Robert W. Siegel

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.

Using an in vivo phagemid system to identify non-compatible loxP sequences.

The site‐specific recombination system of bacteriophage P1 is composed of the Cre recombinase that recognizes a 34‐bp loxP site. The Cre/loxP system has been extensively used to manipulate eukaryotic genomes for functional genomic investigations. The creation of additional heterologous loxP sequences potentially expands the utility of this system, but only if these loxP sequences do not recombine with one another. We have developed a stringent in vivo assay to examine the degree of recombination between all combinations of each previously published heterologous loxP sequence. As expected, homologous loxP sequences efficiently underwent Cre‐mediated recombination. However, many of the heterologous loxP pairs were able to support recombination with rates varying from 5 to 100%. Some of these loxP sequences have previously been reported to be non‐compatible with one another. Our study also confirmed other heterologous loxP pairs that had previously been shown to be non‐compatible, as well as defined additional combinations that could be used in designing new recombination vectors.

Antibodies in proteomics I: generating antibodies

The explosion in genome sequencing, and in subsequent DNA array experiments, has provided extensive information on gene sequence, organization and expression. This has resulted in a desire to perform similarly broad experiments on all the proteins encoded by a genome. Panels of specific antibodies, or other binding ligands, will be essential tools in this endeavour. Because traditional immunization will be unlikely to generate antibodies in sufficient quantity, and of the required quality and reproducibility, in vitro selection methods will probably be used. This review--the first of two--examines the strategies available for in vitro antibody selection. The second review discusses the adaptation of these methods to high throughput and the uses to which antibodies, once derived, can be put.

Flow Cytometric Screening of Yeast Surface Display Libraries

A method to screen and isolate antigen specific clones from a library of single-chain antibodies expressed on the surface of yeast cells is presented. Two rounds of magnetic bead enrichment before flow cytometric sorting enables one to screen libraries of far greater diversity than can be screened by just flow cytometry. The strength of flow cytometric sorting is the ability to follow the selection in real time and to isolate easily the highest affinity antigen-specific clones. A major strength of yeast display as a discovery platform is the ability to characterize the binding properties, the affinity of a clone without the need for subcloning, expression, and purification of the scFv. The methodology for directed evolution of single-chain antibodies to increase the affinity of a clone is also described.

A paracrine signal mediates the cell transformation response to low dose gamma radiation in JB6 cells

The carcinogenic response to radiation is complex and may involve adaptive cellular responses as well as a bystander effect mediated by paracrine or intercellular signaling activities. Using a newly developed co‐culture model we have examined whether low dose gamma radiation induces the transformation of JB6 mouse epidermal cells as well as non‐irradiated bystander cells. Cell transformation response is defined as the acquisition of anchorage‐independent growth properties and is quantified by counting colonies on soft agar. Exposure of JB6 cells to low dose (2–20 cGy) gamma radiation resulted in an approximate 1.9 ± 0.1 and 2.8 ± 0.5‐fold increase in cell transformation response when cells were seeded at 1 × 104 or 1 × 105 cells/dish, relative to respective sham exposed controls. We developed a co‐culture model where sham exposed or irradiated JB6 cells were mixed with non‐irradiated JB6 cells that had been stably transfected with the enhanced yellow fluorescent protein (EYFP) to enable the distinction of fluorescent bystander‐specific colonies. A significant increase in the number of bystander‐specific colonies was observed in co‐culture with 10 cGy irradiated JB6 cells (224 ± 9), relative to the number of bystander‐specific colonies arising in co‐culture with sham exposed JB6 cells (55 ± 16). Our results indicate that low dose radiation induces the transformation of JB6 cells and that a soluble paracrine factor that is secreted by irradiated cells induces the transformation of non‐irradiated bystander cells.