title: Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides.
abstract: Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of alpha-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3'-->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, 414 Wartik Laboratory, University Park, PA 16802, USA.
authors.forename: S
authors.initials: S
authors.lastname: Lutz
authors.forename: M
authors.initials: M
authors.lastname: Ostermeier
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009946:Organothiophosphorus Compounds / Q000378:metabolism*; D019151:Peptide Library; D051026:Phosphoribosylglycinamide Formyltransferase; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics*
chemical_list: D009946:Organothiophosphorus Compounds; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase