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Applications of BBa_K4865002

Introduction

Our aim is to produce a biomaterial that can reduce the complications of skin damage during recovery and support accelerated wound healing. We constructed a composite part, PelB-EGF-R (BBa_K4865002), to achieve this goal. For promoting wound healing, we chose to use the epidermal growth factor, EGF, a single polypeptide which is involved in the regulation of cell proliferation. R is a repetitive region of a spider silk protein gene. And it shows a good mechanical properties and thermal stability, which is a potential medical material (Perry D J. et al, 2010; Tuo Yi, 2019). The N-terminal of this composite part is a secretion signal, pelB, which directs synthesized polypeptides to the E. coli periplasm (Yoon S.H. et al., 2010).

Design

We designed a prokaryotic expression system to express EGF-R fusion protein. The composition part (BBa_K4865002) consist of PelB (BBa_K4223000), EGF (BBa_K4865000), and R (BBa_K4865001) (Fig. 1A). We constructed the PelB-EGF-R_pET-22b prokaryotic expression vector using seamless cloning technique. The recombinant expression vector was transformed into E. coli BL21 competent recipient cells and used for subsequent protein expression.

Fig. 1 Constitution of PelB-EGF-R gene circuits.

Characterization

Protein expression

The protein expression was detected by SDS-PAGE and western blot (Fig. 2). We induced the expression of the protein under two conditions: cultured with 0.5mM IPTG at 15℃ for 16h and with 0.5mM IPTG at 37°C for 4h. As shown in the Fig. 2 A and B, the fusion protein (30 kDa) was successfully expressed and mainly existed in the form of inclusion bodies under both conditions. Then we used 1L bacterial solution to express the protein, and the His-tagged fusion proteins were purified by Ni-NTA resin (Fig. 2C). The concentration of purified EGF-R fusion protein was determined by Bradford, and finally the recombinant protein with mass concentration of 0.74 mg/mL and total amount of 3.7 mg was obtained for subsequent cell proliferation experiments.

Fig. 2 Expression and purification of EGF-R. (A) SDS-PAGE analysis. M1: Protein marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Cell lysate without IPTG induction; 1: Cell lysate with induction for 16 h at 15 ℃; 2: Cell lysate with induction for 4 h at 37 ℃; NC1: Periplasmic space of cell without induction; 3: Periplasmic space of cell with induction for 16 h at 15 ℃; 4: Periplasmic space of cell with induction for 4 h at 37 ℃; NC2: Supernatant of cell lysate without induction; 5: Supernatant of cell lysate with induction for 16 h at 15 ℃; 6: Supernatant of cell lysate with induction for 4 h at 37 ℃; NC3: Pellet of cell lysate without induction; 7: pellet of cell lysate with induction for 16h at 15 ℃; 8: Pellet of cell lysate with induction for 4 h at 37 ℃. (B) Western blot analysis. M2: Western blot marker; 3 to 8: Consistent with the same lane sample in Fig. 3A. (C) Purification results. M: Protein marker; PC: BSA (2 μg); 1: EGF-R (Purity: ≥90%).

EGF-R promotes cell proliferation

In order to characterize the function of EGF-R protein, the in vitro cell proliferation experiments were performed. In detail, human skin cell line HDF were selected and cultured with 5% FBS DMEM at 37℃ in a humidified chamber with 5% CO₂. The cultured cells were divided into four groups (Control, R, EGF, EGF-R) with different proteins added. Cell morphology under different culture conditions is shown in the Fig. 3. Obviously, the EGF significantly promoted the HDF cell growth comparing with the Control and R group. While as shown in Fig. 3D, despite the weakened proliferative capacity, EGF-R retain the mitogen function of EGF, which can promote HDF proliferation.

Fig. 3 Cell morphology of HDF cell under different culture conditions. Images of four groups of HDF cell after cultured 36 h. (A) Control group. (B) R group. (C) EGF group. (D) EGF-R fusion protein group. (Magnification, 10 x) For quantitative analysis, Cell Counting Kit-8 (CCK-8) assay was performed. CCK-8 were added to the cells in 96-well plate, and incubated at 37℃ for 1 hour. Subsequently, the absorbance of the culture medium was measured at a single wavelength of 450 nm using microplate reader to determine their growth rate. The OD450 value were calculated are presented in Fig. 4. The spider silk protein R had a minor impact on HDF cell viability. However, comparing with the Control group, the addition of fusion protein EGF-R can significantly enhance the HDF cell growth as the EGF did.

Fig. 4 Quantitative analysis of HDF cell viability.

Fusion protein can be used to make biomaterial

To further validate the potential of producing fusion protein dressings for treating skin injuries, we added the EGF-R protein to sodium alginate and made the form of hydrogel to prepare for subsequent tests. As shown in Fig. 5, sodium alginate hydrogels can be formed by adding different concentrations of fusion protein (0.5% and 1%) to 2%(W/V) sodium alginate.

Fig. 5 Mixed hydrogel of Sodium alginate and Protein EGF-R. (A) 2% Sodium alginate. (B) 2% Sodium alginate+0.5% protein EGF-R. (C) 2% Sodium alginate+1% protein EGF-R.

We also tried to form the fusion protein directly, different masses of freeze-dried fusion protein (0.024 g, 0.100 g, 0.1677 g) were added to 2 mL of formic acid solution to configure different mass fractions of the protein solution (1%, 4%, 7%), stirring at 300 r/min for 2h. The dissolved protein solution was dried naturally in a fume hood for 12h. A gel-like morphology is formed when the protein concentration is 7% (Fig. 6).

Fig. 6 Formic acid dissolves fusion proteins to produce biomaterials. (A) Stirred samples. (B) A jelly-like simple. (C-E) The morphology and structure of the sample were observed by inverted biological microscope (ECLIPSE TS100-F, Nikon; Magnification, 40 x)

Conclusion

The above results indicate that we have successfully constructed the prokaryotic expression system of PETase-R protein and obtained PETase-R protein by IPTG inducing. In addition, we tested the EGF-R cell proliferation function in vitro, and the results showed that the EGF-R can significantly enhance the HDF cell growth as the EGF did. The biological material produced by using sodium alginate to make hydrogels or dissolving proteins with formic acid presents the gel-like morphology and structure.

Reference

Perry D J, Bittencourt D, Siltberg-liberles J, et al. (2010) Piriform spider silk sequences reveal unique repetitive elements. Biomacromolecules, 11(11): 3000-3006. S.H. Yoon, S.K. Kim, J.F. Kim (2010) Secretary production of recombinant proteins in Escherichia coli. Recent Pat. Biotechnol., 4: pp. 23-29 Tuo Yi (2019) Research on effects of repeat modules on properties of recombinant spidroins. Master's thesis, Donghua University.

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