Library truncation

Andrew Braisted and J.A. Wells prepared phage containing Z domain helices 1 and 2 and restored Fc binding of this 38 residue minidomain in three iterative stages (see Figure 17.10). The truncated peptide was first randomized at four hydrophobic residues which contact helix 3 in the complete Z domain. The consensus sequence from this library maintained the wild-type residues lie 17 and Leu 23 while the hydrophobic residues Leu 20 and... 

Figure 17.10 Construction of a two helix truncated Z domain, (a) Diagram of the three-helix bundle Z domain of protein A (blue) bound to the Fc fragment of IgG (green). The third helix stabilizes the two Fc-binding helices, (b) Three phage-display libraries of the truncated Z-domaln peptide were selected for binding to the Fc. First, four residues at the former helix 3 interface ("exoface") were sorted the consensus sequence from this library was used as the template for an "intrafece" library, in which residues between helices 1 and 2 were randomized. The most active sequence from this library was used as a template for five libraries in which residues on the Fc-binding face ("interface") were randomized. Colored residues were randomized blue residues were conserved as the wild-type amino acid while yellow residues reached a nonwild-type consensus, [(b) Adapted from A.C. Braisted and J.A. Wells,...

FTA, fault free analyzer module, uses SETS and FTAP to reduce fault trees and generate minimal cutsets for storage as minimal cutset libraries. Cutset control uses truncation hy probability or order. The user chooses the codes according to the personal computer s capabilities. The FTA module uses OR, AND, N/M, switch gates and supercomponents. 

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. 

Very recendy a new, physiologic inhibitor of calcineurin has been described by Liu and colleagues at MIT and OriGene (Sun et al., 1998). This protein, called cabin-1 (calcineurin binding protein) is a 2220-residue nuclear protein, which was isolated from a murine T-cell cDNA library in a yeast two-hybrid system with a truncated, catalydcally inac-dve calcineurin used as the bait. Cabin-1 was shown to be a phospho-protein capable of binding to and inhibiting the phosphatase activity of calcineurin, and the interaction between cabin-1 and calcineurin was shown to be sensitive to disruption by FK506 or CsA. In a mammalian... 

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]. 

Fig. 9.6. Fundamental mechanisms of homology-independent fragment swapping by a) ITCHY and b) SHIPREC. In the ITCHY protocol, the two parental genes are truncated independently, creating two separate libraries that correspond to the N- and C-terminal fragments. Ligation of the mixed libraries then generates a comprehensive...

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