title: Bacterial expression, purification and angiogenesis-promoting activity of human thymosin β4.
abstract: Thymosin β4 (Tβ4) is an actin-binding peptide involved in tissue regeneration and angiogenesis. This 43-amino acid peptide is chemically synthesized for research or clinical trials. To overcome the high costs of solid phase synthesis, we developed a genetic engineering procedure of Tβ4 expression in a protease-deficient host strain, Escherichia coli BL21(DE3), transformed with different expression vectors (pRSETA, pET-15b and pEcoli-Cterm6 × HN). The recombinant, non-glycosylated peptide was overexpressed in soluble form and purified by two-step immobilized metal ion affinity chromatography. Use of the pET vector expression system allowed for easy removal of the polyhistidine tag by thrombin. Functional studies revealed that recombinant Tβ4 stimulated angiogenesis via activation of the endothelial proteolytic systems, inhibition of endothelial cell adhesion, promotion of migration and capillary tube formation in Matrigel, and that its activity was similar to that observed for the synthetic peptide. The presented study comprises the first evidence that recombinant Tβ4 promotes angiogenesis in an in vitro endothelial cell model.
authors.affiliation: Institute of Medical Biology, Polish Academy of Science, Lodz, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kozaczuk
authors.forename: Anna
authors.initials: A
authors.lastname: Selmi
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Bednarek
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000302:isolation & purification* / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002846:Chromatography, Affinity; D003094:Collagen; D004338:Drug Combinations; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007797:Laminin; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D011509:Proteoglycans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013947:Thymosin / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D000099201:Thymosin beta(4)
chemical_list: D043925:Angiogenesis Inducing Agents; D003094:Collagen; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D011994:Recombinant Proteins; D013947:Thymosin; D000099201:Thymosin beta(4); C059485:matrigel
keywords: Affinity chromatography; Angiogenesis; Polyhistidine tag; Recombinant peptide; Thrombin; Thymosin β4