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.

[1]  E V Koonin,et al.  Origin of alternative splicing by tandem exon duplication. , 2001, Human molecular genetics.

[2]  W. Stemmer,et al.  Directed evolution of proteins by exon shuffling , 2001, Nature Biotechnology.

[3]  Z. Gu,et al.  Evolutionary analyses of the human genome , 2001, Nature.

[4]  G. Dover,et al.  How genomic and developmental dynamics affect evolutionary processes. , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[5]  G. Winter,et al.  Novel folded protein domains generated by combinatorial shuffling of polypeptide segments. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K Nishigaki,et al.  Whole genome sequence-enabled prediction of sequences performed for random PCR products of Escherichia coli. , 2000, Nucleic acids research.

[7]  Anthony D. Keefe,et al.  Constructing high complexity synthetic libraries of long ORFs using in vitro selection. , 2000, Journal of molecular biology.

[8]  S. Harayama,et al.  An effective family shuffling method using single-stranded DNA. , 2000, Gene.

[9]  C. Voigt,et al.  Rational evolutionary design: the theory of in vitro protein evolution. , 2000, Advances in protein chemistry.

[10]  Y. Husimi,et al.  A novel mutant of green fluorescent protein with enhanced sensitivity for microanalysis at 488 nm excitation. , 1999, Biochemical and biophysical research communications.

[11]  W. Gilbert,et al.  Centripetal modules and ancient introns. , 1999, Gene.

[12]  K D Wittrup,et al.  Selection of functional T cell receptor mutants from a yeast surface-display library. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  N. Fedoroff,et al.  Transposable Elements As a Molecular Evolutionary Force , 1999, Annals of the New York Academy of Sciences.

[14]  K. Yoshida,et al.  Foldability of barnase mutants obtained by permutation of modules or secondary structure units. , 1999, Journal of molecular biology.

[15]  Gerd Folkers,et al.  Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling , 1999, Nature Biotechnology.

[16]  F. Cosset,et al.  In vivo selection of protease cleavage sites from retrovirus display libraries , 1998, Nature Biotechnology.

[17]  F. Arnold,et al.  Random-priming in vitro recombination: an effective tool for directed evolution. , 1998, Nucleic acids research.

[18]  K. Ishimori,et al.  ‘Module’-substituted globins: Artificial exon shuffling among myoglobin, hemoglobin α- and β-subunits , 1997 .

[19]  Y Husimi,et al.  In vitro virus: Bonding of mRNA bearing puromycin at the 3′‐terminal end to the C‐terminal end of its encoded protein on the ribosome in vitro , 1997, FEBS letters.

[20]  A. Plückthun,et al.  In vitro selection and evolution of functional proteins by using ribosome display. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[21]  G. F. Joyce,et al.  Continuous in vitro evolution of catalytic function. , 1997, Science.

[22]  T. Noda,et al.  Creation of libraries with long ORFs by polymerization of a microgene. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[23]  A. Lüchow,et al.  Recovery of nucleic acids from immobilized biotin-streptavidin complexes using ammonium hydroxide and applications in MALDI-TOF mass spectrometry. , 1997, Analytical chemistry.

[24]  S. Brenner,et al.  Surface Display of Proteins on Bacteriophage λ Heads , 1996 .

[25]  T. M. Horiagon,et al.  Deletion mapping of the Aequorea victoria green fluorescent protein. , 1996, Gene.

[26]  Jack W. Szostak,et al.  In vitro evolution of a self-alkylatlng ribozyme , 1995, Nature.

[27]  Y. Kinoshita,et al.  Restriction-Enzyme-Nondependent Recombination and Rearrangement of DNA (RRR) , 1995 .

[28]  M. Illangasekare,et al.  Aminoacyl-RNA synthesis catalyzed by an RNA , 1995, Science.

[29]  G. F. Joyce,et al.  Cleavage of an amide bond by a ribozyme , 1995, Science.

[30]  R. Doolittle.  The multiplicity of domains in proteins. , 1995, Annual review of biochemistry.

[31]  W. Stemmer.  DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[32]  W. Dower,et al.  An in vitro polysome display system for identifying ligands from very large peptide libraries. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[33]  G. Steger,et al.  Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction. , 1994, Nucleic acids research.

[34]  J. Szostak,et al.  In vitro selection of RNA aptamers specific for cyanocobalamin. , 1994, Biochemistry.

[35]  V. Stanton,et al.  Use of denaturing gradient gel electrophoresis to study conformational transitions in nucleic acids. , 1992, Methods in enzymology.

[36]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[37]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[38]  J. Scott,et al.  Searching for peptide ligands with an epitope library. , 1990, Science.

[39]  Gerald F. Joyce,et al.  Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA , 1990, Nature.

[40]  D. Tessier,et al.  Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase. , 1986, Analytical biochemistry.

[41]  W. Gilbert.  Origin of life: The RNA world , 1986, Nature.

[42]  D Botstein,et al.  Strategies and applications of in vitro mutagenesis. , 1985, Science.

[43]  Manfred Eigen,et al.  Evolutionary molecular engineering based on RNA replication , 1984 .

[44]  K. Kaneko,et al.  Strand dissociation and cooperative melting of double-stranded DNAs detected by denaturant gradient gel electrophoresis. , 1984, Journal of biochemistry.

[45]  T Tanaka,et al.  Cellstat-a continuous culture system of a bacteriophage for the study of the mutation rate and the selection process of the DNA level. , 1982, The Review of scientific instruments.

[46]  M. Go.  Correlation of DNA exonic regions with protein structural units in haemoglobin , 1981, Nature.