The Escherichia coli-derived thymosin β4 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 × Tβ 4 concatemer (four tandem copies of Tβ4, termed 4 × Tβ4) 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.

Figure 1

Diagramme of pET28a-6 × his-4 × Tβ4. lac I, lac repressor coding gene; Kan: kanamycin coding gene; 6 × his, a DNA sequence encoding six histidines; 4 × Tβ4, four copies of a DNA sequence which encode Tβ4 arranged in tandem; origin, DNA sequence of pBR322 origin of replication; f1 origin, DNA sequence of phage f1 origin of replication.

Figure 2

(a) Expression of recombinant 4 × Tβ4 in BL21 cells at different induction times. Lane M, middle-molecular mass protein markers; lanes from 1 to 5: induced expression of the recombinant 4 × Tβ4 by IPTG (1 mM) at 0, 2, 4, 6, and 8 hs, respectively; lane 6: induced BL21 containing pET28a by IPTG (1 mM) for 6 hs. Red arrows indicate specific band of E. coli-derived 6 × his-×Tβ4. (b) Western blot analysis of the E. coli-derived 6 × his-4 × Tβ4. Lane M: middle-molecular-mass protein markers; lanes from 1 to 5: BL21 harboring pET28a-6 × his-4 × Tβ4 after IPTG (1 mM) induction at 37°C for 0, 2, 4, 6, and 8 h, respectively; lane 6: induction of expression of the BL21 containing pET28a by IPTG (1 mM) at 37°C for 6 h. Red arrows indicate the specific hybridization signal for the E. coli-derived 6 × his-×Tβ4.

Figure 3

(a) Purification of the E. coli-derived 4 × Tβ4. Lane M: middle molecular mass protein markers; lane 1: unbound proteins were eluted using 1×Ni-NTA buffer B; lane 2: nonspecific proteins were eluted using 1 × Ni-NTA buffer C; lanes from 3 to 9: 4 × Tβ4 was eluted using 1 × Ni-NTA buffer E. Red arrows indicate the specific band of E. coli-derived 4 × Tβ4 on SDS/PAGE gel. (b) Assay of mice splenic lymphocyte proliferation using MTT method. 4 × Tβ4: purified 4 × Tβ4 protein extracted from BL21 (E. coli) stimulate, mice splenic lymphocyte proliferation; Tβ4: commercial Tβ4 protein (GL Biochem) stimulates mice splenic lymphocyte proliferation.

Figure 4

Change of keratinocyte migration in wound bed at different times after application treatment. Tβ4: wounds of the applied standard Tβ4 purchased from GL Biochem; 4 × Tβ4 (BL21): wounds of the applied E. coli-derived 4 × Tβ4; NaCl: wounds of the applied 0.9% physiological saline; 2 d to 10 d: days after application.

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 μg 4 × Tβ4 (dissolved in 50 μL 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 μg Tβ4 (dissolved in 50 μL of the 0.9% physiological saline). (c) Treatment with 0.9% physiological saline. (d) Normal mouse skin tissues (bar = 100 μm).