Incremental truncation

A modified version of this protocol, called THIO-ITCHY, includes the random incorporation of a-phosphothioate nucleotide analogs into the parent genes [27,28]. Exonuclease III activity is inhibited at sites of analog incorporation, which relieves the efforts of producing incremental truncation aliquots. In combination with epPCR, the diversity of the fusion libraries created can be further expanded. 

Lutz, S., Ostermeier, M. and Benkovic, S.J. (2001) Rapid generation of incremental truncation librariesforprotein engineering using alpha-phosphothioate nucleotides. Nucleic Acids Research, 29, E16. 

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY THIO-ITCHY) no requirement for sequence homology operationally easier to implement than SHIPREC only one crossover Oster- meier, 1999b... 

S. J. Benkovic, Incremental truncation as a strategy in the engineering of novel biocatalysts, Bioorgan. Med. Chem. 1999a, 7, 2139-2144. 

M. Ostermeier, A. E. Nixon, J. H. Shim, and S. J. Benkovic, Combinatorial protein engineering by incremental truncation,... 

Incremental truncation, in its simplest form, allows the creation of a library of every one bp deletion of a gene or gene fragment (Fig. 10). Despite its counterintuitive nature (i.e., that by deleting amino acids... 

Fig. 10. Incremental truncation libraries (Ostermeier et al., 1999b). Plasmid DNA is digested with two restriction enzymes one that produces a 3 recessed end (A which is susceptible to Exo III digestion) and the other that produces a 5 recessed end (B which is resistant to Exo III digestion). Digestion with Exonuclease III proceeds under conditions in which the digestion rate is slow enough so that the removal of aliquots at frequent intervals results in a library of deletions of all possible lengths from one end of the fragment. The ends of the DNA can be blunted by treatment with SI nuclease and Klenow so that unimolecular ligation results in the desired incremental truncation library.

The limitations of DNA shuffling, arising from its dependency on sequence identity, have motivated the development of new technologies to facilitate an unbiased sampling of sequence space. At present, the reported approaches for creating sequence-independent hybrid libraries between two parental sequences include a family of methods collectively known as Incremental Truncation for the Creation of Hybrid enzYmes (ITCHY) [72,95,96] and Sequence-Homology Independent Protein RECom-bination (SHIPREC) [91]. 

Incremental Truncation for the Creation of Hybrid enzymes (ITCHY) was the first developed homology-independent recombination method (12). Incremental truncation of two parental... 

ITCHY incremental truncation for the creation of hybrid enzymes... 

Ostermeier M, Nixon A, Shim J, Benkovic S. Combinatorial protein engineering by incremental truncation. Proc Natl Acad Sci USA 1999 96 3562-3567. 

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