Protein Engineering Protocols - Mycobacteriology research center

Protein Library Design and Screening 1291.1. Parameters in Library Creation and ScreeningThere exist a number of parameters that must necessarily be determined inthe process of protein library design and screening: the library size that isdesired, the library representation that is required for a given application, andthe constraints that are imposed by the screening strategy. These parametersmay be addressed intuitively, although this can lead to conceptual or experimentalerrors. The mathematics underlying these parameters are discussed inSubheading 3.1. to help the experimenter better plan and execute library-basedexperimentation.1.1.1. Design of Library SizeWhether the combinatorial biologist relies on a naturally occurring library ora synthetic library, library size must be properly defined to later assess results ofscreening with a measure of accuracy. Naturally occurring libraries include naturalantibody repertoires and whole repertoires of proteins from organelles andorganisms. Synthetic libraries generally stem from a single protein or class ofhomologous proteins, although they can be more highly diversified. The techniquescurrently used in synthetic library generation include saturation mutagenesis;random mutagenesis by the polymerase chain reaction; “gene shuffling,”in which fragments of DNA from similar origins are combined; “directed” mutagenesis,in which specific regions within the encoding DNA are targeted; as wellas strategies for nonhomologous, random recombination of sequences (refer torefs. 1, 2, and 9 for nonhomologous recombination, for example). To design thedesired synthetic library size, the following considerations are essential.For a small peptide library (10 amino acids long), there are 20 10 (= 2 × 10 11 )possible amino acid combinations. The same calculation applies to a proteincontaining 10 randomized amino acids. However, because there are multiplecoding possibilities for any 10 amino acid peptide as a result of codon degeneracy,one must create a library of DNA larger than 20 10 to encode a reasonable proportionof the 20 10 different peptides, which will be represented with varyingdistributions according to the number of coding possibilities per peptide. Forexample, in a random DNA library, there are six possible coding possibilitiesfor Ser, Arg, and Leu; these are maximally redundant. Thus, the coding sequencefor a deca-Ser peptide is highly redundant, with 6 10 = 6 × 10 7 possible combinations,all encoding deca-Ser peptides, relative to the single coding combinationthat exists for the nondegenerate deca-Met peptide. The result of the codon degeneracycharacterizing certain amino acids is that DNA libraries must be largerthan calculated if ignoring degeneracy to carry a reasonable chance of encodingnondegenerate amino acids; these amino acids are encoded by only 1 outof the 64 possible triplet codons. Explicitly, in this example, one should encode