AUTHOR=Yuan Hui , Peng Li , Han Zhong , Xie Juan-Juan , Liu Xi-Peng 

TITLE=Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics

JOURNAL=Frontiers in Microbiology

VOLUME=6

YEAR=2015

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2015.00943

DOI=10.3389/fmicb.2015.00943

ISSN=1664-302X

ABSTRACT=<p>Hyperthermophile <italic>Pyrococcus furiosus</italic> grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of <italic>P. furiosus</italic> proteins at whole genome level, we constructed expression plasmids of each <italic>P. furiosus</italic> 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 <italic>Escherichia coli</italic> Strain Rosetta 2(DE3)pLysS. In summary, this recombinant expression library of <italic>P. furiosus</italic> 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.</p>