Interference No. 105,099 Paper 25
Hannum v. Immunex Corp. Page 5
b) said protein precipitates in ammonium sulfate at 60 to 85%
saturation at 4°C;
c) on hydrophobic interaction chromatography with an
(NH4)2SO4 gradient in 20mM Tris, pH 7.5, on a Phenyl-5PW column, said
protein elutes between 900-750 [sic] mM (NH4)2SO4;
d) on anion exchange chromatography (NaCl gradient in
20 mM Tris, pH 7.5 on Mono Q column), said protein elutes between 130-
250 mM NaCl;
e) on cation exchange chromatography (NaCl gradient in
10 mM citrate, pH 3.0 on Mono S column), said protein elutes between
440-540 mM NaCl;
f) on gel filtration chromatography (Sephacryl S200 column),
said protein runs with an apparent molecular weight of 70 kD;
g) on reversed phase HPLC (water to acetonitrile gradient in
0.1% TFA on a Poros R/H column), said protein elutes between 32-35%
acetonitrile; and
h) said protein comprises a sequence selected from the group
consisting of:
i) Phe Val Gln Thr Asn Ile Ser His Leu Leu Lys (SEQ.
ID No. 1);
ii) Asp Tyr Pro Val Thr Val Ala Val Xaa Leu Gln Asp Glu
(Residues 1-13 of SEQ. ID No. 2); and,
iii) Trp Ile Glu Gln Leu Lys (Residues 1-6 of SEQ. ID
No. 4).
[16] Hannum SEQ ID NO:1 is [3002]:7
Phe Val Gln Thr Xaa Ile Ser His Leu Leu Lys
7 C.H. Hannum, J.A. Culpepper, F.D. Lee & D. Birnbaum, "Purified Mammalian Flt3 Ligands;
Agonists; Antagonists", Appln. No. 08/484,882 (filed 7 June 1995).
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