Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems
Section snippets
Cloning and sub cloning
The genes used for this study were obtained from our repositories of Pseudomonas aeruginosa gene collection (LaBaer et al., in revision). The clones were verified by end terminal sequencing (5′ and 3′) of the genes in pDONR201 and comparing the sequencing results with the sequence in the Pseudomonas aeruginosa database. An LR reaction was performed to sub-clone the genes into the GATEWAY acceptor vector (pDEST17). Briefly, 150 ng of master clone (gene cloned into the plasmid vector, pDONR201)
Expression and purification of 63 proteins in bacterial cell-free system
Sixty-three genes from P. aeruginosa corresponding to response regulators from the 2-component system were tested for in vitro expression. N-terminal 6HIS tag was used based on previous results from our laboratory, which showed that 96-well one-step purification under denaturing conditions provided reasonably pure recombinant protein preparations [4]. 51/63 tested proteins could be expressed and partially purified by Ni2+–NTA affinity chromatography under denaturing conditions from 50 μl of in
Discussion
In this study, we have used a bacterial cell-free system to express and purify 63 P. aeruginosa response regulator proteins using 50 μl reactions in a 96-well format. Furthermore, we have expressed and partially purified a subset of this gene set using a high-throughput cell-based bacterial expression to compare the yield and purity obtained using these two systems. In the in vitro system, 51/63 tested proteins were detected on Coomassie blue-stained gels whereas 10/63 proteins were either
Acknowledgements
We thank Dr. S. Yokoyama for kindly providing the protocols for preparation of bacterial cell-free extracts. We thank all the members of the Harvard Institute of Proteomics for their constant support. We thank Dr. Steve Lory for his help in selecting the genes of the two-component system. This work was funded by a Cystic Fibrosis Foundation grant.
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Present address: Karolinska Institute, Center for Genomics and Bioinformatics, Berzelius Vag 25, 171 77 Stockholm, Sweden.