Molecular Biotechnology. Bernard R. Glick

tagged, for example, with a biotin molecule, and fluorescently labeled streptavidin, which binds specifically to biotin, is added to detect the phosphorylated protein. In a similar manner, glycosylated proteins can be detected with lectins (Fig. 2.53B). Lectins are plant glycoproteins that bind to specific carbohydrate moieties on the surface of proteins or cell membranes, and many different lectins with affinities for different glycosyl groups (glycans) are available.

      Figure 2.53 Detection of post-translational modifications with antibody microarrays. (A) Detection of tyrosine phosphorylation. An antibody microarray (1) is incubated with a protein sample (2). Biotinylated antiphosphotyrosine antibody is added (3) and, for visualization, a streptavidin-fluorescent dye conjugate attaches to the biotin of the antiphosphotyrosine antibody (4). (B) Detection of glycan groups. An antibody microarray (1) is incubated with a protein sample (2). A biotinylated molecule (e.g., lectin) that binds to a specific glycan is added (3) and, for visualization, a streptavidin-fluorescent dye conjugate attaches to the biotin of the lectin (4).

      In another type of microarray, purified proteins representing as many proteins of a proteome under study as possible are arrayed on a solid support and then probed with antibodies in serum samples collected from healthy (control) and diseased individuals. The purpose of these studies is to discover whether individuals produce antibodies that correlate with particular diseases or biological processes. For example, the differential expression of antibodies in serum samples from individuals with and without Alzheimer disease was tested using a microarray consisting of more than 9,000 unique human proteins (Fig. 2.54). After incubation of the serum samples with the protein microarray, bound antibodies were detected using a fluorescently labeled secondary antibody that interacts specifically with human antibodies. The screen resulted in the identification of 10 autoantibodies (i.e., directed against an individual’s own protein) that may be used as biomarkers to diagnose Alzheimer disease. Protein microarrays can also be used to identify proteins that interact with therapeutic drugs or other small molecules (Fig. 2.55). This can aid in determining the mechanism of action of a drug, for assessing responsiveness among various forms of a target protein (e.g., variants produced by different individuals), and for predicting undesirable side effects.

      Figure 2.54 Identification of disease biomarkers with a human protein microarray. Serum samples are collected from diseased and healthy individuals and incubated with microarrays of purified human proteins. Serum autoantibodies bind to specific proteins on the microarray and are detected by applying a fluorescently labeled secondary antibody directed against human antibodies. Autoantibodies present in the serum from diseased individuals but not in serum from healthy individuals are potential biomarkers that can be used in diagnosis of the disease.

      Figure 2.55 Protein microarrays to detect protein-drug interactions. Therapeutic drugs or other small molecules tagged with a fluorescent dye are applied to purified proteins arrayed on a solid support.

      Protein−Protein Interactions

      Proteins typically function as complexes comprised of different interacting protein subunits. Important cellular processes such as DNA replication, energy metabolism, and signal transduction are carried out by large multiprotein complexes. Thousands of protein-protein interactions occur in a cell. Some of these are short-lived, while others form stable multicomponent complexes that may interact with other complexes. Determining the functional interconnections among the members of a proteome is not an easy task. Several strategies have been developed to examine protein interactions, including protein microarrays, two-hybrid systems, and tandem affinity purification methods.

      The two-hybrid method that was originally devised for studying the yeast proteome has been used extensively to determine pairwise protein—protein interactions in both eukaryotes and prokaryotes. The underlying principle of this assay is that the physical connection between two proteins reconstitutes an active transcription factor that initiates the expression of a reporter gene. The transcription factors employed for this purpose have two domains. One domain (DNA-binding domain) binds to a specific DNA site, and the other domain (activation domain) activates transcription (Fig. 2.56A). The two domains are not required to be part of the same protein to function as an effective transcription factor. However, the activation domain alone will not bind to RNA polymerase to activate transcription. Connection with the DNA-binding domain is necessary to place the activation domain in the correct orientation and location to initiate transcription of the reporter gene by RNA polymerase.

      Figure 2.56 Two-hybrid assay for detecting pairwise protein interactions. (A) The DNA binding domain of a transcription factor binds to a specific sequence in the regulatory region of a gene which orients and localizes the activation domain that is required for the initiation of transcription of the gene by RNA polymerase. (B) The coding sequences for the DNA binding domain and the activation domain are fused to DNA X and DNA Y, respectively, and both constructs (hybrid genes) are introduced into a cell. After translation, the DNA binding domain-protein X fusion protein binds to the regulatory sequence of a reporter gene. However, protein Y (prey) does not interact with protein X (bait) and the reporter gene is not transcribed because the activation domain does not, on its own, associate with RNA polymerase. (C) The coding sequence for the activation domain is fused to the DNA for protein Z (DNA Z) and transformed into a cell containing the DNA binding domain-DNA X fusion construct. The proteins encoded by the hybrid genes interact and the activation domain is properly oriented to initiate transcription of the reporter gene demonstrating a specific protein-protein interaction.

      For a two-hybrid assay, the coding sequences of the DNA-binding and activation domains of a specific transcription factor are cloned into separate vectors (Fig. 2.56). Often, the Gal4 transcriptional factor from Saccharomyces cerevisiae or the bacterial LexA transcription factor is used. A DNA (or cDNA generated from a eukaryotic mRNA) sequence that is cloned in frame with the DNA-binding domain sequence produces a fusion (hybrid) protein and is referred to as the “bait.” This is the target protein for which interacting proteins are to be identified. Another DNA sequence is cloned into another vector in frame with the activation domain coding sequence. A protein attached to the activation domain is called the “prey” and potentially interacts with the bait protein. Host yeast cells are transformed with both bait and prey DNA constructs. After expression of the fusion proteins, if the bait and prey do not interact, then there is no transcription of the reporter gene (Fig. 2.56B). However, if the bait and prey proteins interact, then the DNA-binding and activation domains are also brought together. This enables the activation domain to make contact with RNA polymerase and activate transcription of the reporter gene (Fig. 2.56C). The product of an active reporter gene may produce a colorimetric response or may allow a host cell to proliferate in a specific medium.

      For a whole-proteome protein interaction study, two libraries are prepared, each containing thousands of cDNAs generated from total cellular mRNA (or genomic DNA fragments in a study of proteins from a prokaryote). To construct the bait library, cDNAs are cloned into the vector adjacent to the DNA sequence for the DNA-binding domain of the transcription factor Gal4 and then introduced into yeast cells. To construct the prey library, the cDNAs are cloned into the vector containing the sequence for the activation domain, and the constructs are transferred to yeast