The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice

Thymosin β4 (Tβ4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tβ4 will increase. In order to develop a new approach to produce recombinant Tβ4, a 168 bp DNA (termed Tβ4) was designed based on the Tβ4 protein sequence and used to express a 4 × Tβ4 concatemer (four tandem copies of Tβ4, termed 4 × T β4) together with a histidine tag (6 × His) in E. coli (strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tβ4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropyl β-D-thiogalactoside (IPTG). The E. coli-derived 4 × Tβ4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cells in vitro and in vivo wound healing. The results demonstrate that these activities of the E. coli-derived recombinant 4 × Tβ4 were similar or even better than existing commercially obtained Tβ4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tβ4.


Introduction
Thymosin 4 (T4), a small acidic polypeptide (43-amino acid residues), was first discovered and isolated firstly from calf thymus [1], and encoding gene was mapped to the X chromosome [2].T4, which has a molecular weight of 4,982 Da and an isoelectric point of 5.1, is one of the most important thymosin in fraction 5, which participates in the regulation of thymus-dependent immune response [1].
T4 is localized in the cytoplasm, shows a high affinity for G-actin, and serves as an G-actin-sequestering protein that regulates actin polymerization.It forms a 1 : 1 complex with monomeric G actin to maintain a dynamic equilibrium between G actin and F actin, preventing polymerization into actin filaments, supplies a pool of actin monomers when the cell needs filaments, and is thus critical for rapid reorganization of the cytoskeleton [2][3][4][5][6].The amino acid residues of the T4 protein that are critical for action binding were confirmed by chemical cross-linking and shown to correspond to a hydrophobic cluster consisting of residues (M6-I9-F12) in N-terminal -helix, as well as K14 and K18 [7].
Several studies have suggested that T4 contributes to various biological functions in different pathological stages and physiological processes, including the migration of epidermal cell and collagen, as well as formation of bloodvessel in the both wound healing [8,9] and the induction of angiogenesis [3,10,11].In particular, T4 has been associated with healing of diabetic ulcers, bedsores, damaged corneas, and heart muscle injured during heart attacks [12,13].T4 also plays important roles in the inhibition of inflammation [14,15], as well as T-cell maturation, proliferation of cell and differentiation [13,16].
T4 is also present in tumor cells [2], and several research groups have found that aberrant expression of T4 is associated with metastasis [10] and invasion of tumor cells [3], as seen in colorectal carcinoma [17,18], gastrointestinal stromal tumors [2], breast cancer cells [19], melanoma, and lung tumors in mouse [10].It has also been shown that T4 promotes the growth of cancerous tumors by enhancing BioMed Research International new blood-vessel formation [12].However, other studies have resulted in contrary conclusions.For instance, the expression level of T4 is significantly lower in multiple myeloma, and it has been suggested that T4 can function to suppress the proliferation of tumor cells in myeloma development [3].Moreover, another analysis concluded that overexpression of T4 does not cause an increase in cell number in tumors in a transgenic strain of mice [12].
Although there is still considerable debate about the actions of T4 in the context of tumor biology, it was concluded that "the clinical prospects for at least two thymosin proteins are finally looking brighter since first medical experiment at UCSF (University of California, San Francisco) more then [sic] a quarter-century" [12], among which, T4 is one of the most promising prospects for clinical application; the other is thymosin 1.
In addition, T4 is a promising molecular marker or therapeutic agent to serve the diagnosis and prognosis of certain diseases [2,20], or to treat some common and frequently occurring conditions, and has been proposed to have value in the wound healing of diabetic ulcers, bedsores, and damaged corneas [9,15].
In order to meet a potential increase in the clinical demand for T4, the goal of this study was to develop a production pipeline for recombinant T4.A DNA sequence, based on the T4 coding sequences, was designed to generate a 4 × T4 concatemer (four copies of the T4 gene arranged end to end in tandem, thereafter 4 × T4), and two different bioactivities of resulting recombinant 4 × T4 expressed in Escherichia coli were evaluated.

Synthesis of T𝛽4 and Construction of the pET28a-6 × his-4
× T4 Vector.Based on the T4 amino acid sequence, a T4 gene was designed and synthesized [1], and DNA fragment of the T4 gene was subcloned into the pUC57 plasmid (termed pUC57-T4).The pUC57-T4 plasmid was digested with two combinations of restriction enzymes of Spe I/Sac I and Xba I/Sac I, and the resulting DNA was ligated into SpeI/SacI sites of pUC57-T4 to create pUC57-2×T4.The pUC57-4 × T4 containing four repeats of the T4 gene was also generated based on the isocaudamer property of Spe I and Xba I.

