GSJ: Received June 4-5, 2005: http://wbabin.net/saba/saba49.htm

In a previous paper (1), the use of support-affixed protein libraries in the isolation of proteins which selectively bind to one non-conjugated radioactive molecule was described. The use of scintillation proximity and photographic emulsions were the means of detecting the library member which binds.

The ability to use non-conjugated molecules is especially important for small synthetic organic molecules, especially drugs and candidates thereof, which cannot be or cannot easily be conjugated.

Herein is disclosed the use of cell surface display libraries in similar processes, as exemplified in Figure 1.

Therein we contact a cell surface display library with a multitude of identical radioactive chiral molecules, which could be racemic or only one stereoisomer. After a multitude of the radioactive molecules are bound by the cell expressing a binding protein, the cells are spun down onto a glass slide layered with photographic emulsion. Alternatively the cells could be spun down, resuspended in a photographic emulsion, and then the mixture layered on a glass slide.

Subsequent to microscopically identifying the radioactive molecule-binding cell, it is isolated with a micromanipulator and its DNA amplified and sequenced. If the isolated cell is still viable it of course can be grown. If when isolating a labeled cell, the picking process includes nonlabeled neighbors, the process in Figure 1 can be repeated with the small set of library members less densely arrayed.

A similar process utilizing scintillation proximity would involve spinning the cells down on an appropriately layered surface.

Well known is the use of support-conjugated molecules as affinity supports to isolate members of encoded libraries, particular phage-display libraries. Interestingly however, a quick search of MedLine indicates that use of soluble conjugated small molecules in the screening of encoded libraries, especially cell-surface or particle-based protein libraries may not be known. This may be particularly true wherein the small molecules used in the screening are unnatural organic synthetics, a group which includes the vast majority of drug and drug candidates. Labels which could be conjugated to a molecule include those which are radioactive (including single atoms such as tritium); mass spectrometry labels; and fluorescent dyes as exemplified in Figure 2.

Therein we contact the cell-surface display library with a multitude of identical fluorescent dye-conjugated molecules. Alternatively to using a fluorescence-activated cell sorter (FACS), cells could be spun down on a glass slide after, or prior to contacting with fluorescent conjugate, then identified microscopically.

Molecules of interest could alternatively be conjugated to affinity tags, which allow for several different kinds of isolation and detection schemes. Such tags are even useful for encoded libraries other than cells or particles, such as phage-display or cis-display libraries. Examples of such tags include biotin (recognized by avidin or antibody), and a facilitator of host cell binding and infection.

The use of the soluble conjugated molecules in protein library screening avoids interactions of the library proteins with a support, and the need for eluting bound library member proteins from the support.

In the screening processes depicted in Figures 1 and 2 we utilized single cells. However, it is conceivable that each cell could be first randomly positioned on or within a partitioned or nonpartitioned growth media and then clonally expanded to produce microcolonies.

The cells in the processes described in Figures 1 and 2, could be replaced with particles (beads) affixed to library member proteins, preferably fabricated via a cis-expression process.

Lastly it has been appreciated that, just as a particle, a cell expressing a target-specific surface protein could be directly used in the isolation of the target, as shown in Figure 3.

Of course any molecule could be isolated by the process in Figure 3.

A process similar to Figure 3 except using recombinant phage displaying identical surface proteins can be envisioned.

Various means of separating cells or phage from supernatant could be utilized. For example filtration; polyethylene glycol (PEG) precipitation; or the use of supports to which phage or cells are affixed prior to or subsequent to binding target molecule.

This invention is considered valuable and a US patent application is anticipated to be filed in the very near future. However, it is hoped that others with laboratory facilities will investigate its full potential, perhaps even to establish a collaboration with the inventor.

The following condensed provisional claims are an attempt to encompass important aspects of this invention.