On the production of alpha, beta-heterodimeric acyl-coenzyme A: isopenicillin N-acyltransferase of Penicillium chrysogenum. Studies using a recombinant source.
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.
Aplin
R T
RT
Dyson Perrins Laboratory, Oxford, UK.
Baldwin
J E
JE
Cole
S C
SC
Sutherland
J D
JD
Tobin
M B
MB
D000217:Acyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D046915:Penicillin-Binding Proteins; D010408:Penicillium chrysogenum / Q000201:enzymology*; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility
D046911:Macromolecular Substances; D046915:Penicillin-Binding Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase; D004262:DNA Restriction Enzymes