Protein Engineering Protocols - Mycobacteriology research center

130 Denault and Pelletierall 64 10 (= 1.2 × 10 18 ) possible DNA sequences to allow for a reasonable probabilityof encoding the deca-Met peptide. Unfortunately, these 1.2 × 10 18decapeptides would be encoded by approx 2 mg of DNA, a sample too large tobe reasonably produced in most research contexts. It should be noted that onecan consider a longer peptide or protein in which any 10 amino acids (contiguousor not) will be varied, using the same reasoning as the described decapeptide.Thus, codon degeneracy should be considered when planning a peptide orprotein library.1.1.2. Biased LibrariesIt is often of interest to bias the coding DNA library. The most obvious advantagesare to reduce the impact of stop codons, which render nonfunctional a largefraction of unbiased libraries (see Table 1), to skew libraries toward desiredamino acid compositions, and to reduce the difference in codon representationbetween various amino acids, thus allowing a more-uniform representation ofthe amino acids in the library. Libraries with “NNC” or “NNT” repeats (whereN represents any of the 4 nucleotides) serve to reduce the number of codonsfrom 64 to 16 while eliminating the 3 stop codons (see Table 1). However, fiveamino acids (Met, Trp, Gln, Glu, and Lys) are not encoded, resulting in a loss ofencoded diversity. A drawback in using either of these degenerate codons is thatcodon usage in Escherichia coli (the most frequently used host) is poor for certaincodons (10). The “NNC/T” codon is also frequently used; it counters theproblem of poor codon usage but doubles the number of codons at each position,with no increase in the number of different amino acids encoded. The advantageof having no stop codons to contend with may well be worth the loss in aminoacid diversity, because, in a randomly encoded library (“NNN”), the probabilityof obtaining products with no stop codons drops rapidly with increasing numbersof degenerate positions (see Table 1) and can result in a low-quality library.An alternative is to encode “NNC/G” or “NNT/G,” where all 20 amino acids areencoded, including a single stop codon; “NNC/G” offers better codon usageoverall in E. coli. Here, the probability of obtaining products with no stops is significantlygreater than when encoding “NNN.” Thus, when multiple degeneratepositions will be created, inclusion of stop codons can be very deleterious tolibrary quality and should be avoided if the screening strategy to be used is laborintensiveor costly.The experimenter frequently requires a specific array of amino acids at a particularposition. Planning such biased oligonucleotides can be accomplished byhand, although software is available to facilitate this task. For example, the“Mixed Codon Worksheet” by T. J. Magliery (www.chemistry.ohio-state.edu/~magliery/publications.html) is an Excel file in which one lists the desired aminoacids to be encoded at a position; various possibilities are computed, allowing