Modified cDNA of rat bcl-x gene and modified protein

#This application is a 371 of PCT/JP00/05502 filed Aug. 17, 2000.

#1. Field of the Invention

#The present invention relates to a genetically engineered cDNA of rat bcl-x gene and an improved protein. More particularly, it relates to a novel cDNA expressing an improved protein of Bcl-xL having higher apoptosis-inhibiting activity and cell death-inhibiting activity than the protein Bcl-xL expressed by the rat's apoptosis-inhibiting gene bcl-x. The invention also relates to materials for utilizing such cDNA in gene engineering, as well as to an improved protein of Bcl-xL expressed by the cDNA.

#FIG. 1 shows the three-dimensional structure of the wild-type rat Bcl-xL.

#FIG. 2 shows the results of Western blotting analysis of the expression level of the improved protein Bcl-xLFNK in the transfected cell.

#FIG. 3 shows the results of a test for resistance of the transfected cells to apoptosis induced by serum depletion.

#The engineered cDNA of the invention as indicated in the above invention (1), is characterized by having at least one substitution selected from the substitutions that change the codon of residues 22 Tyr (tac) to the codons of Phe (ttt/ttc), the codon of residues 26 Gln (cag) to the codons of Asn (aat/aac) and the codon of residues 165 Arg (cgg) to the codons of Lys (aaa/aag), in the coding region of rat bcl-x cDNA of SEQ ID NO: 1. In a preferred embodiment, the nucleotide substitutions have occurred in all the above 3 sites in the engineered cDNA of the invention (1). The engineered cDNA which has the nucleotide substitutions at the 3 sites produces the improved protein Bcl-xFNK as shown in the amino acid sequence of SEQ ID NO: 3. In this improved protein Bcl-xFNK, three hydrogen bonds between Tyr22 and Asp156, between Gln26 and Ser164 and between Arg165 and Pro116, which are formed in the wild-type rat Bcl-xL as shown in the three-dimensional structure of FIG. 1, are disturbed as a result of the amino acid substitutions (Tyr22Phe; Gln26Asn; Arg165Lys) caused by the above-described nucleotide substitutions.

#The engineered cDNA may be prepared by a known method with a mutation kit or a PCR method as mentioned in Examples using the rat bcl-x cDNA as a template. For the cDNA of rat bcl-x, the plasmid pEF1-BOSbcl-x (J. Biol. Chem. 271(22): 13258-13265, 1996) can be used. An alternative method may be used, in which an oligonucleotide of any parts of the nucleotide sequence of SEQ ID NO: 1 is synthesized to use as a probe for screening of a rat cDNA library. Or an oligonucleotide that hybridizes with both ends of the cDNA fragment to be a subject may be synthesized and used as a primer in an RT-PCR method of preparing the cDNA from the mRNA isolated from rat cells.

#The inventions (2) and (3) relates to a DNA fragment (polynucleotide) in which an oligonucleotide encoding a protein-transduction-domain peptide is ligated to the 5′-end of the engineered cDNA of the above invention (1). This DNA fragment can be used in preparation of the improved protein of Bcl-xL as mentioned below.