Purification of high yields of catalytically active lysyl oxidase directly from Escherichia coli cell culture.
Lysyl oxidase is a highly insoluble enzyme requiring high concentrations of urea to solubilize. A method to obtain lysyl oxidase in high yields directly from an Escherichia coli culture without the need for refolding of inclusion bodies has been developed using nutrient rich media. pET21b was used to overexpress the lysyl oxidase enzyme and to introduce a C-terminal 6X histidine tag for purification. Lysyl oxidase yields of 10 mg of active and properly folded enzyme per liter of media have been obtained. Purification was achieved via affinity chromatography using a Ni-NTA column. Copper content was found to be 19%. LTQ cofactor formation in LOX is a self-processing event in the presence of copper. LTQ content was determined to be 24% based on reaction with phenylhydrazine to form a phenylhydrazone adduct. Quantification of this adduct was attained using the previously reported extinction coefficient of 15.4 mM(-1)cm(-1). LTQ presence was also verified by redox cycling. Specific enzymatic activity was measured to be 0.31 U/mg, one of the highest activities reported.
Herwald
Sanna E
SE
Department of Chemistry, Pomona College, Claremont, CA 91711, United States.
Greenaway
Frederick T
FT
Lopez
Karlo M
KM
D000595:Amino Acid Sequence; D003300:Copper / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010659:Phenylhydrazines / Q000037:antagonists & inhibitors; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011809:Quinones / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D010659:Phenylhydrazines; D011809:Quinones; D011994:Recombinant Proteins; C030299:phenylhydrazine; D003300:Copper; D008249:Protein-Lysine 6-Oxidase