GFPs of insertion mutation generated by molecular size-altering block shuffling.
Insertion and deletion analyses of a protein have been less common than point mutation analyses, partly due to the lack in effective methods. This is the case with the green fluorescent protein (GFP), which is so widely applied in molecular biology and other fields. In this paper we first introduce a systematic approach for generating insertion/deletion mutants of GFP. A new technology of Y-ligation-based block shuffling (YLBS) was successfully applied to produce size-altered GFPs, providing insertion-containing GFPs of fluorescence, though no deletion type of fluorescence was obtained so far as examined. The analysis of these proteins suggested that size alteration (deletion/insertion) is acceptable so far as some type of rearrangement in a local structure can accommodate it. This paper demonstrates that YLBS can generate insertion and deletion mutant libraries systematically, which are beneficial in the study of structure-function relationship.
Kitamura
Koichiro
K
Department of Functional Materials Science, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
Yoshida
Chuya
C
Nishigaki
Koichi
K
D001483:Base Sequence; D003001:Cloning, Molecular; D003198:Computer Simulation; D017931:DNA Primers / Q000235:genetics; D005453:Fluorescence; D015723:Gene Library; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008970:Molecular Weight; D016254:Mutagenesis, Insertional / Q000379:methods*; D017433:Protein Structure, Secondary; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics
D017931:DNA Primers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D011130:RNA Ligase (ATP)