A Tobacco-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice

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.


Introduction
Thymosin 4 (T4), a 43-amino acid peptide that is encoded by the TMSB4X gene on the X chromosome of mice and humans, was initially isolated from thymosin fraction 5 (TF5) as a biologically active component [1].TF5 was originally authorized by the FDA (Food and Drug Administration) to treat the primary immunodeficiency disease, DiGeorge syndrome, in clinical trials involving young children in the US [2].T4 was initially identified as an actin monomer (Gactin) binding protein and has the capacity to sequester Gactin, thereby inhibiting intracellular actin polymerization [3][4][5].In addition to regulating actin formation, extracellular T4 participates in several biological processes, including blood coagulation, osteoblast differentiation, activation and degranulation of platelets, and regulation of cell migration (http://www.ncbi.nlm.nih.gov/gene/7114).T4 therefore plays an important role in medical treatments, such as antiinflammation [6], angiogenesis [7,8], remodeling of dam-aged tissues [9,10], and the prevention of organic fibrogenesis [5,11,12].
Several studies have demonstrated that the expression of T4 is upregulated in injured tissue and differentiating cells [13,14], and high concentrations of T4 protein have been found in blood platelets, wound fluid, and a range of tissues [15,16] and its effects appear to be widespread.It has been extensively employed to treat diabetic ulcers and bedsores [17,18] and damaged corneas [10,19,20] and for cardiac cell survival and the repair of heart muscle injured during heart attacks [9,21,22], as well as antifibrogenesis of the liver [5], kidney [11,23] and lung [12].T4 therefore shows great promise for numerous clinical applications [17] and the resulting increased commercial demand relies on efficient production methods.Traditionally, T4 is obtained from extracts of animal thymus glands or is chemically synthesized; however, genetic engineering approaches, such as expression in Escherichia coli, have also been developed [24,25].It has also been reported that a T4 protein 2 BioMed Research International concatemer expressed in E. coli is able to promote wound healing in mice [24].However, for clinical applications, E. coli derived T4 protein must undergo a complex purification process to eliminate impurities and endotoxins, which represents a significant disadvantage of this expression system.Overall, the disadvantages to the aforementioned methods include high production costs associated with separation and purification of the target protein, as well as the risk of zoonosis [26,27].
Current T4 production capacity cannot meet the clinical demand, and plant-based protein/peptide expression systems represent a potentially attractive solution as they are relatively inexpensive, do not have problems associated with zoonosis, and are scalable.Accordingly, over the last decade plantbased expression systems have been extensively used to synthesize therapeutic proteins, antibodies, and vaccines [26][27][28].In order to meet the rising clinical demand for T4, we wanted to develop a novel expression system to produce T4 protein at a lower cost, in a safe manner, and through a process that is readily scalable.To this end, a T4 protein encoding gene (T4) was designed to take into account tobacco (Nicotiana tabacum) codon usage bias and was integrated into the tobacco genome via Agrobacteriummediated transformation.We describe here the production of tobacco-derived 4×T4 protein and report that it can promote cell proliferation in vitro and wound healing in vivo in mice.Furthermore, we present evidence that the plant-derived T4 protein, which can be directly applied to wounds, is more bioactive than the heterologous form derived from overexpression in E. coli.The study provides important information for the future development of plant expressed pharmaceutical proteins for direct administration.

Materials and Methods
2.1.Biological Materials.Tobacco (N.tabacum cv.Bairihong) seeds and DH5 (E.coli) and EHA105 (A. tumefaciens) cells were obtained from the Plant Biotechnology Research Centre, School of Agriculture and Biology, Shanghai Jiao Tong University, China.Six-to eight-week-old, healthy Balb/c mice were purchased from the Animal Centre, Shanghai Jiao Tong University, Shanghai, China.

