Gel Electrophoretic Procedures Potentially Applicable to the Isolation of Human Growth Hormone from A Transformed Bacterial Source at the Gram-Preparative Scale |
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| Authors: | G Kapadia A J S Jones A Chrambach |
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| Institution: | 1. Laboratory of Theoretical and Physical Biology , National Institute of Child Health and Human Development, National Institutes of Health , Bethesda, MD, 20205;2. Genentech Inc. , 460 Point San Bruno Blvd., South San Francisco, CA, 94080;3. Genentech Inc. , 460 Point San Bruno Blvd., South San Francisco, CA, 94080 |
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| Abstract: | A crude bacterial extract containing approximately 4 mg/ml protein, 25% of which was human growth hormone (hGH), was subjected to two alternative gel electrophoret ic isolation procedures, designated I and II. Procedure I exploits the high electrophoretic net mobility (RM2 larger than 0.127) at pH 7.6, 0°C, of the bacterial contaminants relative to hGH. This allows one to stack 3 the contaminants at a protein load of 31.5 mg/cm2 of gel, using a “non-restrictive” gel concentration. Unstacked hGH is collected from the gel section between 0.3 and 0.6 of relative gel length and extracted electrophoretically as described previously4 Alternatively, the unstacked hGH was concentrated on the gel by dispatching a second moving boundary behind the original stack (“re-stacking”) and a gel section (relative gel length 0.45 to 0.6) between the two moving boundaries was excised and subjected to electrophoretic extraction. The yield of hGH ranged from 70 to 82%, and its purity (weight/Lowry) ranged from 86 to 115%. Procedure II exploits the high electrophoretic net mobility (RM larger than 0.064) at pH 10.5, 0°C, of hGH relative to its bacterial contaminants at a gel concentration of 9 %T, 2 %C_. 2, at a protein load of 2.5 mg/cm2 of gel. The selectively stacked hGH is collected by preparative elution-PAGE, using an apparatus with 17.6 cm2 gel surface area. The yield of hGH was 90% and its purity ranged from 84–92%. |
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