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On the C-terminus a small peptide marker epitope is present. These vectors display the protein of interest flanked on the N-terminus by mating protein Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1 ( Boder and Wittrup, 1997). The available commercial yeast display vector pYD1 (Invitrogen) is based on pCT202 developed by Boder and Wittrup (1997). Over the past 8 years techniques have been developed to increase the affinity and stability of scFv with random mutagenesis coupled with yeast display technology utilizing sorting flow cytometry and magnetic bead selection methodologies ( Colby et al., 2004). The affinity of these scFvs is an important variable and must exceed a certain threshold to be useful. Recombinant single-chain antibodies have wide utility as diagnostic and therapeutic reagents. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand–scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3εγ increased ∼3-fold when displayed on pYD5 compared with pYD1. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3εγ. Consequently we rebuilt pYD1 tethering the scFv off the NH 2 terminus of Aga2. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3εγ was too low to measure.

We have noted that two anti-CD3ε scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH 2 terminal to either truncated toxins or human serum albumin. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs.

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