Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Patent
1984-07-27
1987-06-02
Phillips, Delbert R.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C07K 710
Patent
active
046705392
ABSTRACT:
Two forms of polypeptide growth factors have been purified to homogeneity from lyophilized powder of mature ewe kidneys. From 500 g of powder 8 to 14 mg of kidney derived growth factor (KDGF) was isolated. An 18,000 fold purification was accomplished with a 4 to 7% yield by a 6 step procedure that included an initial acetic acid extraction, heating at 95.degree., Bio-Rad AG50W X8 cation exchange chromatography, 2 sequential DEAE-Sepharose CL-6B anion exchange steps at pH 5.8 and 6.2, respectively, and finally Sephadex G-50 chromatography in 0.1M acetic acid. From the Sephadex molecular sieve separation, the KDGF activity eluted in the same fractions as the single protein peak. Polyacrylamide gel electrophoresis analysis under non-reducing and non-denaturing conditions followed by Coomassie Blue staining confirmed a single band having a M.sub.r 4,200. Molecular sieve HPLC done under acidic conditions confirmed a similar high (i.e. greater than 95%) degree of homogeneity. This preparation of KDGF was shown to be mitogenic for rat pituitary tumor cells, and hamster and rat kidney tumor cells, but not for normal rat diploid fibroblasts or low passage normal rat kidney (NRK) cells. The KDGF preparation that appeared greater than 90% homogeneous by the criteria described above was separated by chromatofocusing into 2 forms, KDGF-I and KDGF-II, of pI 5.2 and 4.8, respectively. Both forms showed M.sub.r 4200. KDGF-II was the more abundant form, being 70% of the total KDGF isolated from the chromatofocusing elution. The G.sub.50s of KDGF-I and KDGF-Ii were 10 ng/ml (2.38.times.10.sup.-9 M) and 19 ng/ml (4.52.times.10.sup.-9 M), respectively, with the MTW9/PL cells in culture without any other additions to the serum free medium used in these bioassays.
REFERENCES:
Ikeda, T. et al., "Isolation and Properties of Endocrine and Autocrine Type Mammary Tumor Cell Growth Factors (Estromedins) in: Proceedings of the Second International Congress on Hormones and Cancer," (F. Bresciani et al., ed.), Raven Press, New York, pp. 171-186, (1984).
Ikeda Tatsuhiko
Sirbasku David A.
Board of Regents The University of Texas
Phillips Delbert R.
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