Construction and Functional Selection of a T4 Lysozyme Gene Library Randomly Mutagenized at Five Specific Sites

Abstract

This chapter discusses construction and functional selection of a T4 lysozyme gene library randomly mutagenized at five specific sites. Random mutagenesis followed by selection or screening for a specific phenotype have been long used to identify functionally important genetic elements and interactions, from the cellular to the amino acid level. The advantage of such an approach is that little prior understanding of the system under investigation is required, other than the means and limitations of the particular mutagenesis and selection employed. A major advantage of using T4 lysozyme as a model for mutational analysis of protein function and stability is the ease of high-resolution crystallographic analysis, and the large amount of existing thermodynamic and structural data. This chapter describes a method using the polymerase chain reaction for generation of mutant genes that are randomized at sites far apart in the DNA sequence but that are proximal in the three-dimensional structure. A library of mutants at five sites was constructed to study interactions in the hydrophobic core of T4 lysozyme. The library was cloned into a phage λ vector, which permitted selection, activity screening, and sequencing of functional genes and subsequent production of the encoded proteins.