Random mutagenesis is a powerful tool for generating enzymes, proteins, entire metabolic pathways, or even entire genomes with desired or improved properties. This technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation. Coupled with the development of powerful high-throughput screening or selection methods, this technique has been successfully used to solve problems in protein engineering. There are many methods to generate genetic diversity by random mutagenesis and to create combinatorial libraries. This can be achieved by treating DNA or whole bacteria with various chemical mutagens, by passing cloned genes through mutator strains, by “error-prone” PCR mutagenesis, by rolling circle error-prone PCR, or by saturation mutagenesis. The next sections of this review article focus on recent advances in techniques and methods used for in vitro directed evolution of enzymes using random mutagenesis. Selected examples, highlighting successful applications of these methods, are also presented and discussed.

A series of small-molecule microbicides has been developed for vaginal delivery to prevent heterosexual HIV transmission, but results from human clinical trials have been disappointing. Protein-based microbicides, such as HIV-specific monoclonal antibodies, have been considered as an alternative approach. Despite their promising safety profile and efficacy, the major drawback of such molecules is the economy of large-scale production in mammalian cells, the current system of choice. Here, we show that an alternative biomanufacturing platform is now available for one of the most promising anti-HIV antibodies (2G12). Our data show that the HIV-neutralization capability of the antibody is equal to or superior to that of the same antibody produced in CHO cells. We conclude that this protein production system may provide a means to achieve microbicide ingredient manufacture at costs that would allow product introduction and manufacture in the developing world.