Characterization of recombinant human aldolase B and purification by metal chelate chromatography.

Abstract

Recombinant human aldolase B and the native enzyme purified from human liver were found to be identical in size, charge, structure, Km constants for fructose-1,6-bis(phosphate) and fructose-1-phosphate, and the activity ratio of the two substrates. Thus recombinant aldolase B is a valid model for the native enzyme and can be used to study mutations that cause hereditary fructose intolerance or others designed in the active site. Addition of six histidine residues to the amino-terminus of the recombinant enzyme did not alter its structural or functional characteristics and allowed for purification by immobilized metal affinity chromatography. This purification protocol does not require a stable or active enzyme and will facilitate the study of mutant aldolase B enzymes that would otherwise be difficult to purify.

• Authors: S A Doyle, D R Tolan
• Journal: Biochemical and biophysical research communications (Biochem Biophys Res Commun) Vol. 206 Issue 3 Pg. 902-8 (Jan 26 1995) ISSN: 0006-291X [Print] United States
• PMID: 7832803 (Publication Type: Comparative Study, Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Recombinant Proteins
• Fructose-Bisphosphate Aldolase

Topics

• Base Sequence
• Electrophoresis, Polyacrylamide Gel
• Fructose-Bisphosphate Aldolase (chemistry, genetics, metabolism)
• Humans
• Kinetics
• Liver (enzymology)
• Molecular Sequence Data
• Molecular Weight
• Mutagenesis, Site-Directed
• Recombinant Proteins (chemistry, isolation & purification, metabolism)
• Substrate Specificity