Construction of the Plant Expression
Vector.Based on the T4 amino acid sequence [1], a 168 bp T4 gene optimized for tobacco codon usage bias was designed, synthesized, and subcloned into the pUC57 plasmid.The resulting pUC57-T4 plasmid was digested with two combinations of restriction endonucleases (Spe I/Sac I and Xba I/Sac I) and the released DNA fragment, with Xba I/Sac I ends, was ligated into the SpeI/SacI sites of pUC57-T4 based on the isocaudameric properties of Spe I and Xba I to create the pUC57-2×T4 plasmid.The pUC57-4×T4 plasmid with four repeats of the T4 gene was generated using a similar approach.A DNA sequence encoding six histidines with a Bam HI restriction site was introduced into the 5  end of the 4×T4 construct via polymerase chain reaction (PCR) using the PCR primers T4 F1: 5 ggggatccatgcaccaccaccaccaccacggtaccatgtctagaatgtctga-3  and T4 R1: 5  -ccgagctcttaact agtcataga-3  .The fused DNA fragment of 6×his-4×T4 was then digested by BamHI and SacI and subcloned into the 35S::hIXF plasmid constructed by the Plant Biotechnology Research Center, Shanghai Jiao Tong University, China, to create the plant binary expression vector, 35S::6×his-4×T4.This plasmid was then introduced into EHA105 (A. tumefaciens) via the freeze thaw method [29].

Tobacco Transformation and PCR Analysis.
Tobacco transformation and extraction of tobacco genomic DNA were conducted as previously described [30], with slight modifications.The concentration of genomic DNA extracted from the regenerated tobacco lines was adjusted to 50 ng/L for PCR analysis, and the 35S::6×his-4×T4 plasmid and genomic DNA derived from untransformed tobacco were used as positive and negative controls, respectively.PCR reactions were performed in 25 L reaction volumes containing the following components: 2 L genomic DNA template, 2.5 L 10x PCR buffer with MgCl 2 , 1.5 L 2.5 mM/L dNTPs, 1 L 10 M each PCR primer (P35S: 5  -ttcgtcaacatggtggagca-3  and Noster: 5  -aagaccggcaacaggattca-3  ), 0.2 L ExTaq DNA polymerase (5 unit/L) (Takara Biotechnology, Dalian, China), and 16.8 L deionized ddH 2 O.The PCR program was one cycle 94 ∘ C for 3 min, followed by 35 cycles (94 ∘ C for 30 s, 54 ∘ C for 30 s, and 72 ∘ C for 1.5 min), and finally 72 ∘ C for 8 min before being held at 10 ∘ C. The PCR products were electrophoresed on a 1.0% (w/v) agarose gel containing 0.5 mg/L ethidium bromide (EB) in 1x TAE buffer (Tris base acetic acid EDTA).Gels were imaged under UV light.

qRT-PCR.
Total RNA was extracted from young leaves of both transgenic and nontransgenic tobacco lines using an RNAprep pure plant kit (TIANGEN, Beijing, China) according to the manufacturer's instructions.The total RNA samples were treated with DNase (RNase-free, TaKaRa, Dalian, China) to eliminate DNA contamination.One g total RNA was used as a template to synthesize complementary DNA (cDNA) using the Prime-ScriptTM RT Master Mix Kit (TaKaRa, Dalian, China) at 37 ∘ C for 15 min and 85 ∘ C for 5 sec.Two L cDNA, diluted 100-fold, was used for qRT-PCR with gene specific primers (T4F: 3  -acggtaccatgtctagaatg-5  ; T4R: 3  -ccgagctcttaactagtcatg-5  ).The qRT-PCR program was initiated at 95 ∘ C for 30 sec, followed by 27 cycles of 95 ∘ C for 15 sec, 58 ∘ C for 15 sec, 72 ∘ C for 25 sec, and finally 72 ∘ C for 5 min, before being held at 4 ∘ C. The UBIQUITIN gene (GenBank: X58253.1) was used as a reference gene for data normalization, using the gene-specific primers UbiF: 3 aagacctacaccaagcccaa-5  and UbiR: 3  -aagtgagcccacacttacca-5  .

