Genome-scale, biochemical annotation method based on the wheat germ cell-free protein synthesis system.
Since the complete genomic DNA sequencing of various species, attention has turned to the structural properties, and functional characteristics of proteins. Current cell-free protein expression systems from eukaryotes are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. This report reviews the high-throughput, genome-scale biochemical annotation method based on the cell-free system prepared from wheat embryos. We first briefly reviewed our highly efficient and robust wheat germ cell-free protein synthesis system, and then showed an application of the system for materialization and characterization of genetic information taking a cDNA library of protein kinase from Arabidopsis thaliana as an example. The procedure consists of: (1) fusion of the gene-of-interest to a purification-tag, amplified by the split-primer PCR method; (2) transcription and purification of mRNA; (3) cell-free protein synthesis in the bilayer system using 96-well titer plate; (4) affinity purification and activity measurement. We took 439 cDNAs encoding kinases among 1064 genes annotated so far, and they were translated in parallel into protein. Subsequent assay revealed 207 products having autophosphorylation activity. Furthermore, seven proteins out of 26 calcium-dependent protein kinase genes tested did phosphorylate a synthetic peptide substrate in the presence of calcium ion, demonstrating that the translation products, retained their substrate specificity. The information on biochemical function of gene products accumulated should revolutionize our understanding of biology and fundamentally alter the practice of medicine and influence other industries as well.
Cell-Free Science and Technology Research Center, The Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
Tatsuya
T
Sawasaki
Yoshinori
Y
Hasegawa
Ryo
R
Morishita
Motoaki
M
Seki
Kazuo
K
Shinozaki
Yaeta
Y
Endo
D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000235:genetics; D002474:Cell-Free System; D015870:Gene Expression; D015723:Gene Library; D016678:Genome; D005854:Germ Cells; D010802:Phylogeny; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013379:Substrate Specificity; D014908:Triticum / Q000235:genetics*
D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; C078571:APK1A protein, Arabidopsis; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases