Expression of mammalian 5-aminolevulinate synthase in Escherichia coli. Overproduction, purification, and characterization

J Biol Chem. 1993 Jan 5;268(1):584-90.


5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes. A cDNA encoding for the mouse erythroid 5-aminolevulinate synthase (Schoenhaut, D. S., and Curtis, P.J. (1986) Gene (Amst.) 48, 55-63) has been expressed in Escherichia coli, using the alkaline phosphatase promoter, to a level of 50-60% of the total bacterial protein. Aminolevulinate synthase was overexpressed in an active form and, therefore, was able to rescue hemA mutants, which are unable to grow in the absence of 5-aminolevulinate. A simple purification from the aminolevulinate synthase-overproducing bacterial strain yielded approximately 50 mg of protein, in a high state of purity, per liter of bacterial culture. Moreover, the expressed aminolevulinate synthase could be easily concentrated up to 6-8 mg/ml. Significantly, recombinant aminolevulinate synthase retained physical and catalytic properties identical to those of natural sources. These include the dimeric structure, subunit molecular mass, and pyridoxal 5'-phosphate as an essential cofactor. Removal of the pyridoxal 5'-phosphate led to complete loss of activity. However, the apoenzyme could be readily reconstituted by incubation with 20 microM 5'-pyridoxal phosphate. The Km values are 51 mM for glycine and 55 microM for succinyl-CoA, in the same range of the Km values determined for the nonrecombinant enzyme. This report describes the overexpression of a mammalian 5-aminolevulinate synthase in E. coli and its purification from an overproducing strain. The ready availability of the pure, cloned, sequenced erythroid 5-aminolevulinate synthase makes it possible now for questions pertinent to the enzyme's structure, mechanism, and regulation to be addressed.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 5-Aminolevulinate Synthetase / genetics
  • 5-Aminolevulinate Synthetase / isolation & purification
  • 5-Aminolevulinate Synthetase / metabolism*
  • Animals
  • Base Sequence
  • Cell Line
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics*
  • Kinetics
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • Oligodeoxyribonucleotides
  • Plasmids
  • Polymerase Chain Reaction
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Restriction Mapping


  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • 5-Aminolevulinate Synthetase