Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions. Most of the expressed proteins showed yields greater than 500ng after a single affinity purification step from 50μl in vitro protein synthesis reactions. The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.
- High-throughput protein expression
- In vitro protein synthesis
- Pseudomonas aeruginosa
- Two-component system
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