A fast mutagenesis procedure to recover soluble and functional scFvs containing amber stop codons from synthetic and semisynthetic antibody libraries.
The selection and production of scFvs from phage display synthetic antibody libraries are frequently delayed by the presence of amber (TAG) stop codons within the sequences corresponding to the variable CDRs. This is due to the use of randomised oligonucleotides for library design and amber mutations for joining the scFv to the phage protein pIII. The screening of such libraries may lead to the selection of scFvs containing stop codons. Then, multiple site-directed mutagenesis is required for their removal or, alternatively, the proteins must be expressed as scFv-pIII fusions, which are not suitable for many functional assays. We describe here an alternative procedure to express soluble scFvs, despite the presence of TAG stop codons, in the currently used Escherichia coli suppressor strain TG1. It is based on a simple mutagenesis protocol that replaces the amber codon between the scFv and the pIII gene by a different stop codon (TAA), functional in E. coli TG1. The expression of soluble scFvs in the suppressor strain TG1 permits their fully functional characterization including the determination of affinity constants, which are critical for selecting the right scFvs for further studies.
Barderas
Rodrigo
R
Protein Technology Unit, Biotechnology Programme, Spanish National Cancer Center CNIO, 28028 Madrid, Spain.
Shochat
Susana
S
Martínez-Torrecuadrada
Jorge
J
Altschuh
Danièle
D
Meloen
Rob
R
Ignacio Casal
Jose
J