Western Blot and ELISA (Enzyme-Linked Immunosorbent
Assay) Analyses.Total soluble protein (TSP) was extracted from young leaves of transgenic and nontransgenic tobacco lines (three biological replicates) using phosphate-buffered saline (PBS) extraction buffer (20 mM sodium phosphate pH 7.4, 137 mM sodium chloride, and 2.7 mM potassium chloride).Approximately 500 mg of each leaf sample was ground to a fine powder using a pestle and mortar with liquid nitrogen and subsequently added to PBS extraction buffer in a 1 : 1 ratio (w/v).The powder and extraction buffer were mixed well using a Vortex-Genie 2 (Scientific Industries, USA) and then transferred to an ice bath for 2 hrs.The extraction mixture was then centrifuged at 12,000 g for 20 min at 4 ∘ C and the supernatant collected.The TSP concentration was determined using the Bradford method [31], using bovine serum albumin (BSA) to create a standard curve.
ELISA assays were carried out as previously described [32].The 4×T4 protein derived from E. coli was used as a positive control and diluted to concentrations of 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 ng/L in PBS buffer [24].The 50 L serial dilutions of the E. coli-derived 4×T4 and the 4×T4 containing TSP extracted from transgenic tobacco (adjusted to 1 g/L) were added to the wells of a 96-well plate in triplicate, and the TSP from nontransgenic tobacco leaves was used as a negative control.The same primary and secondary antibodies were used as for the Western blot analysis.BCIP/NBT was used as substrate for the color reaction and A 410 was measured with a microtiter plate reader (BioTek Instruments, Winooski, VT, USA).4×T4 protein concentrations were calculated using the previously established standard curve.

Cell Proliferation Assay (In Vitro).
Chemically synthesized standard T4 protein was diluted to 10 ng/L and employed as a positive control.The 4×T4 containing TSP extracted from the young transgenic tobacco leaves was filtered through a 0.45 m pore size membrane (Millipore Millex, Shanghai Jinxin Bio.Shanghai, China) and the 4×T4 protein concentration adjusted to 10 ng/L with PBS extraction buffer.TSP (amount equal to the tested sample) derived from nontransgenic tobacco leaves were used as negative control.
The in vitro bioactivity of T4 was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay [33].Spleen cells isolated from 6-8-week-old Balb/c mice were collected via centrifugation at 1,000 ×g for 10 min at room temperature [24].Pellets of spleen cells were subsequently resuspended and diluted to 1 × 10 5 cells/mL in RPMI 1640 medium (Sigma-Aldrich, Shanghai, China).A 100 L spleen cell aliquot was added to each well of a 96-well plate and then 100 L of the diluted standard T4 protein (1 g), and the TSP extracted from the transgenic and nontransgenic tobacco leaves and a PBS vehicle control were added separately to triplicate wells.The 96-well plate was incubated at 37 ∘ C, 5% (v/v) CO 2 , for 24 h in a cell-culture incubator, and then 10 L of MTT reagent purchased from Shanghai Hushi Medical Technology Co., Ltd.(Shanghai, China) was added to each well and incubated for an additional 4 h.The plate was periodically observed using a CKX31 inverted microscope (Olympus, Watford, Herts., U.K.) and 150 L of dimethyl sulphoxide (DMSO) was added to each well when purple precipitate was observed.After swirling gently, the plate was kept in the dark at room temperature for 15 min.Subsequently, the absorbance of each reaction at wavelength of 570 nm was measured with a microtiter plate reader (BioTek, USA).
Spleen cell proliferation was calculated using the following equation:

Wound Healing Experiment (In Vivo
).The 4×T4 containing TSP extracted from young transgenic tobacco leaves were filtered through a 0.45 m pore size membrane (Millipore Millex, Merck KGaA, Darmstadt, Germany) and freezedried (Thermo Fisher Scientific ISS110, USA), before being diluted to 100 ng 4×T4/L and 50 ng 4×T4/L with PBS extraction buffer.Nontransgenic tobacco-derived TSP were used as a negative control.Three, full-thickness, 5 mm punch wounds were inflicted on the dorsal surfaces of each 6-8-week-old Balb/c mouse as previously described [13].Punch wounds were made on eighteen Balb/c mice and twelve healthy mice were chosen for the experiments.Fifty L samples of six treatments were then applied at 24 and 48 h after wounding.The six treatments included tobacco-derived 4×T4 proteins (5 g and 2.5 g), T4 protein (Purchased from GL Biochem, 5 g), E. coliderived 4×T4 protein (5 g), nontransgenic tobacco TSP (5 g and 2.5 g), and PBS as a vehicle control.Three biological replicates were carried out for each experimental treatment.
From days 2 to 10 after treatment, keratinocyte migration from six mice was examined by measuring the distance between epidermal tongues of the wound edges with a Vernier caliper (Endura-Greenlee Tools, E0531, Shanghai, China).Wound closure was calculated using the formula described by Li et al. (2007) [25]: distance of migrated keratinocytes from the wound edge total wound width × 100. ( To examine reepithelialization and vessel counts of the wound, six additional mice (treated as above) were euthanized on day 8 after treatment and tissues from the healing wounds collected and fixed in 4% (v/v) formalin buffer.The fixed tissues were then embedded in paraffin after dehydration in a series of ethanol concentrations (75%, 85%, 95%, and 100%) and 5 m sections from the middle of the wounds were made using a microtome (Leica, RM2200) [24].Sections were then mounted on glass slides and stained with hematoxylin and eosin (Shanghai Dingjie Biotechnology Company, Shanghai, China) after paraffin removal.Vessel counts in the wound beds were determined by identifying vascular spaces distinguished by their endothelial lining, including those at the junction of the dermis and the hypodermis, as angiogenesis within wounds occurs to a great extent from these vessels.Counts were averaged as vessel counts per 10 high-powered fields (40x).

