Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • br Acknowledgments br Protein microarrays were developed to

    2023-01-16


    Acknowledgments
    Protein microarrays were developed to provide miniaturized high-throughput tools to study protein function, K03861 and post-translational modifications. Just a few years ago, the seminal work performed in Stuart Schreiber's laboratory at Harvard University () and at Michael Snyder's laboratory at Yale University () demonstrated that large numbers of proteins can be immobilized in a microarray format and retain their function []. Since that time, there has been an explosion of published applications in the field. Functional protein microarrays can be used to reproduce most major types of interactions and enzymatic activities that take place in biochemical pathways (). In signal transduction pathways, for instance, signal transmission begins with binding of a ligand to a transmembrane protein, followed by signal amplification through a combination of molecular interactions and modifications, often involving the generation of second messengers or kinase cascades, with the signal ultimately being transmitted to its final effector(s). Protein–protein, protein–lipid, protein–DNA and protein–small molecule interactions, multiprotein complexes, as well as kinase activities inherent in such pathways have all been studied using protein arrays. Although many cellular pathways have been characterized to date, much remains to be learned, especially regarding the combinatorial and dynamic interactions of individual pathways. Because of the unique ability to address different aspects of biological pathways, functional protein microarray technology is primed to make significant contributions to the understanding of disease pathways for both basic and drug research. Functional protein microarrays Most, protein microarrays are constructed via direct arraying of purified proteins onto a slide surface [2–5]. In the first example of a proteome microarray, Zhu et al. [2] purified and arrayed proteins from a library of nearly all known yeast open-reading frames (ORFs) expressed as glutathione-S-transferase (GST)-fusions. The use of these arrays for protein–protein and protein–lipid interaction discoveries was successfully demonstrated. Recent proof-of-concept publications have demonstrated the feasibility of additional manufacturing approaches. He and Taussig [6] first demonstrated the production of a protein in situ array (PISA). A PISA is generated by expression of tagged proteins from PCR fragments and their immobilization. It was demonstrated that antibody fragments arrayed using this method retain function through antigen-specific interactions. Using a similar approach, a group led by Joshua LaBaer developed a nucleic acid programmable protein array (NAPPA) technology based on the arraying of DNA on modified glass slides, coupled with an in vitro transcription-translation system [7]. The proteins are expressed with tags and are sequestered on the chip by a co-immobilized affinity capture molecule, such as an antibody. The capture molecule retains the protein at the site of expression. Using RNA-display, Weng et al. [8] and Jung and Stephanopoulos have produced protein microarrays that contain mRNA-linked protein complexes captured on arrays by hybridization to specific cDNA molecules immobilized on the underlying substrate.
    Protein interactions on whole proteome microarrays
    Biochemical assays with functional protein microarrays Performing biochemical assays on arrays requires that the immobilized proteins retain their enzymatic activity in this state. Work by Chen et al. [22], conducted using a variety of fluorescently labeled molecules that covalently attach to proteins (so-called suicide inhibitors), suggested that a variety of enzyme classes (hydrolases, phosphatases and proteases) retain their activities on arrays. In further support of this, we have demonstrated that kinases can both autophosphorylate and phosphorylate substrates on a solid-state support, indicating that protein kinases are also active on slides. An example of this is depicted in Figure 3a, where kinase activity in the presence of γ-33P-ATP results in in situ autophosphorylation. Mirzabekov et al. [23] have also demonstrated enzyme activities for horseradish peroxidase, alkaline phosphatase and β-D-glucuronidase on protein arrays [23].Although these examples K03861 show that several enzyme activities are compatible with arrays, the development of robust microarray assays remains a challenge.