A four-step procedure for the purification of thrombopoietin.
The present work reports the preparation of a highly bioactive and stable thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) by a four-step purification procedure, i.e., Sephadex column chromatography, ethanol precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reverse phase-high performance liquid chromatography. The molecular weight (MW) of the purified product depended upon the method of purification, i.e., using denaturing buffers at 56 degrees C for 10 min, the MW was approximately 30,000 daltons; whereas, after preparing in denaturing buffers and heating to 100 degrees C for 10 min, the purified protein had an apparent MW of approximately 15 kd. Both moieties had significant biological activity. The data indicate that TSF may exist normally as a dimer (30 kd), but can disassociate to 15 kd without loss of bioactivity. The present work illustrates that the purified TSF has an isoelectric pH of 4.47 and exists in trace amounts in human embryonic kidney (HEK) cell culture media. The final product prepared in the presence of Tween-20 had a specific activity of approximately 21,000 U of TSF per mg of protein, representing a purification factor of approximately 164,000. Using this four-step purification procedure, a homogeneous product was obtained as judged by SDS-PAGE and chromatofocusing. This purified material will be suitable for further studies, including amino acid sequencing.
McDonald
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TP
University of Tennessee, College of Veterinary Medicine, Knoxville 37901-1071.
Clift
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RE
Cottrell
M B
MB
D011232:Chemical Precipitation; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D013926:Thrombopoietin / Q000302:isolation & purification*
D006023:Glycoproteins; D000431:Ethanol; D013926:Thrombopoietin