Protein Engineering Protocols - Mycobacteriology research center

Protein Library Design and Screening 131Table 1Probability that no stopcodon is encodedIn a protein In a proteinBiased codon, Amino containing 10 containing 30encodes Codons acids Stops biased codons biased codonsNNN 64 20 3 0.62 a,b 0.24NNC or NNT 16 15 0 1 1NNC/G or NNT/G 32 20 1 0.73 0.39a The value 0.62 signifies that a peptide or protein library containing 10 fully randomizedcodons (NNN) is expected to contain 62% of variants that are full length, with no stop codon.b Calculated as (61/64) 10 = 0.619, where 61 out of the 64 codons do not encode a stop codon,for 10 codons.the experimenter to select the mixed codon best suiting their needs. Certainoligonucleotide suppliers also offer the possibility of including different proportionsof each nucleotide of interest, allowing the experimenter to bias thenucleotide distribution to favor certain amino acids relative to others. This strategycan be used to reduce or enhance differences in codon representation, asrequired.In the ideal case, a biased library can be constructed from trinucleotide codons(11), in which three specific nucleotides are covalently linked into “buildingblocks,” each encoding a single amino acid, and which can be included inoligonucleotide synthesis at specified positions and mixed in the desired proportions.This reduces the DNA library size to the protein library size that is preciselyof interest, because it is nondegenerate. The advantages of trinucleotide use havebeen highlighted (see refs. 12–14,for example) but relatively little work has beenperformed with them because they have only recently become commerciallyavailable (Glen Research Sterling, VA; and Metkinen Oy, Kuusisto, Finland).1.1.3. Limits to Library SizeThe maximal physical library size that can be attained, in practice, is onthe milligram scale, which corresponds to approximately 3 × 10 16 moleculesof double-stranded DNA for a library of 30 basepairs (bp; 10 amino acids).However, protein expression is generally undertaken via vector-based DNA.A typical expression vector may be 3000-bp long. Therefore, on the milligramscale and under ideal conditions, one can maximally produce 3 × 10 14 moleculesof double-stranded DNA for a vector of 3000 bp. Thus, the practical maximalrepresentation of a library with 10 fully randomized positions (1.2 × 10 18possibilities) is but a fraction of all possibilities (more detail is provided in