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Detailed biophysical, biochemical, and structural studies rely on the preparation of milligram amounts of pure recombinant proteins. Many useful overexpression systems have been developed for this purpose (1–3), and of these, bacterial overexpression is still the most convenie ...

Methods for the efficient overexpression and purification of recombinant proteins are of paramount importance for biotechnology. In particular, for the era of functional genomics that we have entered after sequencing complete genomes, this has become a routine matter. High-thro ...

Affinity chromatography, the method of purifying target proteins from complex mixtures using immobilized affinity ligands on chromatographic supports, is perhaps the most common of all affinity techniques (1–3). Many affinity chromatography systems are comprised of activa ...

The use of λ repressor fusions to study protein-protein interactions in E. coli was first described by Hu and others (1). Since then, the repressor system has been employed by several laboratories to screen genomic (2–5) and cDNA libraries (6) for homotypic or heterotypic interactions. λ repress ...

Though widely used, expression of heterologous genes in E. coli can be cumbersome and often fails to yield significant amounts of the desired protein. There are a variety of reasons why a particular heterologous gene fails to yield significant amount of protein, including susceptibility of t ...

Two-hybrid systems are powerful genetic assays that allow the interaction between two proteins to be detected in vivo. Although originally described in yeast (1,2), several bacterial two-hybrid systems have recently been developed (reviewed in 3–6). This chapter will describe the use of ...

The completion of the human genome project has ushered in a new era of life science (1,2) in which the new challenge is to understand functions of the entire collection of the gene products, or the proteome. One important feature of biological research in this post-genomics era is the emphasis on unders ...

Advances and opportunities in drug discovery and functional genomics have put methods for generating molecular diversity at a premium. Both chemical and biological approaches for the production of compound libraries have been pursued. Combinatorial chemistry has been used to syn ...

Rapid transfer of exponentially growing E. coli cultures from physiological to low temperatures (10–15#X00B0;C) has profound consequences on cell physiology: membrane fluidity decreases, which interferes with transport and secretion, the secondary structures of nucleic ac ...

Since its invention in the early 1990s, phage display has revolutionized the generation and engineering of monoclonal antibodies (for review, see ref. 1). Without the need for laboratory animals or hybridomas, it was now possible to create antibodies binding to almost any antigen of choice. A ...

Among secondary metabolites, many interesting compounds including flavors, fragrances, and those with pharmaceutical potential can be found. Once the biosynthetic pathway of an interesting compound or group of compounds has been elucidated and the genes encoding the enzymes of the ...

The advent of combinatorial approaches to problems at the interface between chemistry and biology has had a profound impact on areas ranging from drug discovery to protein chemistry. One area of intense work has been the application of combinatorial libraries of proteins to the discovery of ...

In the near future, the nucleotide sequence of the genomes from many different organisms will be available. The next and more challenging step will be to characterize the biological role of each gene and the way in which the encoded protein functions in the cell. Dual-expression vectors for expres ...

Protein fusions are invaluable tools for the genetic studies involving the mechanisms of protein export in bacteria. In 1985, Hoffman and Wright developed an in vitro fusion approach that allowed for fusions of the gene encoding Escherichia coli alkaline phosphatase to a variety of cloned g ...

The IMPACT (Intein-Mediated Purification with an Affinity Chitin-Binding Tag) vectors are designed for the isolation of pure, functional, recombinant proteins by a single affinity chromatography step. The IMPACT technology was developed at New England Biolabs (NEB) by exploiti ...

Calmodulin is a small (148 amino acids, 17 kDa) ubiquitous protein in eukaryotes, and is considered the primary intracellular calcium sensor, making it a key regulator of intracellular signal transduction. Upon calcium binding, calmodulin can interact with a variety of proteins (1), medi ...

A major impediment to the production of recombinant proteins in Escherichia coli is their tendency to accumulate in the form of insoluble and biologically inactive aggregates known as inclusion bodies. Although it is sometimes possible to convert aggregated material into native, bio ...

Protein purification is an important tool for investigations on protein function, structure analysis, and biotechnological use. Therefore a number of different techniques have been developed for fast, reliable, and reproducible overexpression and purification of relevant ...

Escherichia coli has traditionally been a popular host for the production of heterologous proteins because of its ease of genetic manipulation and growth. Recombinant proteins produced in E. coli have been useful for a variety of purposes, including the study of protein tertiary structu ...

Fusion protein technology has been creatively applied to solve many problems encountered in the study of protein structure and function (1,2). One prevalent application is the use of fusion proteins to improve protein expression in Escherichia coli and provide convenient methods for a ...