Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis.
The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 A resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 A, alpha = gamma = 90.0, beta = 119.1 degrees. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.
Meining
Winfried
W
Karolinska Institutet, Department of Biosciences, Center of Structural Biochemistry, Sweden. wim@csb.ki.se
Scheuring
Johannes
J
Fischer
Markus
M
Weinkauf
Sevil
S
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D003460:Crystallization / Q000379:methods; D013293:Enterococcus faecalis / Q000201:enzymology*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D012997:Solvents; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D012997:Solvents; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins