Protein Engineering Protocols - Mycobacteriology research center

58 Mason et al.Fig. 3. Listed are amino acid frequencies corresponding to the codon randomizationscheme shown in Fig. 2. Additionally, the distribution for NNK is given. This combinationis widely used when all 20 amino acids should be included and stop codonsshould be minimized. “Total” gives the number of amino acids to be used when calculatingthe library size. Hydrophobic amino acid combinations encoded by NTN (blackline in Fig. 2) or VTY (gray long dashes), respectively. To minimize overrepresentationof Ile and Leu, NTN can be reduced to NTK or DTK. Different polar and chargedcombinations are encoded by VAN (gray line), VAR (black long dashes), GAN (blackdots), or MRG (gray short dashes), respectively. Possible loop regions, if desired, canbe obtained by the codon RSY (black short dashes). Please note that, in our examples,the third base is also mixed to increase variety in codon usage, which might be importantfor repetitive sequences. However, if desired, the last base can be kept constant inmost instances. This is especially important in the case of the Gln, Arg, and Lys mixture(short gray dashes), in which the codon MRG yields only one rare Arg codon(AGG), whereas the combination MRR yields three rare Arg codons (CGG, AGA,AGG). Combinations NTN and VAN have been applied previously in the binary designpattern developed in the Hecht group (see refs. 84 and 85 and Chapter 9).2.5.3. Selection SystemsThe most common selection system open to the coiled coil is the proteinfragmentcomplementation assay selection. In this assay, interacting proteins,e.g., two coiled coil fragments, are tethered to two halves of a reporter protein thatonly becomes active after association of the two fused proteins or peptides.This has been used for dihydrofolate reductase (8,31,70,86–88), ubiquitin (89),