Biosensor detection systems: engineering stable, high-affinity bioreceptors by yeast surface display

Abstract

Over the past two decades, the field of biosensors has been developing fast, portable, and convenient detection tools for various molecules of interest, both biological and environmental. Although much attention is paid to the transduction portion of the sensor, the actual bioreceptor that binds the ligand is equally critical. Tight, specific binding by the bioreceptor is required to detect low levels of the relevant ligand, and the bioreceptor must be stable enough to survive immobilization, storage, and in ideal cases, regeneration on the biosensing device. Often, naturally-occurring bioreceptors or antibodies that are specific for a ligand either express affinities that may be too low to detect useful levels, or the proteins are too unstable to be used and reused as a biosensor. Further engineering of these receptors can improve their utility. Here, we describe in detail the use of yeast surface display techniques to carry out directed evolution of bioreceptors to increase both the stability of the molecules and their affinity for the ligands. This powerful technique has enabled the production of stabilized, single-chain antibodies, T cell receptors, and other binding molecules that exhibit affinity increases for their ligands of up to 1 million-fold and expression of stable molecules in E. coli.

Fig. 1

Schematic diagrams of yeast surface display elements. (A) Plasmid map of the yeast surface display vector pCT302 with a single-chain T cell receptor (scTCR) cloned in as a fusion with the yeast mating agglutinin protein Aga-2. Map was prepared with the help of PlasMapper (48). The open reading frame containing Aga-2 and the scTCR is expanded to show the important elements and restriction sites. (B) Diagram of a bioreceptor displayed on the surface of yeast fused to Aga-2. A detection scheme involving biotinylated ligand bound to the bioreceptor and fluorescent streptavidin may be used to analyze bioreceptor libraries on the surface of yeast.

Fig. 1

Schematic diagrams of yeast surface display elements. (A) Plasmid map of the yeast surface display vector pCT302 with a single-chain T cell receptor (scTCR) cloned in as a fusion with the yeast mating agglutinin protein Aga-2. Map was prepared with the help of PlasMapper (48). The open reading frame containing Aga-2 and the scTCR is expanded to show the important elements and restriction sites. (B) Diagram of a bioreceptor displayed on the surface of yeast fused to Aga-2. A detection scheme involving biotinylated ligand bound to the bioreceptor and fluorescent streptavidin may be used to analyze bioreceptor libraries on the surface of yeast.

Fig. 2

Flow diagram for bioreceptor engineering by yeast surface display.

Fig. 3

Light scatter and fluorescence properties of yeast populations stained for FACS. (A) Typical forward light scatter (FSC) versus side scatter (SSC) profile of a yeast population. A gate (R1) can be drawn to exclude any events that do not fall within a given range of FSC and SSC properties. (B) A large library of yeast expressing a single-chain T cell receptor (scTCR) was stained with a fluorescently-labeled anti-TCR Vβ domain antibody, and the most fluorescent cells were collected using FACS. (C) After two rounds of sorting, the yeast population was stained with the anti-Vβ antibody for a third sort, revealing that the population had been enriched for Vβ⁺ scTCR mutants. The most fluorescent cells were again collected using FACS.

Fig. 4

Schematic representation of pre-SOE PCR primers. Forward flanking primer a and reverse overlap primer b are used in pre-SOE 1 to amplify the 5’ portion of the construct. Primer b contains the degenerate codons (saw-tooth), a ~50 nucleotide region of complementarity to forward overlap primer (c) at its 5’ end, and ~30 nucleotides of template complementarity to direct annealing for elongation at its 3’ end. Forward overlap primer c and reverse flanking primer d are used in pre-SOE 2 to amplify the 3’ portion of the construct, with a ~50 nucleotide region at its 5’ end that is complementary to the 3’ end of pre-SOE 1 product. This complementarity or “overlap” mediates the subsequent SOE PCR.