细胞功能测定

The methylotrophic yeast Pichia pastoris has been widely exploited for its high-level expression of heterologous proteins by recombination of gene sequences of interest with the methanol-inducible alcohol oxidase gene (AOX1) promoter (1–3). Secreted and cytoplasmic express ...

In optimizing expression of heterologous protein, the issue of proteolysis is often an important factor, since many peptides and proteins are susceptible to degradation by proteases produced in the host organism. In such cases even if the protein product is expressed at high levels, overall ...

Thorough characterization of recombinant strains of Pichia pastoris requires analysis of genomic DNA and RNA. The protocols in this chapter describe methods for isolation of high-quality genomic DNA suitable for hybridization experiments, crude genomic DNA suitable for use as a PCR ...

The extremely high levels of alcohol oxidase produced from the native AOX1 gene in Pichia pastoris (5–30% of cell protein on induction) suggested that single-copy AOX1 -promoter expression vectors would be sufficient for efficient foreign gene expression. Therefore, the first strate ...

The most commonly used vectors for heterologous protein expression in Pichia pastoris carry its wild-type HIS4 gene and the bacterial ampicillin resistance gene as selectable markers (1–6). The HIS4 gene is relatively large (3 kb), with ill-defined functional boundaries, and its use limi ...

The key to the molecular genetic manipulation of any organism is the ability to introduce and maintain DNA sequences of interest. For Pichia pastoris, the fate of introduced DNAs is generally similar to those described for Saccharomyces cerevisiae. Vectors can be maintained as autonomou ...

Immunohistochemistry is a powerful technique to localize proteins in tissues and cultured cells as well as in fractions of subcellular compartments, like mitochondria, endoplasmic reticulum, vesicles, and membranes. In most cases, the detection of a specific protein occurs by a sand ...

Research in the field of membrane proteins has undergone explosive growth during the last decade, primarily owing to the influence of the powerful techniques of modern molecular biology. Membrane proteins fulfill essential functions, such as communication, selective transport of ...

This chapter reviews the expression of two proteins that are naturally secreted: murine epidermal growth factor (mEGF) and the 120-kDa envelope protein from human immunodeficiency virus (HIV-1 ENV). Although the initial aims of these projects were to examine the secretion in Pichia past ...

The Pichia pastoris system has now been used successfully to express a large number of different intracellular and secreted proteins. In some cases, extremely high levels have been obtained (e.g., tumor necrosis factor, TNF ; human serum albumin, HSA ). The aim of this chapter is to illustrate some of t ...

Antibodies consist of two functional parts, the antigen binding site, a heterodimer composed of the VH and VL domains, and the Fc part, which facilitates in vivo the effector functions and stability of the antibody. Recombinant antibodies were first successfully expressed by secretion fr ...

A significant advantage of Pichia pastoris as an experimental system is the ability to bring to bear readily both classical and molecular genetic approaches to a research problem. Although the recent advent of yeast molecular genetics has introduced new and exciting capabilities, clas ...

The development of efficient recombinant protein production processes can be a critical factor in whether or not a pharmaceutical therapeutic protein can enter human clinical trials and ultimately the marketplace. This is especially true for therapeutic proteins that need to be adm ...

Many subcellular processes present in multicellular eukaryotes have been shown to reside in yeast as well. The ease with which yeasts are genetically manipulated and analyzed by biochemical and ultrastructural techniques has greatly contributed to the present knowledge of these p ...

The purpose of this chapter is to provide sufficient instruction for the reader to implement fermentation strategies to produce recombinant proteins in the yeast Pichia pastoris. P. pastoris utilizes the highly efficient alcohol oxidase 1 gene (AOX1) promoter for high-level express ...

Pichia pastoris has become a highly successful system for the expression of heterologous genes. Several factors have contributed to its rapid acceptance, the most important of which include: 1. A promoter derived from the alcohol oxi

Oligonucleotide-mediated mutagenesis is a predictable and flexible means of introducing very specific changes into cloned DNA sequences to facilitate study of structure-function relationships or modification of restriction endonuclease restriction sites. Several ...

It is now technically possible to create any desired mutation in a given DNA sequence. So-called site-directed mutagenesis allows the introduction of designed mutations into specific locations. This approach is invaluable for studying gene regulation as well as for functional asses ...

Oligonucleotide-directed site-specific mutagenesis is a powerful tool to explore protein structure—function relationships. The single-stranded (M13) method (1,2), the polymerase chain reaction (PCR) (3–6), and the double-stranded plasmids method (7–9) are three basic proc ...

It is often necessary to make multiple amino acid substitutions at a particular site to determine the function of the wild-type amino acid in protein structure and function studies. Each substitution requires a unique mutation at that site. An alternative to making a series of predetermined s ...