细胞功能测定

Dehydrogenases are studied for a variety of reasons. Several dehydrogenases are of use in industrial processes to produce pharmaceuticals and fine chemicals. Scientists are trying to enhance the performance of such enzymes by limiting substrate inhibition (1) and eliminating the n ...

Lipases and esterases are some of the most extensively used enzymes for biotransformations (1–3). This is in part due to their widely applicable chemistry, often exceptional regio- and stereo-selectivity, lack of required cofactors and their functionality in organic as well as aqueous m ...

Microbial hydrolytic haloalkane dehalogenases catalyze the cleavage of halogen-carbon bonds of a variety of aliphatic halogenated compounds, including a broad range of chlorinated (C2–C6) and brominated (C2–C8) alkanes, with water as the sole co-substrate, resulting in the prod ...

In principle there are two ways for the biocatalytic synthesis of α-hydroxyketones. Oxidoreductases may be used to convert diketones or diols into the respective α-hydroxyketones. This kind of reaction is exploited, e.g., in the industrial synthesis of the α-glucosidase inhibitor mig ...

Cellulases degrade cellulose, which is the most abundant biological polymer on the earth (1). Although the chemical composition of cellulose is very simple, consisting of only glucose residues connected by β-1,4-glycosidic bonds, no single enzyme is able to degrade crystalline cellul ...

Disulfide isomerases (EC 5.3.4.1) were first discovered almost 40 years ago by Christian Anfinsen, and have since been shown to occur in numerous organisms from bacteria to man (1). These enzymes play a key role in disulfide bond formation, an essential step in the oxidative folding of secreted prot ...

The directed evolution of enantioselective enzymes (1,2) for use in organic synthesis constitutes an attractive alternative to traditional forms of asymmetric catalysis based on chiral transition metal complexes or catalytic antibodies. It involves the proper combination of ...

A variety of in vivo selection and screening methods have been developed for the directed evolution of protein function. Typically, in vivo selection strategies involve the identification of new binding or catalytic functions based on their ability to confer a selective growth advanta ...

Directed evolution is a powerful technology for the isolation of enzyme variants with enhanced properties such as organic solvent tolerance (1), thermostability (2), glutaraldehyde resistance (3), and altered substrate specificity (4,5). Typically, enzyme variants are isolat ...

Directed evolution has proven to be a powerful route to enhance the stability, catalytic efficiency, or substrate specificity of enzymes. Large repertoires of enzyme mutants are generated, followed by the isolation of those enzyme variants with the desired catalytic properties. In th ...

Recombinant protein expression in heterologous hosts such as Escherichia coli (E. coli) can provide large amounts of a protein of interest. Often, expression can result in the accumulation of the recombinant protein as inactive, insoluble inclusion bodies (1). When attempts at refoldi ...

Directed evolution by sequential cycles of random mutagenesis and screening has proven to be useful for producing new or improved enzyme properties (1,2). The first step is construction of a mutant library, usually accomplished by random point mutagenesis with error-prone PCR (3) or by DNA sh ...

Functional gene expression is a prerequisite for directed evolution with Escherichia coli (E. coli), the preferred host organism. However, bacterial expression of eukaryotic genes can be impossible, or produce proteins with substantially altered properties, because of differ ...

The directed migration of cells (chemotaxis) occurs not only during wound healing and inflammatory responses but also during embryonic development. However, the intracellular signaling pathways that enable a cell to detect a chemoattractant and subsequently migrate toward the ...

One of the most important properties of cells that are derived from multicellular organisms is their ability to adhere to extracellular matrix proteins or other cells. Analysis of cell-extracellular matrix and/or cell-cell adhesion, therefore, is of important value to experimental b ...

A method is provided to quantitate the extent of cell spreading as a function of the expression level of transfected recombinant proteins. This chapter contains protocols for 1) replating and staining transfected cells for immunofluorescence microscopy, 2) optimizing image acquis ...

Cell scattering is used to describe the dispersion of compact colonies of epithelial cells induced by certain soluble factors such as growth factors, cytokines, and phorbol esters. The dispersal of epithelial colonies is a dynamic process usually initiated by membrane ruffling and cen ...

Research in cell biology often is based in tissue culturing cells on artificial substrates, such as plastic or glass. These artificial conditions are prone to distorting findings by persuading cells to adjust to artificial flat rigid surfaces. In contrast, the natural substrate for most ce ...

The migration of endothelial cells (ECs) play an important role in embryonic vasculogenesis, angiogenesis, and post-angioplasty reendothelialization. ECs are constantly subjected to fluid shear stress (the tangential component of hemodynamic forces) from blood flow, but the ...

The migration of neuronal precursor cells is essential for the formation of the embryonic nervous system and for the maintenance of the adult nervous system. Modern approaches have greatly facilitated molecular and cellular studies of mechanisms underlying neuronal migration. He ...