Overexpression of Crithidia fasciculata trypanothione reductase and crystallization using a novel geometry.
TR1, a previously cloned gene for Crithidia fasciculata trypanothione reductase (TR), has been overexpressed in Escherichia coli strain SG5 to produce about 20 mg enzyme 1(-l) of culture. Since natural C. fasciculata TR is heterogeneous, this expression system provides an important source of homogeneous C. fasciculata TR for use in structural studies and drug design. Steady-state kinetic constants of the purified recombinant enzyme are K(m) = 56 micro M and k(cat) = 10 500 min(-1). Four crystal forms of TR1 were grown using this preparation. Synchrotron radiation was crucial to discover the high level of order present in crystal form IV, which diffracts to about 1.4 A resolution. To optimize growth and handling of form IV crystals, a novel crystallization setup called the 'plug drop' was developed.
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
C L
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Strickland
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Puchalski
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Savvides
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Karplus