Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1994-05-13
1996-03-26
Wax, Robert A.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
4352523, 43525233, 4353201, 435200, 536 232, C12N 1529, C12N 1556, C12N 121, C12N 510, C12N 1563
Patent
active
055019706
DESCRIPTION:
BRIEF SUMMARY
This invention refers to DNA sequences coding for proteins present in the plant Dianthus caryophyllus, to expression vectors containing said sequences and to hosts transformed by said vectors.
It is widely known that proteins extracted from various plants are able to inhibit protein synthesis of animal cells by inhibiting their ribosomes.
This group of proteins has been classified as ribosome inactivating proteins type 1 and type 2 (RIP 1 and RIP 2 ) according to the numbers of polypeptide chains they are composed of: RIPs 1 have a single polypeptide chain having enzymatic activity causing ribosome modifications; RIPs 2, besides this chain, have a second chain binding to cells with particular receptor molecules.
RIPs 2 are very toxic because of their capacity to bind most intact cells: examples of RIPs 2 are ricin, abrin, modeccin, viscumin. RIPs 1 are practically non-toxic since they cannot bind to cell surface; they need to enter the cytoplasm to exert their enzymatic activity inhibiting the protein synthesis; examples of RIPs 1 are saporin, gelonin, momordin, dianthin 30 and 32. The last two ones may be extracted from different tissues of the plant Dianthus caryophyllus wherein they are present in two forms having different molecular weight, respectively 30000 and 32000 Daltons; said molecules seem to be two different proteins although no definitive conclusion has been drawn.
According to their molecular weight, they were named dianthin 30 and 32 (Stirpe et al Biochem. J., 1981, 195, pp. 399-405; Falasca et al Biochem. J., 1982, 207, pp. 505-509). The biochemical characteristics of these two proteins are similar: pI is remarkably basic for both (>9.5 pH units ); kms calculated on rabbit reticulocyte lysate are identical whereas kcat are slightly different, that of dianthin 32 being higher than dianthin 30.
It is known that some proteins are expressed together with signal peptides controlling the targeting of proteins to different organelles. Some signal peptides are cleaved once completed their targeting role and are therefore no longer present in the mature protein.
In the case of dianthin 30, the protein is synthesized as a precursor, pre-dianthin, containing the active sequence preceded by a signal peptide characteristic of the secretion protein, targeting the protein to the endoplasmatic reticulum.
This signal sequence is located at the N-terminus of the polypeptide chain and it is characterized by the presence of hydrophobic amino acids helping the translocation of the protein.
We have cloned and expressed cDNA sequences coding for RIP 1 of Dianthus caryophyllus, dianthin 30 and for its precursor pre-dianthin 30.
The DNA sequences of the invention are reported in the Sequence Listings Nos: 1 and 2.
The present invention also relates to DNA sequences that hybridize to the above mentioned DNA sequence and that encode a polypeptide having dianthin-like activity. In this context, the term "hybridization" refers to conventional hybridization conditions.
Most preferably, the term "hybridization" refers to stringent hybridization conditions.
The DNA sequences of the invention may be obtained according to the following method: cDNA library; radioactive phosphorus coding for particular regions of the protein to be identified or by anti-dianthin polyclonal antibodies raised in rabbit so as to identify a clone containing the expressed protein and, finally, identified clone; (v) so as to amplify only the region coding for either pre-dianthin 30 or for the native protein.
The mRNA is extracted from leaves of Dianthus caryophyllus.
The cDNA may be synthesized using commercial kits.
cDNA is inserted in a cloning vector to obtain a cDNA library. The cloning vector may be a plasmid or a bacteriophage.
In the present invention, the lambda gt 11 phage has been used as a cloning vector, in which the different cDNA sequences of the library are inserted in its genome so as to allow the expression of a fusion protein between the beta-galactosidase normally present in the phage and the protein to be cloned.
The express
REFERENCES:
patent: 5317009 (1994-05-01), Lee-Huang et al.
Falasca et al. (1982) Biochem J. 207:505-509.
Legname et al. (1991) Biochim. Biochim. Biophys. Acta 1090: 119-122.
Brocchetti Diego
Gromo Gianni
Legname Giuseppe
Modena Daniela
Bugaisky G. E.
Italfarmaco S.p.A.
Wax Robert A.
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