A novel strategy by the action of ricin that connects phenotype and genotype without loss of the diversity of libraries.
We present a novel strategy for connection of phenotype and genotype in vitro that can be used for the selection of functional proteins even at room temperature. The strategy involves generation of a stable complex between a ribosome, an mRNA, and its translated protein, without removal of the termination codon, as a result of the action of the ricin A chain during translation. We demonstrate the potential selection capacity of this novel strategy by isolating such complexes that contain newly synthesized streptavidin and glutathione-S-transferase (GST) using appropriate ligands. The technique requires no transfection, no chemical synthesis, no ligation, and no removal of the termination codon. Thus our novel "Ribosome-Inactivation Display System (RIDS)" should provide, without loss of the pool population, a reliable, simple, and robust selection system for in vitro evolution of the properties of proteins in a predictable direction by a combination of randomization and appropriate selection strategies.
Zhou
Jing-Min
JM
Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan.
Fujita
Satoshi
S
Warashina
Masaki
M
Baba
Tadashi
T
Taira
Kazunari
K
D020869:Gene Expression Profiling / Q000379:methods*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D019151:Peptide Library; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics*; D012276:Ricin / Q000737:chemistry / Q000378:metabolism*; D019809:Streptavidin / Q000096:biosynthesis / Q000235:genetics
D019151:Peptide Library; D011506:Proteins; D012333:RNA, Messenger; D012276:Ricin; D019809:Streptavidin; D005982:Glutathione Transferase