On the production of alpha, beta-heterodimeric acyl-coenzyme A: isopenicillin N-acyltransferase of Penicillium chrysogenum. Studies using a recombinant source

FEBS Lett. 1993 Mar 15;319(1-2):166-70. doi: 10.1016/0014-5793(93)80060-8.


A high level E. coli expression system has been constructed for the Penicillium chrysogenum penDE gene, which encodes the acyl-coenzyme A: isopenicillin N-acyltransferase (AT) enzyme. Induction of overexpression of recombinant AT (recAT) by increasing the growth temperature of the host adversely affected solubility and activity of the AT enzyme. Addition of isopropylthio-beta-D-galactopyranoside (IPTG) at decreased growth temperatures (less than 32 degrees C) resulted in the overproduction of soluble, active recAT. When purified to homogeneity, recAT was an alpha, beta-heterodimer, comprised of 11 kDa (alpha) and 29 kDa (beta) subunits, derived from a 40 kDa precursor polypeptide by a posttranslational cleavage. The recAT enzyme contained both the acyl-coenzyme A: isopenicillin N-acyltransferase and the acyl-coenzyme A: 6-aminopenicillanic acid acyltransferase activities. The processing event that generated the two subunits of recAT from the 40 kDa precursor polypeptide occurred between Gly102/Cys103. This expression system produced a large amount of soluble, active recAT that is identical to native AT, making it a suitable source of AT enzyme for further characterization.

MeSH terms

  • Acyltransferases / biosynthesis*
  • Acyltransferases / chemistry
  • Acyltransferases / genetics
  • Amino Acid Sequence
  • Base Sequence
  • DNA Restriction Enzymes
  • Escherichia coli / genetics
  • Gene Expression
  • Hot Temperature
  • Isopropyl Thiogalactoside / pharmacology
  • Macromolecular Substances
  • Mass Spectrometry
  • Molecular Sequence Data
  • Penicillin-Binding Proteins*
  • Penicillium chrysogenum / enzymology*
  • Plasmids
  • Protein Precursors / metabolism
  • Protein Processing, Post-Translational
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Solubility


  • Macromolecular Substances
  • Penicillin-Binding Proteins
  • Protein Precursors
  • Recombinant Proteins
  • Isopropyl Thiogalactoside
  • Acyltransferases
  • acyl-CoA-6-aminopenicillanic acid acyltransferase
  • DNA Restriction Enzymes