Production, IMAC purification, and molecular modeling of N-carbamoyl-D-amino acid amidohydrolase C-terminally fused with a six-his peptide.
A six-His peptide was genetically engineered to the C-terminus of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase monomer to facilitate the protein purification with immobilized metal affinity chromatography (IMAC). The fusion enzyme, named as DCaseH, was overexpressed in Escherichia coli and one-step IMAC-purified. The production study showed that DCaseH was optimally produced at 15 degrees C for 25 h by the induction of 0.05 mM IPTG. Both Co(2+)-chelated TANOL gels and Ni(2+)-chelated nitriloacetic acid agarose gels efficiently purified DCaseH, with the former yielding purer enzyme than the latter. Highly pure DCaseH was obtained in the former purification with the addition of 5 mM imidazole in the washing buffer, and the specific enzyme activity was increased more than 11-fold. Denaturing IMAC purification successfully purified DCaseH from inclusion bodies that were mostly composed of the overexpressed DCaseH, while the attempt to refold the purified enzyme by either dialysis or solid-state refolding was not achieved. The purified native enzyme was optimally active at pH 6.5 and 50 degrees C, and the presence of 10% glycerol increased the activity. The molecular modeling of dimeric DCaseH indicated that the six-His tags were freely exposed to the protein surface, resulting in the selective and effective IMAC purification of DCaseH.
Chen
Hsiu-Mei
HM
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan. hsiumei@ch.ntust.edu.tw
Ho
Cheng-Wei
CW
Liu
Jui-Wen
JW
Lin
Kun-Ying
KY
Wang
Yi-Tung
YT
Lu
Chi-Hung
CH
Liu
Hsuan-Liang
HL
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D012231:Rhizobium / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship
D006639:Histidine; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase