TheEscherichia coli-Derived Thymosinβ4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice

Abstract

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.

A Tobacco-Derived Thymosinβ4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice

“…After boiling for 5 min, 10 μ L of the adjusted TSP was mixed with loading buffer (120 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 3% β -mercaptoethanol, and 0.02% bromophenol blue) in equal volumes (v : v = 1 : 1) and separated via sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, electrophoretically separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (filter pore size 0.22 μ m, Bio-Red, USA) for Western blot analysis [ 24 ]. The target protein was detected via incubation of the PVDF membrane with a mouse anti-His-tag monoclonal primary antibody (1 : 5,000 dilutions) purchased from Generon Ltd. (Maidenhead, The United Kingdom) and an alkaline phosphatase- (AP-) conjugated goat anti-mouse IgG secondary antibody (1 : 2,000 dilution) (Shanghai ImmunoGen Biological Technology, Shanghai, China).…”

“…ELISA assays were carried out as previously described [ 32 ]. The 4×T β 4 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/ μ L in PBS buffer [ 24 ]. The 50 μ L serial dilutions of the E. coli -derived 4×T β 4 and the 4×T β 4 containing TSP extracted from transgenic tobacco (adjusted to 1 μ g/ μ 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 in vitro bioactivity of T β 4 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).…”

“…T β 4 therefore shows great promise for numerous clinical applications [ 17 ] and the resulting increased commercial demand relies on efficient production methods. Traditionally, T β 4 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 T β 4 protein concatemer expressed in E. coli is able to promote wound healing in mice [ 24 ].…”

“…Traditionally, T β 4 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 T β 4 protein concatemer expressed in E. coli is able to promote wound healing in mice [ 24 ]. However, for clinical applications, E. coli derived T β 4 protein must undergo a complex purification process to eliminate impurities and endotoxins, which represents a significant disadvantage of this expression system.…”