Strategies for high-throughput gene cloning and expression.
High-throughput approaches for gene cloning and expression require the development of new, nonstandard tools for use by molecular biologists and biochemists. We have developed and implemented a series of methods that enable the production of expression constructs in 96-well plate format. A screening process is described that facilitates the identification of bacterial clones expressing soluble protein. Application of the solubility screen then provides a plate map that identifies the location of wells containing clones producing soluble proteins. A series of semi-automated methods can then be applied for validation of solubility and production of freezer stocks for the protein production group. This process provides an 80% success rate for the identification of clones producing soluble protein and results in a significant decrease in the level of effort required for the labor-intensive components of validation and preparation of freezer stocks. This process is customized for large-scale structural genomics programs that rely on the production of large amounts of soluble proteins for crystallization trials.
Dieckman
L J
LJ
Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439, USA.
Hanly
W C
WC
Collart
E R
ER
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods* / Q000639:trends*; D006801:Humans