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A Tobacco-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice
Rylosona Janarthini1, Xiaolei Wang1, Lulu Chen1
1Joint Tomato Research Institute, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
Abstract:
Thymosin β4 (Tβ4) is a peptide that is known to play important roles in protection, regeneration, and remodeling of injured tissues in humans, and that shows great promise in a range of clinical applications. However, current strategies to Tβ4 are insufficient to meet growing demand and have a number of limitations. In this current study we investigated whether expression of recombinant Tβ4 in plants, specifically in tobacco (Nicotiana tabacum) leaves, represents an effective approach. To address this question, a 168 bp Tβ4 gene optimized for tobacco codon usage bias was constitutively expressed in tobacco as a 4-unit repeat concatemer, fused to a polyhistidine tag. Quantitative polymerase chain reaction and Western blot analyses were used to verify 4×Tβ4 expression in 14 transgenic tobacco lines and enzyme-linked immunosorbent assay analysis indicated 4×Tβ4 protein concentrations as high as 3 μg/g of fresh weight in the leaves. We observed that direct administration of tobacco-derived Tβ4 was more effective than Tβ4 either obtained commercially or derived from expression in Escherichia coli at promoting splenocyte proliferation in vitro and wound healing in mice through an endothelial migration assay. This study provides new insights into the development of plant-derived therapeutic proteins and their application by direct administration.
Insights
Plant-based production of Thymosin β4 (Tβ4) in tobacco leaves offers a promising alternative to current methods. Tobacco-derived Tβ4 demonstrated superior efficacy in promoting splenocyte proliferation and wound healing compared to commercially sourced or E. coli-derived Tβ4.
Area of Science:
- Biotechnology
- Plant molecular biology
- Immunology
Background:
- Thymosin β4 (Tβ4) is crucial for tissue repair and regeneration, with significant clinical potential.
- Existing Tβ4 production methods face limitations in scalability and efficiency.
Purpose of the Study:
- To evaluate the efficacy of producing recombinant Tβ4 in tobacco plants (Nicotiana tabacum).
- To compare the therapeutic effectiveness of plant-derived Tβ4 with conventional sources.
Main Methods:
- Constitutive expression of a codon-optimized Tβ4 gene in tobacco as a 4-unit repeat concatemer fused to a polyhistidine tag.
- Verification of expression using quantitative PCR and Western blot.
- Quantification of Tβ4 protein levels via ELISA.
- In vitro splenocyte proliferation assay and in vivo mouse wound healing model (endothelial migration assay).
Main Results:
- Successfully generated 14 transgenic tobacco lines expressing 4×Tβ4.
- Achieved Tβ4 protein concentrations up to 3 μg/g fresh weight in tobacco leaves.
- Tobacco-derived Tβ4 showed enhanced efficacy in promoting splenocyte proliferation and wound healing compared to commercial or E. coli-derived Tβ4.
Conclusions:
- Plant-based expression in tobacco is a viable strategy for producing functional Tβ4.
- Direct administration of plant-derived Tβ4 offers therapeutic advantages.
- This approach advances the development of plant-derived biopharmaceuticals.
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