Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Charles R. Flynn, Christopher C. Smoke, Elizabeth Furnish, Padmini Komalavilas, Jeffrey Thresher, Zhengping Yi, Lawrence J. Mandarino, Colleen M. Brophy

    Research output: Contribution to journalArticle

    12 Citations (Scopus)

    Abstract

    Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57% ± 4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.

    Original languageEnglish (US)
    Pages (from-to)50-58
    Number of pages9
    JournalProtein Expression and Purification
    Volume52
    Issue number1
    DOIs
    StatePublished - Mar 2007

    Fingerprint

    Phosphorylation
    Escherichia coli
    Chemical activation
    Serine
    HSP20 Heat-Shock Proteins
    Human Immunodeficiency Virus Proteins
    Cyclic GMP-Dependent Protein Kinases
    3T3 Cells
    Proteins
    Cyclic Nucleotides
    Post Translational Protein Processing
    Vascular Smooth Muscle
    Recombinant Proteins
    Smooth Muscle
    Arginine
    Actins
    Catalytic Domain
    Serotonin
    Therapeutics
    Complementary DNA

    Keywords

    • Co-expression
    • HSP20
    • Phosphorylation
    • Protein kinase G (PKG)
    • Protein transduction

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Flynn, C. R., Smoke, C. C., Furnish, E., Komalavilas, P., Thresher, J., Yi, Z., ... Brophy, C. M. (2007). Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli. Protein Expression and Purification, 52(1), 50-58. https://doi.org/10.1016/j.pep.2006.08.015

    Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli. / Flynn, Charles R.; Smoke, Christopher C.; Furnish, Elizabeth; Komalavilas, Padmini; Thresher, Jeffrey; Yi, Zhengping; Mandarino, Lawrence J.; Brophy, Colleen M.

    In: Protein Expression and Purification, Vol. 52, No. 1, 03.2007, p. 50-58.

    Research output: Contribution to journalArticle

    Flynn, CR, Smoke, CC, Furnish, E, Komalavilas, P, Thresher, J, Yi, Z, Mandarino, LJ & Brophy, CM 2007, 'Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli', Protein Expression and Purification, vol. 52, no. 1, pp. 50-58. https://doi.org/10.1016/j.pep.2006.08.015
    Flynn, Charles R. ; Smoke, Christopher C. ; Furnish, Elizabeth ; Komalavilas, Padmini ; Thresher, Jeffrey ; Yi, Zhengping ; Mandarino, Lawrence J. ; Brophy, Colleen M. / Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli. In: Protein Expression and Purification. 2007 ; Vol. 52, No. 1. pp. 50-58.
    @article{80276ee65b4b4080b6e60268bafbeb89,
    title = "Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli",
    abstract = "Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57{\%} ± 4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.",
    keywords = "Co-expression, HSP20, Phosphorylation, Protein kinase G (PKG), Protein transduction",
    author = "Flynn, {Charles R.} and Smoke, {Christopher C.} and Elizabeth Furnish and Padmini Komalavilas and Jeffrey Thresher and Zhengping Yi and Mandarino, {Lawrence J.} and Brophy, {Colleen M.}",
    year = "2007",
    month = "3",
    doi = "10.1016/j.pep.2006.08.015",
    language = "English (US)",
    volume = "52",
    pages = "50--58",
    journal = "Protein Expression and Purification",
    issn = "1046-5928",
    publisher = "Academic Press Inc.",
    number = "1",

    }

    TY - JOUR

    T1 - Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

    AU - Flynn, Charles R.

    AU - Smoke, Christopher C.

    AU - Furnish, Elizabeth

    AU - Komalavilas, Padmini

    AU - Thresher, Jeffrey

    AU - Yi, Zhengping

    AU - Mandarino, Lawrence J.

    AU - Brophy, Colleen M.

    PY - 2007/3

    Y1 - 2007/3

    N2 - Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57% ± 4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.

    AB - Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57% ± 4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.

    KW - Co-expression

    KW - HSP20

    KW - Phosphorylation

    KW - Protein kinase G (PKG)

    KW - Protein transduction

    UR - http://www.scopus.com/inward/record.url?scp=33845957320&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=33845957320&partnerID=8YFLogxK

    U2 - 10.1016/j.pep.2006.08.015

    DO - 10.1016/j.pep.2006.08.015

    M3 - Article

    C2 - 17084643

    AN - SCOPUS:33845957320

    VL - 52

    SP - 50

    EP - 58

    JO - Protein Expression and Purification

    JF - Protein Expression and Purification

    SN - 1046-5928

    IS - 1

    ER -