Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment electrolyzer with Immobiline membranes.
Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.
Department of Biomedical Sciences and Technologies, Univ. Milano, Italy.
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Ettori
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Righetti
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Chiesa
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Frigerio
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Galli
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Grandi
D013006:Growth Hormone / Q000302:isolation & purification*; D006801:Humans; D007525:Isoelectric Focusing; D011498:Protein Precursors / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification
D011498:Protein Precursors; D011994:Recombinant Proteins; D013006:Growth Hormone