Confirmation of the E. coli-Derived 4 × T𝛽4 Production.
The BL21 cell strain harboring pET28a-6 × his-4 × T4 was cultured in a 15 mL flask with 3 mL LB broth liquid medium (pH = 7.5) containing kanamycin (100 g/mL) in a shaker (220 rpm) at 37 ∘ C for overnight.A similar culture of the BL21 cell strain transformed with pET28a was also grown and used as a negative control.Both cultures (200 L) were added to 50 mL fresh LB medium with kanamycin (100 g/mL) and cultured to the OD 600 = 0.5 in a 37 ∘ C shaker (220 rpm).Then IPTG was added to a 1 mM final concentration and incubated as above for 0 h to 8 h.
The BL21 cells from a 1 mL aliquot culture were collected by centrifugation at 8000 g for 10 min then resuspended in 10 L of the 1 × PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , PH 7.4).The cells were then mixed with 10 L of 2 × SDS/PAGE sample buffer (120 mM Tris-HCl pH6.8, 20% glycerol, 4% SDS, 3% mercaptoethanol, 0.02% bromophenol blue) and boiled in a water bath (100∘C) for 5 min.The boiled samples were centrifuged at 8000 g for 5 min at 4 ∘ C, and 7 L supernatant from each sample was loaded a SDS-12% polyacrylamide gels and then subjected to electrophoresis in 1×Tris-Glycine Buffer (0.025 M Tris, 0.25 M glycine, 0.1% (w/v) SDS) at 100 volt at 4 ∘ C for 2 h.Duplicate gels were stained with 1% Coomassie Brilliant Blue (R250), another one, or electrotransferred to 0.22 m PVDF (Polyvinylidene Fluoride, Bio-Rad, USA) membrane using a Bio-Rad transfer equipment (35 volt at 4 ∘ C) for overnight for Western blotting analysis.A mouse anti-His-tag monoclonal antibody (Shanghai ImmunoGen Biological Technology, Shanghai, China) and an AP-conjugated goat anti-mouse antibody (Shanghai ImmunoGen Biological Technology, Shanghai, China) were, respectively, used as primary and second antibodies, respectively, to detect the expression of the recombinant 4 × T4 protein.

