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Cloning, purification, crystallization, and preliminary X‐ray diffraction analysis of cystathionine γ‐synthase from E. coli
Author(s) -
Wahl Markus C,
Huber Robert,
Prade Lars,
Marinkovic Snezan,
Messerschmidt Albrecht,
Clausen Tim
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)01057-0
Subject(s) - escherichia coli , plasmid , crystallography , chemistry , crystallization , restriction enzyme , resolution (logic) , microbiology and biotechnology , gene , biology , biochemistry , artificial intelligence , computer science , organic chemistry
The Escherichia coli metB gene has been PCR‐extracted from genomic DNA and placed under the control of a tac and a T7 promoter in plasmids pCYB1 and pET22b(+), respectively, to produce overexpressing bacterial strains for the gene product, cystathionine γ‐synthase. Efficient purification procedures have been developed for a C‐terminally intein‐tagged version and the wild‐type target protein, yielding the product in a quantity and homogeneity amenable to high‐resolution single‐crystal X‐ray analysis. Crystals have been obtained in space group P1 with unit cell constants a=82.2 Å, b=84.2 Å, c=116.2 Å, α=107.0°, β=96.3°, γ=108.0°, suggesting eight monomers per asymmetric unit (V M =2.23 Å 3 /Da). Crystals diffract to beyond 2.6 Å resolution and a data set complete to 2.8 Å resolution has been collected using a rotating anode X‐ray source. A cryogenic buffer system has been developed to allow synchrotron data collection. Patterson self rotation searches reveal the presence of two independent tetramers with local 222 symmetry in an asymmetric unit. The crystallographic results corroborate and extend previous solution studies regarding the quaternary organization of the enzyme.