Thymosin beta 4 (T beta 4) 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 T beta 4 expression in a protease-deficient host strain, Escherichia coli BL21(DE3), transformed with different expression vectors (pRSETA, pET-15b and pEcoli-Cterm6 x 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 T beta 4 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 T beta 4 promotes angiogenesis in an in vitro endothelial cell model. (C) 2013 Elsevier Inc. All rights reserved.