Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-11-30
2004-09-28
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S514000, C436S161000, C210S198200, C210S656000, C435S007100
Reexamination Certificate
active
06797523
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for identifying protein—protein interactions. The interacting proteins are isolated by affinity chromatography and identified by mass spectrometry. The affinity chromatography method allows for the purification of interacting proteins which are identified using mass spectrometric techniques. The invention provides a method for the high throughput analysis of protein—protein interactions that lends itself to automation.
BACKGROUND OF THE INVENTION
The genome sequencing projects are providing vast amounts of information. With the whole genome of many organisms, including humans, complete or nearing completion, the next challenge involves the characterization of the gene products. Surprisingly, little is known about the functions of most proteins that the genes encode, or how these proteins interact to control cellular functions.
Protein interactions are intrinsic to virtually every cellular process. Most proteins in cells function in multi-subunit complexes of proteins created by specific protein—protein interactions. Many of the protein—protein interactions involved in cellular processes are too weak to allow co-purification of the interacting species by conventional methods from cellular extracts. The relatively weak binding is generally expected as proteins that must reversibly interact with each other in the concentrated intracellular environment will rapidly dissociate in a comparatively protein mixture. As the characterization of protein—protein interactions requires the in vitro reassembly of multi-subunit protein complexes, it is important to have methods for identifying and purifying all of the interacting proteins starting with one member of a protein complex.
The method of the invention uses a form of protein-affinity chromatography for the detection of protein—protein interactions. This method has been used in a non-systematic manner and has distinct advantages over other methods for the detection of protein—protein interactions, such as the two-hybrid method and co-immunoprecipitation. The two-hybrid system consists of two components, a target protein (the “bait”), fused to a DNA binding domain which binds to a specific region of DNA upstream of a reporter gene, and a protein (the “prey”) fused to an activation domain which, when brought in close proximity of the reporter gene, can initiate transcription. Usually the “bait” protein is known and the “prey” protein is derived from genomic or cDNA libraries in order to isolate the interacting partner to the bait. The advantage of the two-hybrid system is that when an interactor is found the gene sequence can be determined directly. This advantage is becoming increasingly less important as the full genomic sequence of many organisms becomes available, making the identification of gene sequence from protein sequence routine. The two-hybrid system yields a very high percentage of false positives, is very labor intensive and does not easily lend itself to automation, making it a poor choice for high throughput analysis.
Protein-protein interactions have commonly been detected by antibody co-immunoprecipitation. Co-immunoprecipitation depends on the strength of a secondary protein—protein interaction, rather than on direct binding to the antibody. The technique is normally limited to relatively strong interactions with K
d
≧10
−9
M. Additionally, it is not as sensitive as protein-affinity chromatography, because the concentration of the antigen is low.
Protein-affinity chromatography offers distinct advantages as a technique for detecting protein—protein interactions. Protein affinity chromatography allows sensitive detection of protein—protein interactions. This method can detect interactions ranging in strength from K
d
10
−5
to 10
−10
M. This limit is within the range of the weakest interactions likely to be physiologically relevant, which is estimated to be about 10
−3
M. Formosa et al., Methods in Enzymology 1991, 208, 24-45. An interacting protein with a K
d
>10
−5
M may not remain bound to the column when the column is washed with buffer in order to lower the nonspecific binding of proteins from the extract to the column material.
Protein-affinity chromatography tests all proteins in an extract equally for binding to the ligand protein. Thus, extract proteins that are detected have successfully competed for the interaction with the ligand protein against the rest of the population of proteins in the extract. Additionally, interactions that are dependent on a multi-subunit complex, including the ligand protein and multiple extract proteins and/or cofactors, can be detected. Both the domains of a protein and critical residues within the protein responsible for a specific interaction can be examined for affinity to extract proteins by the use of mutant derivatives of the ligand protein.
The method of the invention allows for the isolation of specific protein interactors. The interacting proteins are identified by protease digestion followed by mass spectrometry. During the past decade, new techniques in mass spectrometry have made it possible to accurately measure with high sensitivity the molecular weight of peptides and intact proteins. These techniques have made it much easier to obtain accurate peptide masses of a protein for use in databases searches. Mass spectrometry provides a method of protein identification that is both very sensitive (10 fmol-1 pmol) and very rapid when used in conjunction with sequence databases. Advances in protein and DNA sequencing technology are resulting in an exponential increase in the number of protein sequences available in databases. As the size of DNA and protein sequence databases grows, protein identification by correlative peptide mass matching has become an increasingly powerful method to identify and characterize proteins.
Historically, the explosion in gene sequence information has far outpaced the characterization of gene products. The processes of isolation and identification of protein interactors have represented a bottleneck in the characterization of protein—protein interactions. Current methods for the isolation and identification of protein interactors are performed on a protein-by-protein basis with relatively low throughput. The method of the invention provides a process for the high throughput analysis of protein—protein interactions. The use of micro-columns and mass spectroscopy provide the basis for using high throughput methods. The use of multiple ligand concentrations provides the binding curves and assures the reliability of the interactions that are identified.
SUMMARY OF THE INVENTION
The method of the invention provides a process for the identification of interacting proteins that is suitable for high throughput analysis and amenable to automation.
The identification of protein interactions is performed using affinity chromatography followed by mass spectrometric analysis. Cellular extract or extracellular fluid is loaded onto multiple experimental micro-columns, those with bound ligand protein, and a control micro-column with no bound ligand protein. Each of the experimental micro-columns contains a different concentration of ligand bound to the matrix support. A fixed volume of cellular extract is chromatographed through each micro-column. Only affinity chromatography buffer (ACB) is chromatographed on a second control micro-column which contains the highest concentration of ligand bound (coupled) to the matrix support. The components of the eluate are separated, for example, on the basis of apparent molecular weight using SDS-PAGE, and visualized, for example, by protein staining. The interacting protein is observed to vary in amount in direct relation to the concentration of coupled protein ligand. The bands of interest are excised from the gel and analyzed using mass spectrometric techniques.
REFERENCES:
patent: 5705813 (1998-01-01), Apffel et al.
patent: 6281493 (2001-08-01), Vestal et al.
patent: WO 97/01755 (1997-01-01), None
patent: WO 00
Awrey Donald E.
Greenblatt Jack
Affinium Pharmaceuticals, Inc.
Counts Gary W.
Foley & Hoag LLP
Le Long V.
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