Compositions for isolating a cDNA encoding a membrane-bound protein

Abstract

A method for isolating a gene encoding a membrane-bound protein characterized by fusing a functional protein with a fused protein itself, which differs from the existing TMT method in which an epitope recognized by an antibody is carried in a fused protein. This method enabled selective isolation of a gene encoding a membrane-bound protein.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel gene-cloning-method for selectively and efficiently isolating genes encoding membrane-bound proteins. BACKGROUND ART [0002] Proteins synthesized in cells can be categorized by their individual characteristics into those localized in intracellular organelles, such as nucleus, mitochondria, cytoplasm, etc.; those that function by binding to the cell membrane, such as receptors and channeling molecules; and those that function by being secreted to the cell exterior, such as growth factors and cytokines, etc. In particular, protein molecules bound to the cell membrane are responsible for biologically important functions, such as cellular responses towards growth factors and differentiation factors, inflammatory responses, cell-cell interactions, hormone responses, and so on, and therefore, can be target molecules for diagnostic and therapeutic drugs for various types of disorders. [0003] In recent years, as typified by the ...

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Benefits of technology

[0010] The present invention solves the problems of the TMT method and provides a gene cloning method with a superior selectivity.

[0011] A feature of the present invention is to isolate a gene encoding a membrane-bound protein by linking a functional protein to the fusion protein itself, differing from the conventional TMT method that carries an epitope recognizing an antibody. The present method thus enabled the selective isolation of genes encoding membrane-bound proteins.

[0040] In order to increase the cloning efficiency in the invention, for example, when using single-chain Fv as the functional protein, it is preferable that the C terminus contains a small amount of hydrophobic amino acids, and specifically, a single-chain Fv in which the elbow region has been deleted as described in Examples below can be used. Also, it is preferable that, in the present invention, stability and expression efficiency can be increased by ligating further a domain of secretory protein origin, for example, a DNA encoding amino acids of the constant region of an antibody described in Examples below, to the C terminus of single-chain Fv.

[0049] The obtained cDNA is ligated to a vector. At this instance, cDNA can be introduced into the vector by introducing it downstream of the 3′ side of a functional protein encoding-DNA that is already contained in the vector. For this purpose, a suitable restriction enzyme site, for example, a multi-cloning site is designed downstream of the 3′ side of the DNA encoding the functional protein, and the cDNA is introduced into that site. Also, cDNA may be ligated first downstream of the DNA encoding the functional protein, and then the obtained DNA may be introduced into the vector. The DNA construct can be introduced into a suitable restriction enzyme site comprised in a vector DNA. When preparing the vector, the DNA encoding the functional protein and the cDNA located downstream of the 3′ side may be directly ligated, or may be ligated via a DNA encoding a peptide linker to enable easy binding of the functional protein to the antigen.

Problems solved by technology

However, the identification of a protein having a desired function from these ESTs is by no means an easy task, and in order to predict and analyze the function of an encoded-protein from gene sequence information, a great deal of time and efforts are required.

However, even if a gene fragment encoding a protein comprising a signal sequence is obtained by this method, one cannot know whether it is a secretory protein, or whether it is a membrane-bound protein.

Also, this method requires the utilization of a cDNA library comprising a 5′ end, but techniques for efficiently constructing a cDNA library that selectively contains a 5′ end are not necessarily easy, versatile techniques.

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