SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping

Abstract

Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories

“…To construct the trastuzumab expression vectors, the selected RgEs including the CMV promoter were amplified with the primers traz_fwd and -_rev from the beforehand generated expression vectors and cloned into NotI (New England Biolabs) and PmeI (New England Biolabs) restriction sites in the 5′-UTR of the HC gene in the vector (pKTH17_trastuzumab, harboring the genes for heavy and light chain). To generate SUMF1 expression constructs a SacI restriction site on the SUMF1 plasmid, originally established by solid phase cloning ( 43 , 44 ), was first removed to have a single SacI site in the 5′-UTR to allow for directional cloning of RgEs. Therefore, the vector pKTH16_sumf1 was digested with HindIII-HF (New England Biolabs) and XhoI (New England Biolabs) and ligated with beforehand annealed oligos SUMF1_SacIX_fwd and SUMF1_SacIX_rev, that carry a point mutation in the SacI site.…”