[13] Hepatic lipase: High-level expression and subunit structure determination
This chapter describes procedures developed for a large-scale production and purification of recombinant hepatic lipase (HL). The purification of sufficient quantities of this enzyme has enabled the analysis of its subunit structure by three independent means: intensity light scattering, sedimentati...
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| Veröffentlicht in: | Methods in Enzymology 1997, Vol.284, p.232-246 |
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| creator | Hill, John S. Davis, Richard C. Yang, Dawn Schotz, Michael C. Wong, Howard |
| description | This chapter describes procedures developed for a large-scale production and purification of recombinant hepatic lipase (HL). The purification of sufficient quantities of this enzyme has enabled the analysis of its subunit structure by three independent means: intensity light scattering, sedimentation equilibrium, and radiation inactivation. The low protein yields associated with the purification of HL from human postheparin plasma have not permitted a more detailed physical analysis of this enzyme. For example, the only previous estimations of the functional molecular weight of HL have relied on gel-filtration techniques. The primary disadvantage of conventional size-exclusion chromatography methods is that the elution position depends not only on the molecular weight of the molecule, but also on its shape. In addition, if the protein has a tendency to adhere to the column matrix, the molecular weight may be underestimated. |
| doi_str_mv | 10.1016/S0076-6879(97)84015-3 |
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The purification of sufficient quantities of this enzyme has enabled the analysis of its subunit structure by three independent means: intensity light scattering, sedimentation equilibrium, and radiation inactivation. The low protein yields associated with the purification of HL from human postheparin plasma have not permitted a more detailed physical analysis of this enzyme. For example, the only previous estimations of the functional molecular weight of HL have relied on gel-filtration techniques. The primary disadvantage of conventional size-exclusion chromatography methods is that the elution position depends not only on the molecular weight of the molecule, but also on its shape. In addition, if the protein has a tendency to adhere to the column matrix, the molecular weight may be underestimated.</abstract><cop>United States</cop><pub>Elsevier Science & Technology</pub><pmid>9379936</pmid><doi>10.1016/S0076-6879(97)84015-3</doi><tpages>15</tpages></addata></record> |
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| source | Elsevier ScienceDirect Journals Complete - AutoHoldings; MEDLINE; ScienceDirect eBooks |
| subjects | Animals CHO Cells Chromatography Chromatography, Affinity Chromatography, Ion Exchange Cloning, Molecular Cricetinae Durapatite Humans Indicators and Reagents Kinetics Lipase - biosynthesis Lipase - chemistry Lipase - isolation & purification Liver - enzymology Macromolecular Substances Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - isolation & purification Restriction Mapping Transfection - methods |
| title | [13] Hepatic lipase: High-level expression and subunit structure determination |
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