遗传学实验技术

Molecular chaperones and chemical compounds like amino acids and osmolytes share the capability to prevent protein aggregation and can contribute to rescue in vivo aggregated proteins. Therefore, both overexpression of the molecular folding machinery and induced accumulati ...

The preparation of sufficient amounts of high-quality protein samples is the major bottleneck for structural proteomics. The use of recombinant proteins has increased significantly during the past decades. The most commonly used host, Escherichia coli, presents many challenges ...

Over the past two decades, prokaryotic expression systems have been widely exploited for the bioproduction of many therapeutic proteins. Much of the success can be attributed to the implementation of basic principles of prokaryotic protein translation and protein folding to the prob ...

Fluorescent tagging of proteins has become a critical step in optical analysis of protein function in vitro and in living cells. Here we describe a two-tag system for expression and isolation of a protein of interest from Escherichia coli and subsequent site-specific fluorescent labeling ...

Transposon-directed base-exchange mutagenesis (TDEM) is an efficient and controllable method for introducing a mutation(s) into a gene. Each round of TDEM removes a predetermined number of bases (up to 11 base pairs) from a randomly selected site within the target gene and replaces them with ...

The expression of heterologous proteins in robust hosts such as Escherichia coli is often plagued by the tendency of the protein of interest to misfold and aggregate. To engineer and improve the folding properties of virtually any protein of interest, the quality control process inherent to t ...

A method for carrying out protein folding with simultaneous separation by protein folding liquid chromatography (PFLC) is described herein. Furthermore, a two-dimensional chromatographic column, termed a 2D column, which can be independently employed for accomplishing PFLC in ...

Intein-mediated protein ligation (IPL) employs an intein to create a protein possessing a C-terminal thioester that can be ligated to a protein or peptide with an amino-terminal cysteine via a native peptide bond. Here we present a procedure to conduct isolation and labeling of recombinant p ...

Human aromatase (CYP19, P450arom) is responsible for the conversion of androgens to estrogens. In addition to the estrogen biosynthesis in gonads and adrenals in a classical endocrine manner, this enzyme is widely expressed in various tissues and locally regulates the estrogen level or e ...

Answering questions about proteins’ structures and functions in the new era of systems biology and genomics requires the development of new methods for heterologous production of numerous proteins from newly sequenced genomes. Cytochromes c – electron transfer proteins carrying ...

Sufficient quantities of homogeneous samples of post-translationally modified proteins are often not readily available from biological sources to facilitate structure–function investigations. Native chemical ligation (NCL) is a convenient method for the production of ...

After the concept of genetic immunization was first demonstrated by Johnston’s group in 1992 (1), numerous studies have reported the potential prophylactic and therapeutic use of nucleic acid-based vaccines for combating various infectious diseases (2–4). Vaccines of this compos ...

The use of the MPL� immunostimulant, a monophosphoryl lipid A preparation derived from the lipopolysaccharide (LPS) of Salmonella minnesota R595, began with the studies of Johnson et al. (1). It was shown that LPS was a potent adjuvant for protein antigens, even if administered at a different site a ...

In 1992, the era of DNA vaccines began with the report of antibody production upon intradermal injection of mice with a plasmid vector expressing a foreign antigen (1). A rapid succession of subsequent manuscripts showed stimulation of immune responses, including cytolytic T cells, upon in ...

The term “adjuvant” originates from the Latin word adjuvare, which means to help or aid. An immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens (1). In a broad se ...

Various studies have used DNA vaccination as a method of immunizing against tumors (1–12). As with any tumor vaccine, one challenge is to find a truly tumor-specific antigen (13,14). The majority of immunologically targeted tumor antigens are also expressed on a subset of normal host cells. Exam ...

Current technologies for the purification of supercoiled plasmids are limited. The use of cesium chloride gradients in the presence of ethidium bromide is time consuming, labor intensive, requires the use of known mutagens and is not conducive to large scale. As a result, first-generation h ...

Although the injection of DNA into tissues was originally reported in the 1950s, the technology has gained more attention in recent years as a safe means of mimicking in vivo protein production normally associated with natural infection (1–3). Nucleic acid or DNA inoculation is an important va ...

The induction of a potent and long-lasting immunity is one of the most important elements to consider in developing an effective vaccine. DNA vaccines induce markedly stronger CD8+ cytotoxic T lymphocyte (CTL) activity than do traditional peptide vaccines through their particular mec ...

In the late 1980s, Jon Wolff of the University of Wisconsin and Phil Felgner here at Vical were screening cationic lipids for their ability to encapsulate and deliver purified plasmid DNA into mouse tissues. They discovered that direct injection of lipid-DNA complexes into muscle resulted in ...