If we are to understand normal and pathophysiological processes at the molecular level in the central nervous system it is essential to develop tools to characterize proteins in individual cells. The cellular heterogeneity of the brain, in which glial cells generally surround and outnumber neurons at a ratio of 10 to 1 and the existence of different types of neurons in close proximity to one another requires a technology that permits the study of proteins from single neurons. High-resolution two-dimensional protein electrophoresis should help facilitate such studies. In high-resolution two- dimensional protein electrophoresis, proteins present in cell extracts are first separated by charge, and then by mass to produce a two dimensional array of purified proteins. However, two-dimensional protein electrophoresis methodology, as it is currently practiced, has two major inadequacies that have hindered this endeavor. The first problem is caused by the limits of sensitivity for protein detection. One of the most sensitive methods currently available is silver staining. While silver staining can detect as little as 0.01 nanogram of protein, for an average protein of molecular weight 30kD this represents approximately 200 million molecules. At this level of detection it is not possible to detect many proteins from a single cell, such as those in the brain, since the average number of molecules of any specific cellular protein is considerably below this range. The second problem is that it has proven to be difficult, and in some instances impossible, to characterize proteins that appear to be biologically interesting but present as silver stained “spots” at the 0.1 to 0.01ng level. While the use of mass spectrometric analysis of the silver stained spots is beginning to alleviate this problem at this time in the development of this technology the analysis of trace proteins spots is still a problem. Given these problems, we recognized that both of these inadequacies could be overcome by using either bacterial viruses (phage) or bacteria as protein detection reagents. These organisms have the potential to serve as detection reagents with high sensitivity because of their ability to replicate exponentially and with high specificity because of their capacity to display specific ligands on their surface. It is currently possible to incorporate ligand or antibody genes into phage or bacterial genomes in such a manner that these ligands or antibodies are expressed as fusion products with normal surface proteins on these viruses or bacteria. The display of such ligands or antibodies on the surface of these organisms can facilitate their binding to appropriate protein-binding motifs. The use of such fusion product displays can be employed both for general and for specific protein detection by using organisms displaying a specific ligand or a collection of organisms displaying a ligand library respectively. As both phage and bacteria can grow exponentially they can provide a detection system that has the theoretically possibility of detecting as few as 1 to 10 molecules of a specific protein. We have tested this approach by using the phage M13 to detect serial dilution of proteins on membranes and blots of serial dilutions of proteins separated on electrophoretic gels. Preliminary experiments have demonstrated senstitivities close to the theoretical limits of 1 to 10 molecules of protein. We have also used E.coli displaying ligand on flagella to detect proteins directly on electrophoretic gels. When a ligand library is used to provide an amplification detection system for protein separated from cells on two dimensional electrophoretic gels, each phage plaque or bacterial colony will contain specific phage or bacteria that bind to a specific protein “spot”. These agents could then be replicated to provide for assay systems for the identification of these specific proteins. They may also be used as reagents to aid in the purification of quantities of the specific proteins necessary for structural characterization. The development of such sensitive detection systems may facilitate the study of molecular events in complex organs such as the brain where individual cells play a critical role Based on these preliminary results a patent for the use of organisms capable of exponential growth and ligand display for high sensitivity detection of proteins has been filed.
| Zullo, S J; Parks, W T; Chloupkova, M et al. (2005) Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy. Rejuvenation Res 8:18-28 |
