细胞技术

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 ...

Oligonucleotide-directed in vitro mutagenesis is an established tool for the investigation of protein structure and function, and increasingly for studying the role of DNA sequences in gene expression. The method described here is based on the phosphorothioate technique develo ...

Site-directed mutagenesis has become an extremely powerful means by which the nature and function of critical amino acids within a protein can be assessed and altered. However, the ability to exploit this technique requires that detailed structural information about the protein (or re ...

Oligonucleotide-directed mutagenesis has become the primary method for testing theories of protein structure/function and control of gene expression by specifically altering DNA sequences. Random oligonucleotides can be used to extend this approach to genes where less stru ...

Randomly induced point mutations provide an effective strategy to recognize and characterize functionally significant features of cloned DNAs. When coupled with sensitive assays for gene function, random mutagenesis permits one to resolve active DNA motifs within a gene’s regu ...

Generating random mutations in a DNA fragment of a specific size is often the method of choice for changing specific properties of an encoded protein or for studying the functional properties of a specific DNA fragment. In all these cases it is helpful and in some cases essential to have a screening met ...

Classically, studies on the relationships between structure and function have focused on the synthesis of individual molecular species and then analyzed their functions in cells. Recently, an entirely different approach has become feasible. A series of techniques has made it possib ...

Polymerase chain reaction (PCR) is a powerful technique for the amplification of specific DNA sequences, for the generation of chimeric molecules, and for site-directed mutagenesis. It allows the rapid generation of relatively large quantities of mutated DNA. Drawbacks are the costs of ...

Saturation mutagenesis is one approach for determining the contributions of individual base pairs or amino acids to the structure and function of defined DNA sequence elements or proteins, respectively (1). If a simple phenotypic screen is available, saturation mutagenesis can be used ...

Cassette mutagenesis methods that introduce site-specific sequence changes into a target gene are powerful tools for the manipulation of proteins to analyze then structure and function (1–3). Typically, a small target region is excised by cleavage at two constructed or naturally occu ...

In Chapter 25 we described linker scanning mutagenesis by oligonucleotide ligation. In this chapter we describe a more recent and versatile procedure that makes use of amplification by the polymerase chain reaction (PCR). For a description of the traditional method of generating linker ...

The purpose of linker-scanning mutagenesis is to create a series of mutant molecules in which individual sections are sequentially replaced with the same “neutral” mutant sequence (see Fig. 1). Typically the technique has been applied to DNA and has most commonly been used to determine the cis- ...

Site-directed mutagenesis techniques allow selective engineering of gene sequences. In in vitro site-directed mutagenesis, base alterations are introduced into a target sequence by incorporating DNA base changes within a primer utilized in the DNA synthesis step. Several site- ...