Method of preparing a protein array based on biochemical...

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Reexamination Certificate

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C435S397000, C435S396000, C435S395000, C435S091500, C435S091500

Reexamination Certificate

active

06743630

ABSTRACT:

BACKGROUND
Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding Sequence Listing and the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Fas (APO-1/CD95) and its ligand have been identified as important signal-mediators of apoptosis (Itoh, et al. 1991) The structural organization of Fas (APO-1/CD95) has suggested that it is a member of the tumor necrosis factor receptor superfamily, which also includes the p75 nerve growth factor receptor (NGFR) (Johnson, et al. 1986), the T-cell-activation marker CD27 (Camerini, et al. 1991), the Hodgkin-lymphoma-associated antigen CD30 (Smith, et al. (1993), the human B cell antigen CD40 (Stamenkovic, et al. 1989), and T cell antigen OX40 (Mallett, et al. 1990). Genetic mutations of both Fas and its ligand have been associated with lymphoproliferative and autoimmune disorders in mice (Watanabe-Fukunaga, et al. 1992; Takahashi, et al. 1994). Furthermore, alterations of Fas expression level have been thought to lead to the induction of apoptosis in T-cells infected with human immunodeficiency virus (HIV) (Westendorp, et al. 1995).
Several Fas-interacting signal transducing molecules, such as Fas-associated phosphatase-1 (FAP-1)(
FIG. 1
) (Sato, et al. 1995), FADD/MORT1 (Chinnaiyan, et al. 1995; Boldin, et al. 1995; Kischkel, et al. 1995) and RIP (Stanger, et al. 1995), have been identified using yeast two-hybrid and biochemical approaches. All but FAP-1 associate with the functional cell death domain of Fas and overexpression of FADD/MORT1 or RIP induces apoptosis in cells transfected with these proteins. In contrast, FAP-1 is the only protein that associates with the negative regulatory domain (C-terminal 15 amino acids) (Ito, et al. 1993) of Fas and that inhibits Fas-induced apoptosis. FAP-1 (PTPN13) has several alternatively-spliced forms that are identical to PTP-BAS/hPTP1E/PTPL1, (Maekawa, et al. 1994; Banville, et al. 1994; Saras, et al. 1994) and contains a membrane-binding region similar to those found in the cytoskeleton-associated proteins, ezrin, (Gould et al. 1989) radixin (Funayama et al. 1991) moesin (Lankes, et al. 1991), neurofibromatosis type II gene product (NFII) (Rouleau, et al. 1993), and protein 4.1 (Conboy, et al. 1991), as well as in the PTPases PTPH1 (Yang, et al. 1991), PTP-MEG (Gu, et al. 1991), and PTPD1 (Vogel, et al. 1993). FAP-1 intriguingly contains six GLGF (PDZ/DHR) (SEQ ID NO:34) repeats that are thought to mediate intra-and inter-molecular interactions among protein domains. The third GLGF (SEQ ID NO:34) repeat of FAP-1 was first identified as a domain showing the specific interaction with the C-terminus of Fas receptor (Sato, et al. 1995). This suggests that the GLGF (SEQ ID NO:34) domain may play an important role in targeting proteins to the submembranous cytoskeleton and/or in regulating biochemical activity. GLGF (SEQ ID NO:34) repeats have been previously found in guanylate kinases, as well as in the rat post-synaptic density protein (PSD-95) (Cho, et al. 1992), which is a homolog of the Drosophila tumor suppressor protein, lethal-(1)-disc-large-1 [dlg-1] (Woods, et al 1991; Kitamura, et al. 1994). These repeats may mediate homo- and hetero-dimerization, which could potentially influence PTPase activity, binding to Fas, and/or interactions of FAP-1 with other signal transduction proteins. Recently, it has also been reported that the different PDZ domains of proteins interact with the C-terminus of ion channels and other proteins (
FIG. 1
) (TABLE 1) (Kornau, et al. 1995; Kim, et al. 1995; Matsumine, et al. 1996).
TABLE 1
Proteins that interact with PDZ domains.
C-terminal
Associated
Protein
sequence
protein
Reference
Fas (APO-1/CD95)
SLV
FAP-1
2
NMDA receptor
SDV
PSD95
3
NR2 subunit
Shaker-type K+
TDV
PSD95 & DLG
4
channel
APC
TEV
DLG
5
A recent trend in biology, biotechnology and medicine is the use of arrays of immobilized biological compounds in studies of immunoassays and enzymatic reactions (see Mendoza, et al. 1999; Arenkov, et al. 2000). For example, mass-sensing, multianalyte microarray immunoassays have been performed (Rowe, et al. 1998; Silzel, et al. 1998). The use of arrays allows for large scale and high-throughput studies of multiple samples in parallel. Integration of microarray technology into the experimental methodology also may increase efficiency in many instances, such as through reducing the volume of samples and reagents required.
It would be desirable to have high-throughput and low cost methodologies for preparing protein arrays based on protein-protein interaction, and which keep the proteins in a functionally active state and allow, for example, multiple drug screenings under physiological conditions.
SUMMARY OF THE INVENTION
This disclosure provides a method of preparing a protein array based on biochemical protein-protein interaction, comprising (a) depositing on a substrate an array of a first protein, the first protein comprising a PDZ domain, and (b) applying a second protein, which comprises an amino acid sequence (S/T)—X—(V/I/L)—COOH, to the first protein array, the amino acid sequence (S/T)—X—(V/I/L)—COOH of the second protein binding to the PDZ domain of the first protein, wherein each hyphen represents a peptide bond, each parenthesis encloses amino acids which are alternatives to one other, each slash within such parentheses separates the alternative amino acids, and the X represents any amino acid which is selected from the group comprising the twenty naturally occurring amino acids.
This disclosure also provides a method of preparing a protein array, (a) depositing on a substrate an array of first proteins, each first protein comprising a corresponding PDZ domain, and (b) applying a second protein, which comprises an amino acid sequence (S/T)—X—(V/I/L)—COOH, to the array of first proteins, the amino acid sequence (S/T)—X—(V/I/L)—COOH of the second protein, for each of the first proteins, binding to the PDZ domain of the first protein, wherein each hyphen represents a peptide bond, each parenthesis encloses amino acids which are alternatives to one other, each slash within such parentheses separates the alternative amino acids, and the X represents any amino acid which is selected from the group comprising the twenty naturally occurring amino acids.
This disclosure also provides a method of preparing a protein array, (a) depositing on a substrate an array of a first protein, the first protein comprising a PDZ domain, and (b) applying a plurality of second proteins, each of which comprises a corresponding amino acid sequence (S/T)—X—(V/I/L)—COOH, to corresponding elements of the first protein array, for each of the second proteins, the amino acid sequence (S/T)—X—(V/I/L)—COOH of the second protein binding to the PDZ domain of the first protein in the corresponding array element, wherein each hyphen represents a peptide bond, each parenthesis encloses amino acids which are alternatives to one other, each slash within such parentheses separates the alternative amino acids, and the X represents any amino acid which is selected from the group comprising the twenty naturally occurring amino acids.
This disclosure also provides a method of preparing a protein array, comprising (a) depositing on a substrate an array of a first polypeptide, the first polypeptide comprising a PDZ domain, and (b) applying a second polypeptide which comprises an amino acid sequence (S/T)—X—(V/I/L)—COOH to the first polypeptide array, the amino acid sequence (S/T)—X—(V/I/L)—COOH of the second polypeptide binding to the PDZ domain of the first polypeptide, wherein each hyphen represents a peptide bond, each parenthesis encloses amino acids which are alternatives to one other, each slash within such pa

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