purification step. In a number of trials, the tags did not alter the function of various test proteins.
Figure 2.58 Tandem affinity purfication to detect multiprotein complexes. The coding region of a cDNA (cDNA X) is cloned into a vector in frame with two DNA sequences (tag 1 and tag 2), each encoding a short peptide that has a high affinity for a specific matrix. The tagged cDNA construct is introduced into a host cell, where it is transcribed and the mRNA is translated. Other cellular proteins bind to the protein encoded by cDNA X (protein X). The complex consisting of protein X and its interacting proteins (colored shapes) is separated from other cellular proteins by the binding of tag 1 to an affinity matrix which is usually fixed to a column. The protein complex is retained on the column and the noninteracting proteins flow through. The complex is then eluted from the affinity matrix by cleaving off tag 1 with a protease, and a second purification step is carried out with tag 2 and its affinity matrix. The proteins of the complex are separated by one-dimensional PAGE. Single bands are excised from the gel and identified by MS.
A DNA–two-tag construct is introduced into a host cell, where it is expressed and a tagged protein is synthesized (Fig. 2.58). The underlying assumption is that the cellular proteins that normally interact with the native protein in vivo will also combine with the tagged protein. After the cells are lysed, the tagged protein and any interacting proteins are purified using the affinity tags. The proteins of the complex are separated according to their molecular weight by PAGE and identified with MS. Computer programs are available for generating maps of complexes with common proteins, assigning proteins with shared interrelationships to specific cellular activities, and establishing the links between multiprotein complexes.
Metabolomics
Metabolomics is a technique that provides a snapshot of the small molecules present in a complex biological sample. The metabolites present in cells and cell secretions are influenced by genotype, which determines the metabolic capabilities of an organism, and by environmental conditions such as the availability of nutrients and the presence of toxins or other stressors. Metabolite composition varies depending on the developmental and health status of an organism, and therefore, a comprehensive metabolite profile can identify molecules that reflect a particular physiological state. For example, metabolites present in diseased cells but not in healthy cells are useful biomarkers for diagnosing and monitoring disease. Metabolic profiles can also aid in understanding drug metabolism, which may reduce the efficacy of a treatment, or in understanding drug toxicity, which can help to reduce adverse drug reactions. Metabolomic analysis can be used to determine the catalytic activity of proteins, for example, by quantifying changes in metabolite profiles in response to mutations in enzyme coding genes and to connect metabolic pathways that share common intermediates.
Biological samples for metabolite analysis may be cell or tissue lysates, body fluids such as urine or blood, or cell culture media that contain a great diversity of metabolites. These include building blocks for biosynthesis of cellular components such as amino acids, nucleotides, and lipids. Also present are various substrates, cofactors, regulators, intermediates, and end products of metabolic pathways such as carbohydrates, vitamins, organic acids, amines and alcohols, and inorganic molecules. These molecules have very different properties, and therefore comprehensive detection and quantification using a single method based on chemical characteristics presents a challenge.
Metabolomics employs spectroscopic techniques such as MS and nuclear magnetic resonance (NMR) spectroscopy to identify and quantify the metabolites in complex samples. Often, multiple methods are used in parallel to obtain a comprehensive view of a metabolome. In a manner similar to protein identification described above, MS measures the m/z ratio of charged metabolites. The molecules may be ionized by various methods before separation of different ions in an electromagnetic field. MS is typically coupled with chromatographic techniques that first separate metabolites based on their properties. For example, MS may be coupled with gas chromatography to separate volatile metabolites. Some nonvolatile metabolites, such as amino acids, are chemically modified (derivatized) to increase their volatility. Liquid chromatography separates metabolites dissolved in a liquid solvent based on their characteristic retention times as they move through an immobilized matrix.
NMR spectroscopy is based on the principle that in an applied magnetic field, molecules (more precisely, atomic nuclei with an odd mass number) absorb and emit electromagnetic energy at a characteristic resonance frequency that is determined by their structure. Thus, the resonance frequencies provide detailed information about the structure of a molecule and enable differentiation among molecules with different structures, even when the difference is very small, such as between structural isomers. In contrast to MS, an initial metabolite separation step is not required, and NMR measures different types of molecules. In addition, NMR is not destructive, and in fact, it has been adapted to visualize molecules in living human cells in the diagnostic procedure magnetic resonance imaging (MRI). A drawback of NMR is low sensitivity, which means that it does not detect low-abundance molecules.
An illustration of the application of metabolome analysis is the identification of metabolites that are associated with the progression of prostate cancer to metastatic disease. Researchers compared more than 1,000 metabolites in benign prostate tissue, localized prostate tumors, and metastatic tumors from several tissues using MS combined with liquid and gas chromatography. Sixty metabolites were found in localized prostate and/or metastatic tumors but not in benign prostate tissue, and six of these were significantly higher in the metastatic tumors. The metabolite profile indicated that progression of prostate cancer to metastatic disease was associated with an increase in amino acid metabolism. In particular, levels of sarcosine, a derivative of the amino acid glycine, were much higher in the metastatic tumors than in localized prostate cancer tissue and were not detectable in noncancerous tissue (Fig. 2.59). Moreover, sarcosine levels were higher in the urine of men with prostate tissue biopsies that tested positive for cancer than in that of biopsy-negative controls, and higher in prostate cancer cell lines than in benign cell lines. Benign prostate epithelial cells became motile and more invasive upon exposure to sarcosine than did those treated with alanine as a control. From this analysis, sarcosine appears to play a key role in cancer cell invasion and shows promise as a biomarker for progression of prostate cancer and as a target for prevention.
Figure 2.59 Metabolite profiles of benign prostate, localized prostate cancer, and metastatic tumor tissues. The relative levels of a subset of 50 metabolites are shown in each row. Levels of a metabolite in each tissue (columns) were compared to the median metabolite level (black); shades of yellow represent increased levels, and shades of blue indicate decreased levels. Metastatic samples were taken from soft (A), rib or diaphragm (B), or liver (C) tissues. Modified with permission from Macmillan Publishers Ltd. from Sreekumar et al., Nature. 457:910–914, 2009.
summary
Molecular biotechnology comprises a large number of fundamental techniques to identify, isolate, transfer, and express specific genes in a variety of host organisms. The tools for these processes were developed from an understanding of the biochemistry, genetics, and molecular biology of cells, especially prokaryotic cells, and viruses. Molecular cloning is the process of inserting a gene or other DNA sequence isolated from one organism into a vector and introducing it into a host cell. The discovery of restriction endonucleases was essential for this process, as it enabled predictable and reproducible cleavage of both target (insert) and vector DNAs in preparation for joining the two molecules. A restriction endonuclease is a protein that binds to double-stranded DNA at a specific nucleotide sequence and cleaves a phosphodiester bond in each of the DNA strands within the recognition sequence. Digestion of target and vector DNA with the same restriction endonuclease
