Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization
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Leona Wagner
Abstract
In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4–6, activity staining resulted in at least 17 isoforms between pH 4.8–6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5–6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.
©2011 by Walter de Gruyter Berlin Boston
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- GENES AND NUCLEIC ACIDS
- Sequence elements outside the catalytic core of natural hairpin ribozymes modulate the reactions differentially
- PROTEIN STRUCTURE AND FUNCTION
- Cooperative and independent activities of Sgt2 and Get5 in the targeting of tail-anchored proteins
- Novel neutrophil inhibitory factor homologue in the buccal gland secretion of Lampetra japonica
- MEMBRANES, LIPIDS, GLYCOBIOLOGY
- Generation of artificial channels by multimerization of β-strands from natural porin
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- Gender-related differences in the oxidant state of cells in Fanconi anemia heterozygotes
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