_name
ACS Comb Scijournal_title
ACS combinatorial scienceauthors
Bacon K,Burroughs M,Blain A,Menegatti S,Rao BMdoi
10.1021/acscombsci.9b00147subject
Has Abstractpub_date
2019-12-09 00:00:00pages
817-832issue
12eissn
2156-8952issn
2156-8944journal_volume
21pub_type
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