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Pichia pastoris has proven to be a valuable model for examining the molecular events of the selective degradation of peroxisomes by a process called pexophagy. We have developed a protocol to rapidly identify genes essential for glucose-induced pexophagy. This method utilizes the rand ...

This chapter describes the different methods used for localization of proteins and organelles in Pichia pastoris. A series of plasmids and a modified immunofluorescence protocol for localization and co-localization of proteins and organelles are described. Also included are pr ...

With the approaching completion of the Pichia pastoris genome, a greater emphasis will have to be placed on the proteome and the protein-protein interactions between its constituents. This chapter discusses methods that have been used for the study of such interactions among both soluble ...

Intracellular structures in Pichia pastoris can be visualized by the complementary methods of fluorescence microscopy and electron microscopy. An improved immunofluorescence protocol yields better optics and more reliable antigen preservation than conventional met ...

Selection of both an appropriate expression vector and corresponding strain is crucial for successful expression of heterologous proteins in Pichia pastoris. This chapter explores both the standard and new vector/strain options available for protein expression in this yeast. I ...

This chapter provides rational approaches to design and optimize fed-batch and continuous fermentations of both Mut+ and Muts (methanol utilization plus and slow) Pichia pastoris strains. The methods are described in detail for glycerol batch, glycerol fed-batch, transition, and me ...

Several methods for DNA-mediated transformation of Pichia pastoris have been developed which vary in type of DNA that is transformable (e.g., linear versus circular) efficiency, cost, and labor and each is described in detail. As in Saccharomyces cerevisiae, gene replacement (also known ...

Flavor compounds synthesis by biotechnological processes today plays an increasing role in the food industry. This is the result of scientific advances in biological processes, making use of microorganisms or enzymes as an alternative to chemical synthesis, combined with recent de ...

Enzymes have been shown to operate successfully under nonphysiological conditions, e.g., in water-poor media consisting of almost pure nonpolar solvents. These new reaction conditions offer interesting possibilities to expand the scope of enzyme technology. An essential issue ...

The enzymatic interconversions of the various hexoses found in polysaccharides and glycoproteins normally take place either on the unsubstituted sugars, their phosphate esters, or at the sugar nucleotide levels. In a few cases, however, epimerization reactions take place after the ...

The phase diagram delineates the potential working area for a particular two-phase system and is a “fingerprint” unique to that system under set conditions of, for example, pH, temperature and salt concentration. Information that can be generated from such a diagram (see Fig. 1) includes: the co ...

Phase separation in solutions containing polymer mixtures is a very common phenomenon; in fact, miscibility of the polymer mixtures is an exception (1). Most hydrophilic polymer pairs are incompatible in aqueous solutions yielding two coexisting phases in equilibrium with each othe ...

The most common aqueous two-phase systems (ATPS) are created using either two incompatible polymers or one polymer and a salt (1). These mixtures

Partitioning in aqueous two-phase system (ATPS) provides a rapid and gentle means of separation of soluble as well as particulate biomaterials, e.g. proteins, nucleic acids, cells, viruses, organelles, and membranes. Partitioning between the two phases is a complex phenomenon, guided m ...

The ability of aqueous two-phase systems to support bioseparations obviously depends intimately on the physicochemical properties of the polymers and yhe phases they form. Measurement of some of these properties can provide insight into the mechanisms responsible for the patition ...

A crucial step in many biochemical studies is the separation and purification of the individual structures of interest. For cells, cell organelles, and membrane fragments, the use of aqueous two-phase partition has become an important tool. The basis for separation in these phase systems is t ...

Liquid-liquid partition chromatography (LLPC) is a column chromato-graphic approach of aqueous two-phase partitioning that offers new and unique possibilites to solve separation and fractionation problems (1–3, for review,) see ref. 4). The bottom phase of the two-phase system is ads ...

Literature related to the partitioning of biological materials in aqueous polymer two-phase systems encompasses over two thousand articles and almost half a century of experience (1). Much of this effort has involved eukaryotic cells or other biological particles. Over the past 15 years ...

Two-phase extraction has found its platform in several aspects of virus research. With the rapid development of rational design systems for the production of biotechnology products (1), development of large-scale systems (see Chapter 36) and methods to recycle the polymers (see Chapter ...

To understand the function of a biological membrane like that of chloroplast thylakoids, it is important to understand the arrangement of its different protein and lipid components. Preparations that have proven to be particularly suited for such studies are those consisting of membra ...