A procedure for high-throughput protein expression analysis has been finalized. Briefly, Dr. Marc Vidal at the Massachusetts General Hospital supplied 96-well plates containing purified plasmid DNA of 88 unique C. elegans genes in the form of entry vectors. These genes were directly sub-cloned into the expression vector pDEST-17.1 by recombinational techniques (Gateway system, Life Technologies) and transformed into the expression bacteria strain BL21-SI. Both the sub-cloning and transformation procedures have been optimized in our lab for 96-well plates. The pDEST-17.1 expression vector is a variation of the initial pDEST-17 expression plasmid that now contains a 3-phase stop codon after the 3’ recombination site. The vector also contains the N-terminal Histidine tag.

The bacteria are grown in 1 ml, 96-well block plates and protein expression is carried out in 5 ml media, 10 ml 24-well block plates. Initially, the bacteria are lysed with 8 M urea, denaturing all proteins. An ELISA is used to identify expression of recombinant proteins using an anti-penta Histidine antibody (Qiagen). Histidine tag was provided by the expression vector pDEST-17.1. Visualization is performed with an alkaline phosphatase conjugated secondary antibody (Pierce), and color change of p-nitrophenyl phosphate (ICN). When clones are identified which produce recombinantly expressed proteins, these genes are used in a second round of small-scale expression analysis. Here, two temperatures (18°C and 37°C) are screened for soluble protein expression.

The bacteria are lysed by the freeze-thaw method using lysozyme, resulting in soluble proteins. A second ELISA is performed. The ELISA results compared with the original ELISA determines which recombinant proteins are expressed as inclusion bodies or as soluble protein at various temperatures. All experiments were performed in either 96-well or 24-well format, which are readily amendable to our current Beckman Saigen system with the BioMak robotic arm.

All membrane proteins from C. elegans will be included in the expression analysis. This is not due to any lack of computational analysis. The prediction of membrane insert domains will be done automatically for all genes. In fact, we purposely use the inclusion body expression in E.coli as one of the new approaches to producing membrane proteins and subsequent refolding. This is one of our innovative high throughput approaches to solve the current scarcity of membrane protein structures. In collaboration with Dr. Lin of OMRF, we will develop a high throughput refolding protocol.

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