title: Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics.
abstract: Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of P. furiosus proteins at whole genome level, we constructed expression plasmids of each P. furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3)pLysS. In summary, this recombinant expression library of P. furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University Shanghai, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University Shanghai, China.
authors.forename: Li
authors.initials: L
authors.lastname: Peng
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University Shanghai, China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Han
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University Shanghai, China.
authors.forename: Juan-Juan
authors.initials: JJ
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University Shanghai, China.
authors.forename: Xi-Peng
authors.initials: XP
authors.lastname: Liu
keywords: Pyrococcus furiosus; high-throughput cloning; ligation-independent cloning; phosphorothioate modification; recombinant expression library