Exploration of human ORFeome: high-throughput preparation of ORF clones and efficient characterization of their protein products.
In this study, we established new systematic protocols for the preparation of cDNA clones, conventionally termed open reading frame (ORF) clones, suitable for characterization of their gene products by adopting a restriction-enzyme-assisted cloning method using the Flexi cloning system. The system has following advantages: (1) preparation of ORF clones and their transfer into other vectors can be achieved efficiently and at lower cost; (2) the system provides a seamless connection to the versatile HaloTag labeling system, in which a single fusion tag can be used for various proteomic analyses; and (3) the resultant ORF clones show higher expression levels both in vitro and in vivo. With this system, we prepared ORF clones encoding 1,929 human genes and characterized the HaloTag-fusion proteins of its subset that are expressed in vitro or in mammalian cells. Results thus obtained have demonstrated that our Flexi ORF clones are efficient for the production of HaloTag-fusion proteins that can provide a new versatile set for a variety of functional analyses of human genes.
Nagase
Takahiro
T
Department of Human Genome Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan. nagase@kazusa.or.jp
Yamakawa
Hisashi
H
Tadokoro
Shinichi
S
Nakajima
Daisuke
D
Inoue
Shinichi
S
Yamaguchi
Kei
K
Itokawa
Yasuhide
Y
Kikuno
Reiko F
RF
Koga
Hisashi
H
Ohara
Osamu
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011506:Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins