Genome-scale expression of proteins from Bacillus subtilis.
We have applied high throughput methods for cloning and expression of more than 850 genes from the Bacillus subtilis genome. The process uses 96-well plates and is automated from the level of primer design to the detection of soluble protein by a tag detection screen. This process was applied to a set of cytoplasmic targets from Bacillus subtilis to produce clones expressing soluble protein for incorporation into the structure determination pipeline of the Midwest Center for Structural Genomics. We also evaluated the feasibility of these plate-based methods for domain-based cloning and expression of secretory proteins and putative soluble domains of membrane proteins. This approach shows promise for implementation in a high throughput format and could provide additional target resources for structure determination. The continued development of new technologies that can be implemented in an automated format will be essential for continued success in the structural genomic programs.
Moy
S
S
Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Dieckman
L
L
Schiffer
M
M
Maltsev
N
N
Yu
G-X
GX
Collart
F R
FR
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D015870:Gene Expression; D016680:Genome, Bacterial; D023281:Genomics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015394:Molecular Structure; D016133:Polymerase Chain Reaction; D040901:Proteomics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins