Expression of thymosin alpha1-thymopentin fusion peptide in Pichia pastoris and its characterization.
Thymopentin plays an important role in improving imbalanced immune systems of patients, however, it has a limited half-life in plasma. To get more stable and active thymopentin analogs, a fusion thymosin alpha1-thymopentin (Talpha1-TP5) gene was synthesized and cloned into vector pGAPZalphaA. Talpha1-TP5 fusion peptide was expressed in pichia pastoris and purified by metal chelating chromatography and gel filtration chromatography. The circular dichroism spectra (CD) indicated that the secondary structure of Talpha1-TP5 fusion peptide is dominated by a-helix and random coil. In vitro analysis showed that the plasma half-life of Talpha1-TP5 fusion peptide is 140 +/- 14 min, which is longer than that of TP5 (5.6+/-0.7 min) and Talpha1 (127+/-11 min). The in vitro activity assay presented that Talpha1-TP5 fusion peptide has greater activity in promoting proliferation of Kunming mouse splenocytes, and in vivo experiment it showed better activity in promoting the phagocytosis of macrophages and secretion of IL-2 than both Talpha1 and TP5. Our findings suggest that Talpha1-TP5 fusion peptide might be a potential therapeutic agent.
Gao
Demin
D
Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China.
Zhang
Xulong
X
Zhang
Jian
J
Cao
Jichao
J
Wang
Fengshan
F
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002614:Chelating Agents / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005822:Genetic Vectors; D006207:Half-Life; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D010587:Phagocytosis / Q000187:drug effects; D010843:Pichia / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013154:Spleen / Q000166:cytology / Q000187:drug effects; D000077596:Thymalfasin; D016305:Thymopentin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013947:Thymosin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
D002614:Chelating Agents; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D013947:Thymosin; D016305:Thymopentin; D000077596:Thymalfasin