Combinatorial Chemistry & High Throughput Screening - Volume 9, Issue 4, 2006

The central aim of protein engineering is the efficient creation of novel and practical biocatalysts and to understand structure/function relationships. The ultimate goal would be to create proteins designed to order on the lab bench. An efficient protein engineering strategy is necessary to design enzymes with improved properties. The basic strategies for protein engineering include rational design and directed Read More

Cofactor regeneration is an important solution to the problem of implementing complex cofactor requiring enzymatic reactions at the industrial scale. NAD(P)H-dependent oxidoreductases are highly valuable biocatalysts, but the high cost of the nicotinamide cofactors necessitates in situ cofactor regeneration for preparative applications. Here we report the use of directed evolution to enhance the industrially important pr Read More

Naturally occurring enzymes are remarkable biocatalysts with numerous potential applications in industry and medicine. However, many of their catalyst properties often need to be further tailored to meet the specific requirements of a given application. Within this context, directed evolution has emerged over the past decade as a powerful tool for engineering enzymes with new or improved functions. This review summarizes Read More

We have been investigating the creation of novel proteins by means of block shuffling, where the term block refers to an amino acid sequence that corresponds to particular features of proteins, such as secondary structures, modules, functional motifs, and so on. Block shuffling makes it possible to explore the global sequence space, which is not feasible with conventional methods, such as DNA shuffling or family shuffling Read More

Over the past decade, we have witnessed a bloom in the field of evolutive protein engineering which is fueled by advances in molecular biology techniques and high-throughput screening technology. Directed protein evolution is a powerful algorithm using iterative cycles of random mutagenesis and screening for tailoring protein properties to our needs in industrial applications and for elucidating proteins' structure f Read More

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-tri Read More

Crucial to the success of directed evolution of enantioselective enzymes for use as catalysts in synthetic organic chemistry is the availability of high-throughput assays for determining the enantiopurity of thousands of samples. Although several such ee-assays are available, they entail time and effort, which means that pre-tests for activity have been developed to eliminate non-active mutants prior to ee-screening. Pre-selection Read More

We have developed two bacterial one-hybrid systems for interrogating and selecting zinc finger-DNA interactions. Our systems utilize two plasmids: a zinc finger-plasmid containing the gene for the zinc finger fused to a fragment of the alpha subunit of RNA polymerase and a reporter plasmid where the zinc finger-binding site is located upstream of a reporter gene-either the gene encoding the green fluorescent protein (GFP) o Read More

Our long-term goal is to direct the evolution of novel protease variants. To this end we have engineered a new type of protease-activated reporter enzyme. Many protease-activated enzymes evolved in nature, but the introduction of novel regulatory mechanisms into normally unregulated enzymes poses a difficult design challenge. Random Elongation Mutagenesis [1] was used to fuse the p6 peptide, which is recognized an Read More

Directed evolution is a reliable method for protein engineering and as a tool for investigating structure/function relationships. A key for a successful directed evolution experiment is oftentimes the screen. Fluorescence activated cell sorting (FACS) is powerful high-throughput screening approach to isolate and identify mutants from large protein libraries. FACS has been successful in isolating proteins with improved or altered bindi Read More

Dr. Edgardo T. Farinas did his undergraduate work at Loyola University of Chicago (B.S. in 1990) and pursued graduate studies in bioinorganic chemistry at the University of California at Santa Cruz (Ph.D. in 1997) under Pradip Mascharak. After postdoctoral studies with Lynne Regan at Yale University, Frances Arnold at the California Institute of Technology, and Brent Iverson and George Georgiou at University of Texas at Aust Read More