Biomedical subjects

Michael J Feldhaus

Publications and source records attributed to Michael J Feldhaus.

8 recordsLinked to original sources

PubMed2005

Directed evolution for the development of conformation-specific affinity reagents using yeast display.

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.

Amino Acid Motifs↗

PubMed2005

Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli.

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.

Cloning, Molecular↗

PubMed2004

Yeast mating for combinatorial Fab library generation and surface display.

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.

Antibody Affinity↗

PubMed2004

High efficiency recovery and epitope-specific sorting of an scFv yeast display library.

In order to more productively utilize the rich source of antigen-specific reagents present in the previously described non-immune single chain fragment variable (scFv) yeast display library, one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi Macs system to recover greater than 90% of the antigen-specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen-specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope-specific scFv. Combining these two improvements in library screening allowed isolation and characterization of three epitope-specific scFv, including a previously uncharacterized epitope to a 6-kDa protein, epidermal growth factor.

Antibodies, Monoclonal↗

PubMed2004

Yeast display of antibody fragments: a discovery and characterization platform.

Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single-chain Fv (scFv) antibodies for increased affinity and thermal stability and, more recently, for the display and screening of a non-immune scFv and immune Fab libraries. A major strength of yeast display as a novel antibody discovery platform is the ability to characterize the binding properties, i.e., the affinity and epitope binding characteristics, of a clone without the need for subcloning, expression and purification of the scFv. This review focuses on novel attributes of yeast display for antibody engineering endeavors.

Animals↗

PubMed2003

Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library.

A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.

Cells, Cultured↗

PubMed2002

Expression levels of transdominant peptides and proteins in Saccharomyces cerevisiae.

From libraries of peptides and protein fragments, several inhibitors that block pheromone response in Saccharomyces cerevisiae have been isolated previously. In many cases, the inhibitors are displayed as part of a scaffold, such as green fluorescent protein. Each of the inhibitors has a characteristic physiological strength or genetic penetrance. In this report, the roles of expression level and display scaffold on the activities of a subset of pheromone-response pathway inhibitors were examined. Special consideration was given to the relationship between expression levels of specific inhibitors, which may exceed 50 microM in some instances, and penetrance.

Adaptor Proteins, Vesicular Transport↗

PubMed2001

Ceramide generation in situ alters leukocyte cytoskeletal organization and beta 2-integrin function and causes complete degranulation.

Ceramide levels increase in activated polymorphonuclear neutrophils, and here we show that endogenous ceramide induced degranulation and superoxide generation and increased surface beta(2)-integrin expression. Ceramide accumulation reveals a bifurcation in integrin function, as it abolished agonist-induced adhesion to planar surfaces, yet had little effect on homotypic aggregation. We increased cellular ceramide content by treating polymorphonuclear neutrophils with sphingomyelinase C and controlled for loss of sphingomyelin by pretreatment with sphingomyelinase D to generate ceramide phosphate, which is not a substrate for sphingomyelinase C. Pretreatment with the latter enzyme blocked all the effects of sphingomyelinase C. Ceramide generation caused a Ca(2+) flux and complete degranulation of both primary and secondary granules and increased surface beta(2)-integrin expression. These integrins were in a nonfunctional state, and subsequent activation with platelet-activating factor or formyl-methionyl-leucyl-phenylalanine induced beta(2)-integrin-dependent homotypic aggregation. However, these cells were completely unable to adhere to surfaces via beta(2)-integrins. This was not due to a defect in the integrins themselves because the active conformation could be achieved by cation switching. Rather, ceramide affected cytoskeletal organization and inside-out signaling, leading to affinity maturation. Cytochalasin D induced the same disparity between aggregation and surface adhesion. We conclude that ceramide affects F-actin rearrangement, leading to massive degranulation, and reveals differences in beta(2)-integrin-mediated adhesive events.

Actins↗