Design, Transformation, and Molecular Examination of the T𝛽4 Transgene.
Based on the T4 amino acid sequence (Genpept accession, P62326.2),an 168 bp T4 gene optimized for tobacco codon usage bias was designed and synthesized, including the restriction endonuclease sites (colored below) Kpn I/Xba I and Spe I/Sac I at the 5  and 3  ends, respectively, protection bases, and both an initiation codon (ATG) and a termination codon (TAA) (bold).The sequence was as shown in Figure 1.
4×T4 and 35S::6×his-4×T4 constructs were subsequently created (Figure 2) and transformed into tobacco and the resulting putative transgenic tobacco lines were screened by PCR.This revealed an 1,140 bp DNA band, corresponding to the 4×T4 gene, in the positive control and in 14 of the putative tobacco lines, while this DNA band was not present in nontransformed tobacco samples (Supplementary Appendices 1-d to f and Appendix 2 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/1973413).We concluded that the 4×T4 gene had been successfully integrated into the tobacco genome of the positive lines.

Expression of the 4×T𝛽4 Gene. Expression of the 4×T𝛽4
gene was evaluated in eight of the positive transgenic tobacco lines by qRT-PCR.The expression levels varied substantially, between lines, with lines 3, 5, and 13 having high  levels of 4×T4 transcript accumulation, and line 3 showing particularly high expression.The expression level in other lines ranged from relatively low (lines 2, 6, 7, and 15) to nondetectable (line 4) (Figure 3).These differences may be attributed to the copy number of the integrated 4×T4 gene, as well as their sites of integration in the tobacco genome [34,35].

Verification of Successful Expression of the Recombinant 4×T𝛽4
Protein in Transgenic Tobacco.The TSP extracted from young leaves of both transgenic and nontransgenic tobacco (negative control) was examined by Western blot and ELISA analyses using anti-His-tag monoclonal primary antibody.The TSP content derived from young tobacco leaves ranged from 4.92 mg/g fresh weight (FW) (line 2) to 6.18 mg/g FW (line 13).Western blot analysis showed a 23.2 kDa band in transgenic tobacco lines, which corresponded to the predicted size of the recombinant protein and which was absent from the nontransgenic tobacco extracts (Supplementary Appendix 4).
The 4×T4 protein content in the transgenic young tobacco leaves was determined by ELISA.The concentrations of 4×T4 protein in transgenic tobacco extracts ranged from 0.492 g/g FW in line 6 to 2.946 g/g FW in line 5 (Supplementary Appendix 3).Differences in the expression  level of the 4×T4 recombinant protein between the lines may be attributed to the number of the 4×T4 copies and position of integration, as well as to posttranscriptional processes [36,37].

Tobacco-Derived 4×T𝛽4 Protein Promotes Healing Wound in Balb/c Mice (In Vivo).
The efficiency with which tobaccoderived 4×T4 protein healed wounds and promoted keratinocyte migration was examined using a full thickness cutaneous mouse wound model.The lengths of the epidermal tongues from the wound edges were measured, and we observed that reepithelialization rates were higher in all of the treatment groups (transgenic tobacco-derived 4×T4, commercial T4, and recombinant E. coli-derived 4×T4) than in the negative controls (nontransgenic tobacco crude protein and PBS buffer) and that the rate of reepithelialization sharply increased during days 6-8 after treatment.Moreover, on day 8, the rate of keratinocyte migration on the wound bed treated with transgenic tobacco-derived 4×T4 (5 g) was the highest of all six treatments (Figure 5(a)).The reepithelialization rate in the transgenic tobacco-derived 4×T4 (2.5 g) treatment group was slightly lower than both commercial T4 and E. coli-derived 4×T4 during days 2-8 after treatment.However, by day 10 the keratinocyte migration rate in tobacco-derived 4×T4 (2.5 g) exceeded that of the positive controls (commercial T4 and E. coliderived 4×T4) (Figure 5(a)).
Histological examination of tissue sections collected at day 8 after application revealed that tobacco-derived 4×T4 protein promoted an increase in blood vessels in the wound bed.We observed that its angiogenic effects significantly exceeded those of both the positive (E.coliderived 4×T4 and commercial T4 protein) and negative controls (nontransformed tobacco-derived crude protein and PBS) (Figure 5(b)).

