期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 52, 期 1, 页码 50-58出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2006.08.015
关键词
protein kinase G (PKG); HSP20; phosphorylation; co-expression; protein transduction
类别
资金
- NHLBI NIH HHS [R01 HL058027] Funding Source: Medline
- NIDDK NIH HHS [R01 DK066483-06, R01 DK047936, R01 DK066483, R01 DK047936-13] Funding Source: Medline
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL058027] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R01DK066483, R01DK047936] Funding Source: NIH RePORTER
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 HPLGESI-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. Published by Elsevier Inc.
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