In Vitro 4 × T𝛽4 Stimulates Cell Proliferation.
The cells harboring pET28a-6 × his-4 × T4 were collected by centrifugation after IPTG (1 mM) induction at 37 ∘ C for 6 h, and the cells pellet was resuspended in PBS buffer (v : v = 1 : 5) and then sonicated at 200-300 W in an ice bath 6 times (pause/sonication = 10 s/10 s per time).The sonicated crude lysate was centrifuged under 4 ∘ C at 10 000 g for 30 min and the supernatant was discarded.The precipitated cells pellet (per gramme) was resuspended in 5 mL buffer B (8 M urea, 0.1 M sodium phosphate buffer, 0.01 M Tris-Cl, pH 8.0) and shaken gently at room temperature for approximately 30 min until the solution became translucent.The mixture was then mixed with Ni-NTA resin (Qiagen Gmbh, Germany) and loaded into a column.The flow-through from the column was collected.Nonbound proteins were collected by passing 20 mL buffer C (8 M urea, 0.1 M sodium phosphate buffer, 0.01 M Tris-Cl, pH 6.3), and the proteins adsorbed with Ni-NTA resin were eluted with 5 mL buffer E (8 M urea, 0.1 M sodium phosphate buffer, 0.01 M Tris-Cl, pH 4.5).
A 100 L aliquot of the diluted spleen cells was added to each well of a 96-well plate.Samples of the E. coli-derived 4 × T4 and commercially obtained T4 protein (GL Biochem, Shanghai, China) were diluted to 250 ng/L in 1 × PBS buffer, and a 100 L of the diluted 4 × T4 was added to each well (six replications per treatment).Aliquots (100 L) of the diluted commercial T4 protein and 1 × PBS buffer were used as positive and negative controls, respectively.The 96-well plate was incubated in a cell culture incubator at 5% CO 2 at 37 ∘ C for 24 h.After incubation, 10 L of MTT reagents was added into each well.Then the plate was then returned to the cell culture incubator for an additional 4 h.The cells in the 96-well plate were periodically examined with a CKX31 inverted microscope (Olympus, Watford, Herts, UK).When a purple precipitate was clearly visible, 100 L of dimethyl sulfoxide (DMSO) was added and the plate was then gently swirled and placed in the dark at room temperature for 15 min.The absorbance was read at 570 nm with a Microplate Reader (Bio-Tek, Power Wave XS, USA).The mouse splenic lymphocyte proliferation rate was calculated according to formula: proliferation rate = (OD test − OD control )/OD test .
In the first experimental group of three mice, keratinocyte migration was determined by measuring the lengths of the epidermal tongues from wound edges with a vernier caliper (Endura-Greenlee Tools, E0531, Shanghai, China) from 2 to 10 days after application treatment, and the percentage wound of closure was calculated by formula: percentage wound closure = distance of migrated keratinocytes from the wound edge/total wound width × 100% [23].
A second experiment group of three mice was used to examine reepithelialization and vessel counts (angiogenesis) in the wound bed.The mice were euthanized on day 8 after wounding, and the skin tissues were taken from wounds, fixed in 4% (v/v) formalin buffer, and then embedded in paraffin after dehydration in series of increasing ethanol concentrations (75%, 85%, 95%, and 100%).The embedded skin tissue samples were sliced into 5 m thick sections using a microtome (Leica, RM2200), and the sections from middle of the wounds were mounted on glass slides and then were stained with hematoxylin and eosin (Shanghai Dingjie Biotechnology Company, Shanghai, China) after paraffin removal, and the samples were evaluated using a microscope (Olympus BX51, Japan).
Histological sections were used to measure the vessel counts as previously described by Malinda et al. [22].Vessel counts were determined by first identifying vascular spaces by their endothelial lining.All such vessels in the wound bed were counted, including those at the junction of the dermis and the subcutis, since angiogenesis in the wounds occurs to a great extent from these vessels.The vessel counts were taken from mean value of 10 view fields (40×) per treatment.Statistical analysis pf the data was performed using Statistical Product and Service Solutions (SPSS) with a  value <0.05 [24].All the above analyses were performed using double blind trials.

Synthesis of T𝛽4 Gene and Construction of pET28a-6 × his-4 × T𝛽4.
According to amino acid sequence of T4 protein, a 168 bp of T4 gene was designed and synthesized, which includes restriction enzyme sites (underlined), protection bases, and initiation codon (ATG) and the termination codon (TAA) (bold), as shown below: The 4 × T4 was created using isocaudamer property of Xba I and Spe I, and a short DNA sequence (18 base pair) encoding six histidines was introduced at 5  -end of the 4 × T4 sequence by PCR, yielding a DNA sequence of the 6 × his-4 × T4 (619 bp).The DNA fragment was subsequently inserted into the Bam H I and Sac I sites in the pET28a plasmid to generate pET28a-6 × his-4 × T4 (Figure 1).

Induced Expression of 4 × T𝛽4 Protein.
To confirm the induced expression of 4 × T4 protein in E. coli, rude proteins extracted from BL 21 cell pellets were separated by SDS/PAGE gels, and the expected 30.87 kDa recombinant 4 × T4 protein was monitored following IPTG induction for 0 h to 8 h in BL 21 harboring pET28a-6 × his-4 × T4 the expected band was detected at all time increments other than 0 h (Figure 2(a)).In order to further confirm the presence of 4 × T4 protein, the mouse anti-His-tag was used as a primary antibody in a Western blot analysis of the protein extracts.The expected hybridization signal at 30.87 kDa was detected at different induction times from 0 h to 8 h in BL 21 harboring pET28a-6 × his-4 × T4; however the signal was much weaker in 0 h sample.As expected, no immunoreactive band corresponding 4 × T4 was detected in the negative control BL21 samples form cultures transformed with pET28a (Figure 2(b)).the E. coli-derived 4 × T4 protein was purified by the Ni-NTA resin and examined by SDS-PAGE gel.The results showed that both unbound and nonspecific bound proteins were eluted from the column using 1×Ni-NTA buffer B and C, respectively; however the 4 × T4 protein with 6 × his fusion was successfully obtained by elution of bound proteins from the Ni-NTA resin (Figure 3(a)).Both E. coli-derived 4 × T4 proteins and commercial T4 were subsequently diluted to 250 ng/L in 1×PBS buffer and then used to in a MTT assay.The MTT results showed that the E. coli-derived 4 × T4 promotes mice lymphocyte proliferation (18.13 ± 3.65%), to a significantly higher degree than the commercial T4 (8.49 ± 3.32%) (Figure 3(b),  < 0.01).

