Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1998-08-12
2000-08-22
Horlick, Kenneth R.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 2431, 536 2433, C07H 2104
Patent
active
061074748
DESCRIPTION:
BRIEF SUMMARY
The present invention refers to a cDNA sequence encoding a 14 kDa protein from goat liver.
WO 92/10197 discloses perchloric acid extracts of mammalian organs, in, particular of goat liver, consisting of at least three different proteins and characterized by unusual pharmacological and immunological properties.
More recently, in WO 96/02567, the partial aminoacid sequence of a 14 kDa protein purified from the extracts disclosed in WO 92/10197 has been described. This protein shows marked antineoplastic activity either in vitro and in vivo and the serum of animals immunized with this protein displays cytotoxic activity against human tumor cell lines.
In 1993, Levy-Favatier et al. [Eur. J. Biochem. 212 (3), 665-673 ] have reported the cDNA sequence coding for a 23 kDa dimeric protein purified by 5% perchloric acid from rat liver and kidney; the corresponding aminoacid sequence shows an high degree of homology with the sequence of the 14 kDa protein disclosed in WO 96/02567. The cDNA sequence has been submitted to the EMBL Data Bank with Accession no. X70825.
In 1995, another cDNA sequence coding for a 14 kDa protein purified by perchloric acid from rat liver, with an high degree of homology with that published by Levy-Favatier et al., has been submitted to the EMBL Data Bank with Accession no. D49363. This sequence has been reported by Oka et al. in J. Biol. Chem. (1995) 270 (50), 30060-30067.
Furthermore, in 1996, two novel mRNA sequences with an high degree of homology with the cDNA published by Levy-Favatier et al. and by Oka et al. have been submitted to the EMBL Data Bank. One of these (Accession no: X95384) codes for a 14.5 kDa human protein and it has been recently published by Schmiedeknecht et al. [Eur. J. Biochem. (1996) 242 (2), 339-351]. The other sequence (Accession no: U50631) codes a "Mus Musculus heat-responsive protein"; up to now, no extensive paper concerning this sequence has been published.
We have now found a new cDNA sequence, encoding the entire 14 kDa protein extracted by perchloric acid from goat liver and disclosed in WO 96/02567. The complete nucleotide sequence is reported in SEQ ID NO: 1.
The cDNA sequence coding for the 14 kDa protein extracted by perchloric acid from goat liver and disclosed in WO 96/02567 (SEQ ID NO: 2), is useful for the preparation of said protein or of muteins thereof by means of recombinant DNA methods or for diagnostic applications based on nucleotide probes.
The cDNA sequence of the invention has been obtained by the following method: two mixtures of degenerate oligonucleotides have been synthesized on the basis of the aminoacid sequence disclosed in WO 96/02567. One mixture (named PG-1) consists of 2.048 oligo-20-mers, corresponding to aminoacid sequence extending from Met-1 to Gln-7. The other mixture (named PG-2) consists of 192 oligo-20-mers corresponding to aminoacid sequence extending from Ala-46 to Xaa-52.
Using these oligonucleotide mixtures as primers and cDNAs obtained by reverse transcription of total RNA purified from goat liver as template, a PCR reaction has been performed. The reverse transcription reaction has been carried out at 42.degree. C. for 60 min. using oligo(dT).sub.15 as primer. After 35 cycles of amplification at the following conditions: 95.degree. C. for 2 min.-55.degree. C. for 2 min.-72.degree. C. for 1 min. in 2 mM MgCl.sub.2, the DNA fragment amplified (155 bp) has been subcloned in the plasmid vector pCRII and the insert of three different clones has been sequenced using T7 and Sp6sequencing primers. The nucleotide sequence of this fragment (corresponding to region extending from nt. 215 to nt. 369 of the Sequence Id n. 1) confirms the aminoacid sequence disclosed in WO 96/02567 and identifies the Xaa-33 as Cys.
After the characterization of the complete aminoacid sequence of the 14 kDa protein reported by Ceciliani et al. [FEBS Lett. (1996) 393, 147-150] and following a procedure similar to that described above, the nucleotide sequence extending from nt. 215 to nt. 511 of the SEQ ID NO: 1 has been determined. Briefly, d
REFERENCES:
Schmiedeknecht et al., Eur. J. Biochem. 242, 339-351 (Dec. 1996).
Colombo et al., Biochim. Biophys. Acta 1442, 49-59 (1998).
Levy Favatier et al., Eur. J. Biochem. 212, 665-673 (1993).
Journal of Biological Chemistry, vol. 270, No. 50, Dec. 15, 1995, pp. 30060-30067, XP002031276, T. Oka et al, "Isolation and Characterization of a Novel Perchloric Acid-soluble Protein Inhibiting . . . ".
FEBS Letters, vol. 393, No. 2,3, Sep. 16, 1996, pp. 147-150, XP002031277, F. Ceciliani et al, "The primary structure of UK114 tumor antigen".
Bartorelli Alberto
Berra Bruno
Colombo Irma
Ronchi Severino
Horlick Kenneth R.
Zetesis S.p.A
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