Appeal No. 2001-0733 Page 2
Application No. 09/095,429
15. An isolated nucleic acid molecule encoding a recombinant ribonuclease
from Rana pipiens oocytes (rOnc protein), comprising:
a polypeptide of SEQ ID NO: 1 having a glutamine residue at position 1, a
hydrophobic residue other than methionine at position 23, a lysine at position 9,
a histidine at position 10, a histidine at position 97, a lysine at position 31, a
phenylalanine at position 98, and a threonine at position 35, and no more than a
single conservative amino acid substitution at other than positions 1, 9, 10, 23,
31, 35 and 98, wherein the conservative amino acid substitution does not
introduce a methionine into the polypeptide; and
a methionine at position -1 that can be subsequently cleaved to allow
cyclization of the glutamine residue at position 1 to form a pyroglutamyl residue,
such that the resulting protein is ribonucleolytic and cytotoxic.
Claims 17-19 are drawn to a method of making the polypeptide by
expressing the claimed nucleic acid. Claim 20 is drawn to a host cell that
expresses the nucleic acid molecule of claim 15, and claims 21 and 22 are
drawn to expression vectors. Claims 16, 18 and 22 specify that the encoded for
polypeptide has a leucine at position 23.
The examiner relies upon the following references:
Ardelt et al. (Ardelt) “Amino Acid Sequence of an Anti-Tumor Protein from Rana
Pipiens Oocytes and Early Embryos,” The Journal of Biological Chem.
Vol. 266, No.1 pp. 245-251 (1991)
Mosimann et al. (Mosimann) “Refined 1.7 Å X-ray crystallographic structure of P-
30 protein, an amphibian ribonuclease with anti-tumor activity,” J. Mol. Biol
Vol. 236 pp.1141-1153 (1994)
Creighton “Protein: Structure and Molecular Properties,” second edition, W.H.
Freeman and Company, New York pp. 9, 62 and 63 (1993)
Rybak et al. (Rybak) “Cytotoxic Onconase and Ribonuclease A Chimeras:
Comparision and in Vitro Characterization,” Drug Delivery, Vol. 1, pp. 3 –10
(1993)
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