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.
N1 - Funding Information:
We thank Dr. Thomas Lincoln and the members of his group at the University of South Alabama for the PKG catalytic subunit cDNA clone and technical expertise. This work was supported by the National Institute of Health Grants RO1HL58027-01 to C. Brophy and R01DK47936-11 to L. Mandarino.
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
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U2 - 10.1016/j.pep.2006.08.015
DO - 10.1016/j.pep.2006.08.015
M3 - Article
C2 - 17084643
AN - SCOPUS:33845957320
SN - 1046-5928
VL - 52
SP - 50
EP - 58
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -