An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase.

Abstract

Random-sequence or highly degenerate oligonucleotides have been useful for defining functionally important sequences both in proteins and in nucleic acids. In this approach, such oligonucleotides are used to replace a segment of DNA required for a desired function, and functional sequences are identified by an appropriate genetic or biochemical selection. Here, a collection of 105 altered .-lactamase proteins was generated by cloning a mixed-base oligonucleotide in place of the sequences coding for a 17-amino acid portion of the enzyme's active site. Approximately 2000 enzymes from this collection were able to confer ampicillin resistance on Escherichia coli. Fifty-eight of these were chosen for further study after characterization with various 13-lactam substrates. (3-Lactamases having altered specificity against different antibiotics, resistance to the suicide inhibitors clavulanic acid and sulbactam, and temperature-dependent activities were obtained. The amino acid residues responsible for these altered properties as well as for basic enzyme activity are defined. This approach should prove to be an effective and general tool for creating proteins with novel properties, especially in situations in which a high-resolution structure of the protein is not known.A frequent goal of genetic engineering is to create proteins with novel functional properties. Although x-ray crystallography often provides an accurate definition of protein structure, the relationship of a protein's structure to its function is still poorly understood. Mutagenesis of a gene allows one to generate subtle variants of an enzyme or to design proteins with new properties. However, with an incomplete understanding of structure-function relationships, it is often difficult to choose variants that will be informative or have a desired property. In contrast, the immune system creates binding antibodies, without foreknowledge of the antigens, by generating many potentially functional structures and selecting and amplifying those that demonstrate the desired function. In this way, antibodies have been generated that recognize reaction intermediates and can function as enzymes (1, 2).The structural diversity required to generate a particular function can also be created in vitro by replacing wild-type DNA sequences with a random-sequence or highly degenerate oligonucleotide (3)(4)(5)(6)(7)(8)(9). Many functional structures can be obtained from such a collection by using genetic or biochemical selections. A comparison of these structures allows one to define a consensus sequence analogous to those obtained more typically for DNA elements. This "random selection" approach has distinct advantages over comparing evolutionarily related enzymes because many more variants can be studied and their function and structure is relatively independent of the evolutionary history of the organism.The basic principle of random selection should also permit the isolation of proteins with novel properties that would otherwise be difficult to obtain by conventi...

Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM beta-lactamases

“…Many other TEM mutations have been constructed that are able to increase resistance to extended-spectrum cephalosporins (21)(22)(23). It remains a puzzling fact that these mutations have not yet been recovered either in clinical isolates or in stepwise in vitro selection tests.…”

Viewing Protein Fitness Landscapes Through a Next-Gen Lens

“…Directed evolution (Oliphant and Struhl 1989) can identify highly functional variants from a library of gene variants and it has provided important insights into the landscape of available adaptive mutations ( Figure 1C). The contributions of directed evolution toward understanding adaptive evolution have recently been reviewed (Bloom and Arnold 2009) and include the observation that mutations that increase folding stability can play an important role in adaptation by increasing the tolerance to secondary adaptive mutations that are destabilizing.…”

Inhibitor resistant class A beta-lactamases

“…Using degenerate oligonucleotides in an attempt to understand the effect of multiple substitutions on protein activity, Oliphant and Struhl discovered that certain key substitutions in the vicinity of the active site of E coli RTEM conferred resistance to beta -lactam beta-lactamase inhibitor combinations (21). Using chemical mutagenesis Mavanathu, Lerner and Mobashery also discovered the TEM enzyme with single amino acid substitutions at the 244 position (Arg 244Ser and Arg 244Cys) could render E. coli resistant to beta−lactam beta−lactamase inhibitors combinations (22).…”