Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli.
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.
Flynn
Charles R
CR
Arizona State University, Harrington Department of Bioengineering, Tempe, AZ 85287-9709, USA. robb.flynn@asu.edu
Smoke
Christopher C
CC
Furnish
Elizabeth
E
Komalavilas
Padmini
P
Thresher
Jeffrey
J
Yi
Zhengping
Z
Mandarino
Lawrence J
LJ
Brophy
Colleen M
CM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000502:physiology; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D050886:HSP20 Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D050886:HSP20 Heat-Shock Proteins; C494382:HSPB6 protein, human; D010446:Peptide Fragments; D011994:Recombinant Proteins