Construction of block-shuffled libraries of DNA for evolutionary protein engineering: Y-ligation-based block shuffling.
Evolutionary protein engineering is now proceeding to a new stage in which novel technologies, besides the conventional point mutations, to generate a library of proteins, are required. In this context, a novel method for shuffling and rearranging DNA blocks (leading to protein libraries) is reported. A cycle of processes for producing combinatorial diversity was devised and designated Y-ligation-based block shuffling (YLBS). Methodological refinement was made by applying it to the shuffling of module-sized and amino acid-sized blocks. Running three cycles of YLBS with module-sized GFP blocks resulted in a high diversity of an eight-block shuffled library. Partial shuffling of the central four blocks of GFP was performed to obtain in-effect shuffled protein, resulting in an intact arrangement. Shuffling of amino acid monomer-sized blocks by YLBS was also performed and a diversity of more than 10(10) shuffled molecules was attained. The deletion problems encountered during these experiments were shown to be solved by additional measures which tame type IIS restriction enzymes. The frequency of appearance of each block was skewed but was within a permissible range. Therefore, YLBS is the first general method for generating a huge diversity of shuffled proteins, recombining domains, exons and modules with ease.
Kitamura
Koichiro
K
Department of Functional Materials Science, Saitama University, 255 Shimo-Okubo, Saitama 338-8570, Japan.
Kinoshita
Yasunori
Y
Narasaki
Shinsuke
S
Nemoto
Naoto
N
Husimi
Yuzuru
Y
Nishigaki
Koichi
K
D020650:Combinatorial Chemistry Techniques / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D020860:Subtilisin / Q000235:genetics
D017931:DNA Primers; D008164:Luminescent Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; D020860:Subtilisin; D011130:RNA Ligase (ATP)