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High-throughput screening of soluble recombinant proteins

Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, Republic of China

Reprint requests to: Ting-Fang Wang, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, Republic of China; e-mail: tfwang{at}gate.sinica.edu.tw; fax: 886-2-27889759.

The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure–function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well-expressed soluble fusion protein, total cell lysates of bacteria culture (1.5 mL) were subjected to high-speed centrifugation in a 96-tube format and analyzed by multiwell denaturing SDS-PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis.

Keywords: Structural genomics; functional genomics; proteomics; protein expression

Abbreviations: HTP, high throughput • IPTG, isopropyl ß-D-thiogalactoside • LB, Luria-Bertani • RC, recombinational cloning • PCR, polymerase chain reaction • Trx, thioredoxin • MBP, maltose-binding protein • GST, glutathione S-transferase • CBP, calmodulin-binding protein • CAP, cellulose-associated protein • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel

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