Protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction

Abstract

The invention relates to protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction. Using overlapping hexapeptides that encode for the entire amino acid sequences of the linker domains of human P-glycoprotein gene 1 and 3 (HP-gp1 and HP-gp3), a direct and specific binding between HP-gp1 and 3 linker domains and intracellular proteins was demonstrated. Three different stretches (617EKGIYFKLVTM627, (SEQ ID NO: 1) 658SRSSLIRKRSTRRSVRGSQA677 (SEQ ID NO: 2) and 694PVSFWRIMKLNLT706 (SEQ ID NO: 3) for HP-gp1 and 618LMKKEGVYFKLVNM631 (SEQ ID NO: 4), 648KAATRMAPNGWKSRLFRHSTQKNLKNS674 (SEQ ID NO: 5), and 695PVSFLKVLKLNKT707 (SEQ ID NO: 6) for HP-gp3) in linker domains bound to proteins with apparent molecular masses of ˜80 kDa, 57 kDa and 30 kDa. The binding of the 57 kDa protein was further characterized. Purification and partial N-terminal amino acid sequencing of the 57 kDa protein showed that it encodes the N-terminal amino acids of alpha and beta-tubulins. The method of the present invention was further validated with Annexin. The present invention thus demonstrates a novel concept whereby the interactions between two proteins are mediated by strings of few amino acids with high and repulsive binding energies, enabling the identification of high affinity binding sites between any interacting proteins.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 010,310, filed Nov. 13, 2001, entitled, “Protein-Protein Interactions and Methods for Identifying Interacting Proteins and the Amino Acid Sequence at the Site of Interaction,” now U.S. Pat. No. 7,176,035, which claims the benefit of PCT Appl. No. PCT / CA00 / 0587, filed May 12, 2000, which in turns claims priority from U.S. Provisional Appl. No. 60 / 134,259, filed May 14, 1999, the entire contents of all of which are hereby incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to proteonomics. More specifically, the invention relates to protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction. BACKGROUND OF THE INVENTION [0003] Specific protein-protein interactions are critical events in biological processes. Protein-protein interactions govern biological processes tha...

AI Technical Summary

Benefits of technology

[0035] It is an advantage of the present invention that the detection of the interaction between proteins and / or peptides be dependent on a label. Such labels provide sensitivity and often enable automation. In one embodiment of the present invention, automation is performed using CHIP technology. For example, the overlapping peptides spanning a chosen sequence of a protein, are bound to a CHIP which can then be used to automate a test for interaction with proteins or peptides. Of course, it should be understood that the present invention is not strictly dependent on a design and synthesis of the overlapping set of peptides spanning a chosen protein sequence. Indeed, banks of peptides are available, from which this set of overlapping peptides could be constructed.

[0036] Protein labelling is well known in the art. Non-limiting examples of labels include 3H, 14C, 32P, and 35S, Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the protein.

[0037] The identification of the interaction is not specifically dependent on labelling of the proteins, since for example, this interaction could be assessed using proteomic approaches (such as 2-D gels and mass spectrometry) or using a library of antibodies.

[0038] As commonly known, radioactive amino acids can be incorporated into peptides or proteins of the invention by several well-known methods. A non-limiting example thereof includes in vitro or in vivo labelling of proteins using 35SMet.

[0039] The term “vector” is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art.

[0040] The term “expression” defines the process by which a gene is transcribed into mRNA (transcription), the mRNA then being translated (translation) into one polypeptide (or protein) or more.

Problems solved by technology

A major obstacle in drug development for the treatment of diseases has been the identification of target proteins and their functional sites.

In fact, most research and development (R&D) projects in pharmaceutical companies take several years to identify a valid target protein.

The selection of drugs that bind to and inhibit the functions of these proteins takes several years and is generally non-specific and random.

Such drugs thus often lead to major side-effects.

Therefore, it is not surprising that many R&D projects never lead to the development of specific drugs even after three to five years of intensive research efforts.

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