Expression systems utilizing autolyzing fusion proteins and a novel reducing polypeptide
AssigneeSankyo Company, Limited
InventorsTohru Takahashi, Nobufusa Serizawa, Ryuta Koishi, Ichiro Kawashima
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Abstract
The present invention provides expression systems for exogenous polypeptides wherein the polypeptide is expressed as a fusion protein together with clover yellow virus Nuclear Inclusion a (NIa), the NIa component serving to autolyze the fusion protein after expression. This system can be used to express a novel polypeptide which we have designated KM31-7 protein and which is capable of reducing dichloroindophenol and reduced glutathione. This polypeptide is useful in the treatment of disorders caused by oxidative stress.
Claims46
An isolated polynucleotide sequence encoding a fusion protein and comprising, in the 5′ to 3′ direction and in the same open reading frame:
- (a) a sequence encoding the clover yellow vein virus Nuclear Inclusion a protein;
- (b) a sequence encoding a cleavage peptide recognizable by and cleavable by said clover yellow vein virus Nuclear Inclusion a protein;
- and
- (c) at least one sequence encoding an exogenous polypeptide, wherein said sequence does not encode a protein or peptide as set forth in (a) or (b).
A polynucleotide sequence encoding clover yellow vein virus Nuclear Inclusion a protein as given by nucleotide numbers 10 to 1311 in SEQ ID NO:
- 1.
An isolated polynucleotide sequence encoding a polypeptide which comprises the amino acid sequence of amino acid numbers 1 to 526 of SEQ ID NO:
- 12, provided that the polypeptide encoded by the polynucleotide sequence is capable of catalyzing the reduction of dichloroindophenol and oxidized glutathione.
An isolated polynucleotide sequence which has a nucleotide sequence consisting of nucleotides 70 to 1647 of SEQ ID NO:
- 11, wherein said isolated polynucleotide sequence encodes a polypeptide which catalyzes the reduction of dichloroindophenol and oxidized glutathione.
An isolated DNA which hybridizes under high stringency conditions to nucleotides 10 to 1311 of SEQ ID NO:
- 1, wherein the corresponding sense strand of said isolated DNA encodes a protein having protease activity and wherein said conditions for hybridization comprise hybridization in 6×SSC at 60 to 70° C. and washing in 0.1×SSC at 60 to 70° C.
An isolated DNA which hybridizes under high stringency conditions to SEQ ID NO:
- 11, wherein the corresponding sense strand of said isolated DNA encodes a polypeptide having oxidoreductase activity and wherein the conditions for hybridization comprise hybridization in 6×SSC at 60 to 70° C. and washing in 0.1×SSC at 60 to 70° C.
Dependencies are inferred from the claim text.