RESUMO
Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca(2+) binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca(2+)-bound (Ca(2+)-CaM) or Ca(2+)-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (K(d) = 0.8 nM) and specificity (>1000-fold) for Ca(2+)-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (K(d) = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform.
Assuntos
Afinidade de Anticorpos/genética , Evolução Molecular Direcionada/métodos , Conformação Proteica , Saccharomyces cerevisiae/genética , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/genética , Calmodulina/imunologia , Calmodulina/metabolismo , Clonagem Molecular , Citometria de Fluxo , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas S100/imunologia , Proteínas S100/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Troponina C/imunologia , Troponina C/metabolismoRESUMO
Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0 to 99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mgL(-1) culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was subsequently used to study three scFv variants engineered to determine structure-function relationships.
Assuntos
Clonagem Molecular , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Escherichia coli/genética , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genéticaRESUMO
Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library.