An approach to prevent aggregation during the purification and crystallization of wild type acyl coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum.
Acyl coenzyme A: isopenicillin N acyltransferase (AT) from Penicillium chrysogenum is an enzyme of interest for the biosynthesis of beta-lactam antibiotics. Severe aggregation problems with wild type AT have, however, prevented significant progress in the structure-function analysis of this enzyme for a decade. In this study, we show an approach to solve this aggregation problem by using dynamic light scattering (DLS) analysis to probe the aggregation state of the protein in the presence of various additives. After a one-step purification of recombinant wild type AT with a C-terminal His-tag using Ni2+ affinity chelate chromatography, addition of a combination of 5 mM DTT, 250 mM NaCl, and 5 mM EDTA to the purified AT effectively prevented aggregation. In the presence of these additives, the DLS profile of AT shows a narrow size distribution indicative of a homogeneous protein solution and the absence of aggregation. The purity and mono-dispersity of wild type AT was sufficient for the growth of high quality crystals diffracting to 1.64 A resolution.
Yoshida
Hiromi
H
Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Hensgens
Charles M H
CM
van der Laan
Jan Metske
JM
Sutherland
John D
JD
Hart
Darren J
DJ
Dijkstra
Bauke W
BW
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D008027:Light; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010408:Penicillium chrysogenum / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012542:Scattering, Radiation
D046912:Multiprotein Complexes; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase