Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-11-01
2004-08-31
Le, Long (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C435S007900, C435S007920, C435S814000, C436S164000, C436S177000, C436S518000, C436S524000, C436S528000, C210S600000, C210S634000, C210S644000, C210S645000, C210S649000, C210S650000, C210S651000, C210S656000
Reexamination Certificate
active
06783929
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to techniques and materials for the purification of biological samples and the differential display and analysis of components of such biological samples. In one embodiment, the invention is directed to protein purification and proteome differential display.
Current methodologies for purifying biological sample components (e.g., protein, nucleic acid, etc.) rely on a few commercially available hydrophilic solid supports. Conventional chromatography supports include anionic exchange supports, such as diethylaminoethyl (DEAE) Sepharose, etc., cationic exchange supports, for example having carboxymethyl functional groups, reverse phase and hydrophobic interaction supports. These materials are able to distinguish between biological sample components, such as proteins, only very generally and are therefore limited in their ability to separate biological sample components, particularly proteins of similar anionic, cationic, etc. character.
Other currently available separation techniques are very specific. For example, affinity chromatography supports based on antibodies, proteins, sugars, etc., are available, such as described by
Use of antibody fragments in immunoaffinity chromatography: comparison of FV fragments, VH fragments and paralog peptides
. Berry, M. J.; Davies, J. J. Chromatogr. (1992), 597(1-2), 239-45. However, just as the conventional chromatography supports are too general to provide more than limited separation of the complex mixtures of chemicals that typically comprise biological samples, conventional affinity supports are too specific to provide useful separation of the many components of such mixtures beyond a specific corresponding target or antigen.
Thus, neither of these currently available separation alternatives is particularly effective in the separation of complex mixtures of biological sample components typically found in biological cell and tissue samples. The current development of new affinity chromatography materials is based on the screening of peptide-, protein- or small molecule-resin complexes. However, these approaches have limitations in terms of diversity, biological/chemical stability and large-scale production.
Another potential approach to the separation of biological sample components is through the use of electrophoresis, particularly two-dimensional gel electrophoresis. Two-dimensional gels may be run for biological samples, such as cell lysates or tissue samples, separating on the basis of pH in one dimension and on the basis of molecular weight in the other. However, this technique has severe specificity limitations due to the fact that very abundant proteins in the mixture often obscure less abundant proteins. In addition, this technique has low throughput and is not amenable to large scale production.
In addition to separating and purifying biological sample components from complex biological samples, it is also useful to characterize the sample components. A comparison of the components of two biological samples having different phenotypes may allow for the identification of the source(s) of the difference between the samples. For example, by conducting such a differential analysis of a healthy tissue sample and a diseased tissue sample of the same type, it may be possible to identify the cause of the disease and/or characterize the phenotypic symptoms of the disease. This approach has received significant attention at the genetic (nucleic acid) level, and has seen the development of the field of “genomics.”
One particular application of this genomics approach is described in M. R. Martzen et al.
A Biochemical Genomics Approach for Identifying Genes by the Activity of Their Products
, Science, Vol. 286, Nov. 5, 1999, incorporated by reference herein. However, this approach may not lend itself to diagnostic and drug development applications.
This approach has received less attention at the gene product (protein) level. However, more recently the field of “proteomics” has begun to develop. The proteomics approach to analyzing differential gene expression is attractive. While there may be correlation between genetic differences in different biological samples and the different phenotypes observed in their associated cells, tissues, or organisms (e.g., healthy vs. diseased states), genomic differences are not do not necessarily manifest themselves as differences in gene expression. Proteomics, on the other hand, directly addresses differences in gene expression by focussing the analysis on the protein constituents of the sample under study. The effectiveness of proteomic approaches, however, are limited by the ability of currently available materials and techniques to provide high resolution differential displays of biological samples under study. For example, Kauvar et al. (e.g., U.S. Pat. No. 5,599,903, Preparation of glutathione analogs and paralog panels comprising glutathione mimics as affinity ligands and glutathione transferase inhibitors; International Patent Application No. WO 9106356, Methods and kits to identify analyte-binding ligands using paralog panels; International Patent Application No. WO 8909088, Paralog substrates for affinity chromatography, their selection, and their use; and
Paralog chromatography
. Kauvar, Lawrence M. et al. BioTechniques (1990), 8(2), 204-9; ) have described the use of peptides mounted on chromatography supports for use in separating protein mixtures. These peptide-based separation materials are, however, subject to proteolytic degradation and are therefore of limited utility.
Accordingly, the development of techniques and materials that facilitate the improved separation of biological sample component mixtures as well as enhance the effectiveness of proteomic analysis would be desirable.
SUMMARY OF THE INVENTION
To achieve the foregoing, the present invention provides affinity support materials having intermediate binding affinity for biological samples, relative to conventional materials having only very general or very specific binding affinities. Among the materials provided by the present invention are hydrophilic solid supports composed of hydrophilic ligands coupled to hydrophilic matrixes which are compatible with biological samples. Suitable biological samples applicable to the present invention may be of virtually any type or source including for example, cells derived from defined cell lines (e.g., tumor cell lines, bacteria, virus-infected cells, replicons, and the like); biological fluids, such as blood, urine, saliva, or mucus; cells collected from biological surfaces such as from washes (e.g., lavages) or swabes (e.g., pap smears or throat cultures); and—cells derived from tissue samples (e.g., cells obtained from biopsies of diseased tissues and/or undiseased “normal” tissues). The biological samples can homogeneous (ie., from a single source) or heterogeneous (i.e., two or more unrelated sources). The ligands described herein may include affinity property groups and hydrophilic groups pendent from a backbone, and be configured to at least partially resolve components of a biological sample as defined above. Such affinity property groups and hydrophilic groups can be selected using methods described hereinbelow.
Affinity supports in accordance with the present invention may be used in a variety of techniques and apparatuses to achieve improved separations of complex biological samples and thereby enhance the results of biological sample component resolutions, fractionations, enrichments, purifications, expression product determinations and comparisons, and other biological sample processing and analysis techniques. In addition, the affinity supports may be included in kits useful in processing biological samples for applications such as novel protein purification.
In some embodiments of the present invention, the hydrophilic ligands of the affinity supports may be peptoids with both affinity property groups and hydrophilic groups pendent from their peptoid backbones. A variety of peptoid structures, as well as paramet
Beausoleil Eric
Kothakota Srinivas
Wachowicz Matthew
Zuckermann Ronald N.
Austin James E.
Blackburn Robert P.
Chiron Corporation
Collier Steven W.
Le Long
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