Transmembrane trapping methods
OwnerHiromitsu Nakauchi, Yukio Nakamura +2inventors
InventorsHiromitsu Nakauchi, Yukio Nakamura, Sousuke Miyoshi, Dong ku Kim
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Abstract
The present invention provides a method of cloning a protein coding for a membrane protein without the need of using anyantibody or ligand. The method of cloning a membrane protein comprises selecting a protein having a signal sequence, using a DNA coding for the signal sequence and N-terminal sequence of the protein as the reporter gene, and screening for a gene for a protein having a transmembrane domain(s). The present invention is concerned also with the CDNA obtained by using such method, a vector containing the DNA, a transformant as transformed with the vector, and the transcription product of the DNA as produced by the transformant.
Claims14
A method of cloning DNA coding for a target transmembrane protein, which comprises:
- a) selecting a known membrane protein comprising a signal sequence, an N-terminal sequence, and a transmembrane domain;
- b) preparing a reporter gene coding for the signal sequence and the N-terminal sequence of said membrane protein by elimination of the transmembrane domain and downstream thereof;
- c) constructing a DNA library comprising the DNA coding for a target transmembrane protein;
- d) constructing an expression vector by joining the DNA library obtained in c) to the downstream of the reporter gene obtained in b);
- e) transforming a host with the expression vector obtained in d);
- f) selecting cells in which the reporter gene is expressed on the cell surface;
- g) recovering the expression vector from the positive cells obtained to give a clone comprising a part or the whole of the DNA coding for a target transmembrane protein;
- and
- h) determining the DNA sequence of the expression vector obtained in g).
Dependencies are inferred from the claim text.