In Vivo 4 × T𝛽4 Promotes Wound
Healing.The full thickness cutaneous mouse wound model was employed to examine the efficiency of the E. coli-derived 4 × T4 in wound healing.Keratinocyte migration was determined by measuring the lengths of the epidermal tongues from the wound edges.The results showed that reepithelialization is increased in all treatments and that rate was higher following treatment with E. coli-derived-4×T4 than that with both positive control (commercial T4) and negative control (0.9% physiological saline).However, the difference is insignificant between treatments from 2 days to 6 days after application treatment.Keratinocyte migration was significantly higher following treatment with the E. coli-derived 4 × T4 than the negative control from day 8, and the percentage of wound closure reached 76.72±5.54%.On day 10, the closure was significantly higher ( < 0.01) than the negative control (54.05 ± 4.55%) (Figure 4, see Supplementary Figure 1 available online at http://dx.doi.org/10.1155/2013/241721);however, there was no significant difference between treatments with the E. coliderived 4 × T4 and commercial T4.The E. coli-derived 4 × T4 also promoted an increase in the number of blood vessels in wound bed, as determined by observing tissue sections on day 8 after wounding.The blood vessel number (10.33±0.58vessel number/9 × 10 4 m 2 ,  < 0.01) was significantly greater than that of the negative control (5.00 ± 1.00 vessel number/9 × 10 4 m 2 ); however, there was no significant difference between the E. coli-derived 4 × T4 and the commercial T4 (10.00 ± 1.00 vessel number/9 × 10 4 m 2 ) samples (Figure 5).

Discussion
The T4 protein has been proposed as one of the two most promising thymosins (the another being thymosin 1) for future clinical applications [12].Several research groups have suggested that T4 protein has the potential to cure or prevent many diseases associated with the immune system and can be used to treat inflammation [14,15] and tumors [2,3] and promote wound healing [8].The number of patients with diseases such as diabetes is gradually rising in conjunction with an aging population and suboptimal life style often resulted in diabetic ulcers and bedsores.T4 protein is therefore likely to have increasing importance as a therapeutic agent, which in turn suggests that there will be an increasing demand for T4 production.Currently, the T4 is produced commercially by two approaches: extraction from animal thymus [1] and chemical synthesis [25], but there are challenges with meeting clinic demand due to risks of zoonosis and higher production costs [25,26].
We therefore sought to produce T4 protein via a genetic engineering approach, and a gene encoding T4 was designed and synthesized.To enhance expression and facilitate purification, a recombinant T4 concatemer protein fused to a histidine tag was expressed in and purified from E. coli cultures.
Several studies have revealed that T4 functions by binding to G-actin [3][4][5][6], and the bioactivity of T4 protein was related to its structure and amino acid components [7,27].As of yet, we have no evidence that the E. coli-derived 4 × T4 has similar binding characteristics equal to the commercial T4, which is generated by chemosynthesis or extraction of animal thymuses.However, the results showed that the E. coli-4 × T4 protein promotes cell proliferation in vitro (Figure 3(b)), as well as wound healing and vessel increase in vivo (Figures 4 and 5), and its capabilities in these regards are at least similar, or even better than commercial T4.Further research focuses on producing T4 using other expression systems including yeast and plant [28][29][30][31][32].

Figure 5 :
Figure 5: Histological sections of reepithelialization and angiogenesis at day 8 after wounding.Arrows indicate the formation of blood vessel in the wound bed.(a) The newly formed blood vessels and reepithelialization of the wound epidermis in wound location by topical treatment with 5 g 4 × T4 (dissolved in 50 L of the 0.9% physiological saline).(b) The newly formed blood vessels and reepithelialization of the wound epidermis in the wound location by topical treatment with 5 g T4 (dissolved in 50 L of the 0.9% physiological saline).(c) Treatment with 0.9% physiological saline.(d) Normal mouse skin tissues (bar = 100 m).