Molecular Biotechnology. Bernard R. Glick

are typically screened for bait-prey protein interactions in one of two ways. In one method, a prey library of yeast cells is arrayed on a grid. The prey library is then screened for the production of proteins that interact with a bait protein by introducing individual bait constructs to the arrayed clones by mating (Fig. 2.57A). Alternatively, each yeast clone in a bait library is mated en masse with a mixture of strains in the prey library, and then positive interactions are identified by screening colonies on plates for activation of the reporter gene (Fig. 2.57B). Challenges with using the two-hybrid system for large-scale determination of protein−protein interactions include the inability to clone all possible protein coding genes in frame with the activation and DNA-binding domains, which leads to missed interactions (false negatives), and the detection of interactions that do not normally occur in their natural environments within the original cells and therefore are not biologically relevant (false positives). Nonetheless, this approach has been used to successfully identify interacting proteins in a wide range of organisms from bacteria to humans.

      Figure 2.57 Large-scale screens for protein interactions using the yeast two-hybrid system. Two libraries are prepared, one containing genomic DNA fragments fused to the coding sequence for the DNA-binding domain of a transcription factor (bait library) and another containing genomic DNA fragments fused to the activation domain of the transcription factor (prey library). Two methods are commonly used to screen for pairwise protein interactions. (A) Individual yeast strains in the bait library are mated with each yeast strain in an arrayed prey library. Resulting strains in the array that produce bait and prey proteins that interact are detected by assaying for reporter gene activation (activated cells growing in a multiwell plate are indicated in green). (B) Yeast strains in the prey library are mated en masse with individual strains in the bait library. The mixture of strains are screened for reporter gene activity that identifies strains with interacting bait and prey proteins (green).

      Instead of studying pairwise protein interactions, the tandem affinity purification tag procedure is designed to capture multiprotein complexes and then identify the components with MS (Fig. 2.58). In this method, a DNA (or cDNA) sequence that encodes the bait protein is fused to a DNA sequence that encodes two small peptides (tags) separated by a protease cleavage site. The peptide tags bind with a high affinity to specific molecules and facilitate purification of the target protein. A “two-tag” system allows two successive rounds of affinity binding to ensure that the target and its associated proteins are free of any nonspecific proteins. Alternatively, a “one-tag” system with a small protein tag that is immunoprecipitated with a specific antibody requires only a single 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