A novel dimeric thymosin beta 4 with enhanced activities accelerates the rate of wound healing

Tian-Jiao Xu,^1,2,* Qi Wang,^1,* Xiao-Wen Ma,¹ Zhen Zhang,³ Wei Zhang,¹ Xiao-Chang Xue,¹ Cun Zhang,¹ Qiang Hao,¹ Wei-Na Li,¹ Ying-Qi Zhang,¹ Meng Li<sup>1

1</sup>State Key Laboratory of Cancer Biology, Biotechnology Center, School of Pharmacy, Fourth Military Medical University, Xi’an, People’s Republic of China; ²The Institute of Medicine, Qiqihar Medical University, Qiqihar, People’s Republic of China; ³Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA

*These authors contributed equally to this work

Objective: Thymosin beta 4 (Tβ4) is a peptide with 43 amino acids that is critical for repair and remodeling tissues on the skin, eye, heart, and neural system following injury. To fully realize its utility as a treatment for disease caused by injury, the authors constructed a cost-effective novel Tβ4 dimer and demonstrated that it was better able to accelerate tissue repair than native Tβ4.
Methods: A prokaryotic vector harboring two complete Tβ4 genes with a short linker was constructed and expressed in Escherichia coli. A pilot-scale fermentation (10 L) was performed to produce engineered bacteria and the Tβ4 dimer was purified by one-step hydrophobic interaction chromatography. The activities of the Tβ4 dimer to promote endothelial cell proliferation, migration, and sprouting were assessed by tetramethylbenzidine (methylthiazol tetrazolium), trans-well, scratch, and tube formation assays. The ability to accelerate dermal healing was assessed on rats.
Results: After fermentation, the Tβ4 dimer accounted for about 30% of all the bacteria proteins. The purity of the Tβ4 dimer reached 98% after hydrophobic interaction chromatography purification. An average of 562.4 mg/L Tβ4 dimer was acquired using a 10 L fermenter. In each assay, the dimeric Tβ4 exhibited enhanced activities compared with native Tβ4. Notably, the ability of the dimeric Tβ4 to promote cell migration was almost two times higher than that of Tβ4. The rate of dermal healing in the dimeric Tβ4-treated rats was approximately 1 day faster than with native Tβ4-treated rats.
Conclusion: The dimeric Tβ4 exhibited enhanced activity on wound healing than native Tβ4, and the purification process was simple and cost-effective. This data could be of significant benefit for the high pain and morbidity associated with chronic wounds disease. A better strategy to develop Tβ4 as a treatment for other diseases caused by injuries such as heart attack, neurotrophic keratitis, and multiple sclerosis was also described.

Keywords: thymosin beta 4, dimer, wound healing, tissue repair, peptide, genetic engineering