Overproduction and crystallization of tryptophanase from recombinant cells of Escherichia coli.
We have cloned the tryptophanase structural gene from Escherichia coli B/1t7-A into E. coli K-12 MD55 with a vector plasmid, pBR322. The cloned cells produced a large amount of the enzyme corresponding to more than 30% of the total soluble protein. With the enzyme obtained by this overproduction system, we have prepared three different crystals of tryptophanase, apo-enzyme, holo-enzyme, and a complex of holo-enzyme and L-alanine, by using polyethylene glycol 4000 or potassium phosphate as a precipitant and the hanging drop method. These single crystals appeared to be suitable for X-ray diffraction analysis.
Tani
S
S
Department of Chemistry, Faculty of Science, Kyoto University, Japan.
Tsujimoto
N
N
Kawata
Y
Y
Tokushige
M
M
D000409:Alanine; D001051:Apoenzymes; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D008190:Lyases / Q000096:biosynthesis*; D010710:Phosphates; D011092:Polyethylene Glycols; D011188:Potassium; D017680:Potassium Compounds; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014169:Transformation, Bacterial; D014368:Tryptophanase / Q000096:biosynthesis* / Q000235:genetics; D014961:X-Ray Diffraction
D001051:Apoenzymes; D010710:Phosphates; D017680:Potassium Compounds; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C013216:potassium phosphate; D008190:Lyases; D014368:Tryptophanase; D000409:Alanine; D011188:Potassium