Medium-scale structural genomics: strategies for protein expression and crystallization.
While high-throughput methods of protein production and crystallization are beginning to be well documented, owing to the output of large structural genomics programs, medium-throughput methods at the laboratory scale lag behind. In this paper, we report a possible way for an academic laboratory to adapt high-throughput to medium-throughput methods, on the basis of the first results of two projects aimed at solving the 3D structures of Escherichia coli and Mycobacterium tuberculosis (Tb) proteins of unknown function. We have developed sequential and iterative procedures as well as new technical processes for these programs. Our results clearly demonstrate the value of this medium-throughput approach. For instance, in the first 14 months of the E. coli program, 69 out of 108 target genes led to soluble proteins, 36 were brought to crystallization, and 28 yielded crystals; among the latter, 13 led to usable data sets and 9 to structures. These results, still incomplete, might help in planning future directions of expression and crystallization of proteins applied to medium-throughput structural genomics programs.
Vincentelli
Renaud
R
AFMB, UMR 6098, CNRS & Universités Aix-Marseille I & II, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France.
Bignon
Christophe
C
Gruez
Arnaud
A
Canaan
Stephane
S
Sulzenbacher
Gerlind
G
Tegoni
Mariella
M
Campanacci
Valérie
V
Cambillau
Christian
C
D003460:Crystallization; D023281:Genomics / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry*; D012107:Research Design
D011994:Recombinant Proteins