Discussion
T4, which has been described as the second most biologically active peptide in thymosin fraction 5, after thymosin 1 [1,17], is a type of actin regulating protein that forms a complex with the actin monomer in a 1 : 1 ratio.This complexation prevents polymerization and so inhibits the formation of actin filaments.Actin monomers released from the T4/actin complex, however, can drive polymerization reactions as a normal function of the cytoskeleton in cell scaffolding and motility [38,39].The sequence LKKTET of the 43-amino acid T4 protein, which is strongly conserved between all -thymosins, represents the "actin-binding motif" and is similar to the sequence of WH2 domains (Wasp Homology Domain 2, a name derived from the Wiskott-Aldrich syndrome protein) [40].Previous research has suggested that T4 may be useful for treating hard-to-heal wounds, including diabetic ulcers, bedsores and damaged corneas, and heart muscle injured by heart attacks and tumor biomarkers, as well as for curing various skin, central nervous system, and lung diseases [5,10,14,17,18,[21][22][23].Consequently, there is great interest in the use of T4 in clinical applications.
Genetically engineered T4 proteins have been produced in prokaryotic expression systems, and E. coli-derived 4×T4 proteins have been reported to promote wound healing in vivo [24].However, high production costs and difficulties in extraction and purification of the protein still limit its practical application.Plant based expression systems provide an attractive alternative as they are typically less expensive and capable of yielding high protein expression levels, as well as providing a system in which protein folding and modification are more similar to equivalent processes in humans than in prokaryotes [22, 26-28, 34, 37].In this study, we expressed recombinant 4×T4 protein in transgenic tobacco lines and observed that it was more effective in healing wounds in Blab/c mice than were either commercial or E. coli derived T4.Additionally, the tobacco-derived 4×T4 protein was more effective at increasing the number of blood vessel counts in wound beds than were the other T4 proteins tested (Figure 5(b)).Another prominent feature of the tobaccoderived 4×T4 is that it can be applied directly to the wound, while 4×T4 derived from E. coli expression systems requires extraction and purification prior to any clinical applications.We propose that plant-derived 4×T4 may be more effective when treating acute injuries, such as burns, diabetic complications, bedsores, and corneal transplantations, than T4 derived from other expression systems.Much of the cost associated with plant expression systems comes from extraction and purification of the target protein, which is the major factor limiting the development of plant expression systems to produce therapeutic proteins.In the present study, we used plant-derived 4×T4 to heal wounds in Blab/c mice via a direct application approach and showed that it was more efficient than both standard T4 (commercial) and E. coli-derived 4×T4 in promoting cell proliferation and wound healing in mice.

Conclusions
A 168 bp T4 gene was designed and synthesized according to tobacco codon usage, and a fused gene, comprising 4×T4 and a polyhistidine tag, was overexpressed in tobacco.Fourteen positive tobacco lines were obtained via Agrobacteriummediated transformation.The successful expression of the T4 protein in transgenic tobacco lines was confirmed by Western blot and ELISA analyses, and 4×T4 protein concentrations as high as 3 g/g of fresh weight were detected in the transgenic tobacco leaves.The tobacco-derived 4×T4 protein was more effective than either T4 derived from E. coli or the chemically synthesized form at promoting splenic lymphocyte proliferation and wound healing when applied directly to the wounds of mice.This research lays the foundation for the development of therapeutic proteins using plant expression systems, particularly in the context of direct delivery administration methods.

Figure 3 :
Figure 3: The differences in relative expression levels between the transgenic tobacco lines.Bars represent the expression levels of different transformed tobacco lines ( = 3).