Expression and purification of mammalian 5-aminolevulinate synthase.
We have described a procedure for production and purification of recombinant, mature-length mouse ALAS-2. The fact that E. coli utilizes the C5 path for ALA production means that there is no problem with contamination of the recombinant ALAS-2 by host cell enzyme, such as one may have with a yeast expression system. While the detailed procedure produces enzyme in good yield with relatively common protein purification techniques, future expression systems may be developed to take advantage of the rapid purification achieved by the use of a 6-histidine (His6) aminoterminal tag and metal chelate chromatography. Such approaches in this laboratory with protoporphyrinogen oxidase, coproporphyrinogen oxidase, and uroporphyrinogen decarboxylase have resulted in the production and purification of enzymes whose kinetic and physical parameters are essentially identical to those of proteins lacking the His6 tag.
Dailey
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Department of Microbiology, University of Georgia, Athens 30602-7229, USA.
Dailey
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D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000622:Aminolevulinic Acid / Q000378:metabolism; D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D011994:Recombinant Proteins; D000622:Aminolevulinic Acid; D000624:5-Aminolevulinate Synthetase