Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-05-07
2000-01-04
Ketter, James
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 15, C12Q 168
Patent
active
060108584
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to an expression cloning method of a gene that codes for protein kinase substrate protein. According to the present invention, a gene can be efficiently cloned that codes for a protein which is a substrate of protein kinase having useful biological activity.
BACKGROUND ART
When cells are stimulated with a growth factor and so forth, information is transmitted inside the cells via receptors on the cell surface layer. In many cases, this transmission of information is known to be performed by phosphorylation of protein. Many receptors are protein kinases or form complexes with protein kinases. Information of growth factor etc. activates protein kinase in this manner resulting in phosphorylation of the target protein, which is then transmitted inside the cell so that the response of the cell begins.
Protein kinase is an enzyme that transfers the phosphoric acid at the .gamma.-position of ATP to a hydroxyl group of serine, threonine, tyrosine and so forth, and plays the role of a central mechanism in the transmission of information into cells. Protein kinases play an important role in the function regulatory mechanisms of almost all cells. For example, they have been clearly shown to be involved in cell movement, cell growth, metabolic response, immune response and so forth. In this way, in order to understand the function regulatory mechanisms of cells, it is essential to determine the target proteins of protein kinases. However, since the substrate proteins of protein kinases are not always clear, many unknown aspects remain regarding their details.
Efforts have been made in the past to explain the phosphorylation reaction in which protein kinases are involved. For example, Carmel, G. & Kuret, J. reported that the substrate selectivity of protein kinase was analyzed by expressing a DNA library containing a mutant gene fragment of a protein kinase substrate protein constructed using the cassette mutation induction method in an E. coli expression system, and performing a phosphorylation reaction in the solid phase in the presence of protein kinase (Analytical Biochemistry (1992), 203, 274-280). However, this method is limited to analysis of the site that recognizes the substrate protein on the protein kinase, and there is no mention of expression cloning of a gene that codes for protein kinase substrate protein. Thus, a method for efficiently cloning a gene that codes for protein kinase substrate protein was unknown.
DISCLOSURE OF THE INVENTION
As a result of earnest studies conducted by the inventors of the present invention on a cloning method of a gene that codes for a protein kinase substrate protein, it was found that a protein coding for protein kinase substrate protein can be efficiently and easily acquired by expressing a DNA library containing various genes and directly identifying protein kinase substrate protein by using a phosphorylation reaction, thereby leading to completion of the present invention.
Thus, the present invention provides a method for cloning a gene that codes for protein kinase substrate protein at a high level of efficiency unattainable with conventional methods.
In order to solve the above-mentioned problems, the present invention provides an expression cloning method of a gene that codes for protein kinase substrate protein comprising: plate-culturing a host into which DNA containing an expression vector has been introduced, expressing said DNA, transferring protein produced by contacting a film onto said plate from said plate to said film, removing said film from said plate, adding a phosphate donor and protein kinase to said film to phosphorylate said protein, detecting phosphoric acid bonded to said protein, and isolating DNA from the host clones on the plate corresponding to the sites on the film that exhibit a positive reaction.
In the case said protein kinase substrate protein is a protein having phosphorylating ability on its own (a protein kinase substrate having a self-phosphorylating ability), it is not necessary to
REFERENCES:
Carmel et al., Analytical Biochemistry, 1992, vol. 203, pp. 274-280.
LandOfFree
Method for expression cloning of gene encoding protein kinase su does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for expression cloning of gene encoding protein kinase su, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for expression cloning of gene encoding protein kinase su will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1071661