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PEDS Advance Access published online on June 23, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi036
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received January 10, 2005
Revised May 11, 2005
Accepted May 13, 2005

Article

A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities

Z. Wang 1, A. Mathias 1, S. Stavrou 1, and D. M. Neville Jr1*

1 Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Building 10, 36 Convent Drive, Bethesda, MD 28092-4034, USA

* To whom correspondence should be addressed.
D. M. Neville Jr, E-mail: davidn{at}mail.nih.gov


  Abstract

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. 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. We have noted that two anti-CD3{varepsilon} scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH2 terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3{varepsilon}{gamma} was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH2 terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3{varepsilon}{gamma}. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3{varepsilon}{gamma} increased ~3-fold when displayed on pYD5 compared with pYD1. 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.

Keywords: CD3; FN18; pYD5; scFv; yeast display.
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