Engineering Success
As the largest organ in the human body, the skin is the first line of defense against the invasion of external pathogens. However, scald, burn and other accidents often lead to skin damage, large skin defects caused by trauma, resulting in partial or complete loss of skin function, to patients bring unbearable and lasting physical and mental pain and economic pressure. In the past decades, research has focused on the bio-inspired material spider silk in the use of medical applications. There have been many reports on the research of spider silk in skin injury repair. Due to its non-immunogenicity, easy degradation into non-toxic by-products, and strong toughness of spider silk, it has been applied to films, fibers and hydrogels. PySp1 is one pyriform silk-cement for joints and attachments. A repetitive region (R) of PySp1 also shows good mechanical properties and thermal stability, which is a potential medical material (Perry D J. et al, 2010; Tuo Yi, 2019). For promoting wound healing, we chose to use the epidermal growth factor (EGF). EGF is a single polypeptide which is involved in the regulation of cell proliferation. We constructed a fusion vector of EGF-R, and transformed it in E. coli BL21(DE3) to express protein, and the fusion protein shows potential for repairing skin damage.
Design
We designed a prokaryotic expression system to express EGF-R fusion protein. The composition part (BBa_K4865002) consist of PelB (BBa_K4223000, directs the protein of interest to the E. coli expression into periplasm), EGF (BBa_K4865000), and R (BBa_K4865001) (Fig. 1A).
In order to improve the prokaryotic expression efficiency, we optimized the codon of R, and the DNA sequence was synthesized by GenScript (Nanjing GenScript Biotechnology Co., Ltd.). We obtained PelB-EGF sequence information and the plasmids DNA from Professor Tan's lab. Firstly, we linearized the EGF plasmid using a single enzyme digestion method, and designed primers with homologous arms to obtain the R gene using PCR. Then, we connected the R gene to the linearized EGF plasmid using seamless cloning method (Fig. 1B). Finally, we successfully obtained the PelB-EGF-R_pET-22b plasmid (Fig. 1C) and transformed it into Escherichia coli (BL21 strain).
Fig. 1 Plasmid design. (A) Constitution of PelB-EGF-R gene circuits. (B) Fusion plasmid PelB-EGF-R construction strategy map. (C) Fusion plasmid PelB-EGF-R map (Drawing software: Snapgene).
Build
Using plasmids containing R as templates, PCR reactions were performed using upstream and downstream primers with homologous arms (Table 1).
From the results of nucleic acid electrophoresis, the target band size was 639 bp, and the band size was correct (Fig. 2A). Xho I was chosen as the cleavage site and design primers for the construction of PelB-EGF-R_pET-22b prokaryotic expression vector. The electrophoresis results shows that the plasmid was successfully linearized by restriction endonuclease (Fig. 2B). Colony PCR results (Fig. 2C), sequencing (Fig. 2D) and sequence alignment (Fig. 2E) showed that the correct R was successfully inserted into C-terminal of EGF. We constructed the prokaryotic expression system of EGF-R.
Table 1. Primers in construction expression vector.
Fig. 2 Plasmid Construction. (A) Gene fragment of interest for spider silk protein R was amplified by PCR. M: DNA Marker; 1 to 4: PCR results of positive clone; Product size: 639 bp. (B) Linearization of PelB-EGF_pET-22b plasmids. M: DNA Marker; 1: Linearized EGF vector. (C) Colony validation of clone strains of EGF-R fusion plasmids. M: DNA Marker; 1: positive control (PelB-EGF_pET-22b plasmids); 2 to 11: PCR results of positive clone. (D) Sequencing peak diagram (Drawing software: Chromas V2.6.5). (E) Sequence alignment between EGF-R and positive clone sequencing (Drawing software: DNAman V5.2.2).
Test
1. Expression and purification of EGF-R
PelB-EGF-R_pET-22b expression vector was transformed into E. coli BL21(DE3), and the protein expression was detected by SDS-PAGE and western blot (Fig. 3). 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. 3 C and D, 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. 3E). 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. 3 Expression and purification of EGF-R. (A) Picked the correct bacterial colony for culture. (B) Prepared the SDS-PAGE gel. (C) 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 ℃. (D) Western blot analysis. M2: Western blot marker; 3 to 8: Consistent with the same lane sample in Fig. 3A. (E) Purification results. M: Protein marker; PC: BSA (2 μg); 1: EGF-R (Purity: ≥90%).
2. 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 (Fig. 4). 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. 5. Obviously, the EGF significantly promoted the HDF cell growth comparing with the Control and R group. While as shown in Fig. 5D, despite the weakened proliferative capacity, EGF-R retain the mitogen function of EGF, which can promote HDF proliferation.
Fig. 4 Cell proliferation experiment in vitro. (A) Clean benches. (B) Cells were cultured in a humidified atmosphere with 5% CO 2 at 37℃. (C) Inverted microscope.
Fig. 5 Cell morphology of HDF cell under different culture conditions. Images of four groups of HDF cell after cultured 36h. (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. 6. 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. 6 Quantitative analysis of HDF cell viability.
2. Using fusion proteins to produce biomaterials
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. 7, sodium alginate hydrogels can be formed by adding different concentrations of fusion protein (0.5% and 1%) to 2%(W/V) sodium alginate. It can also be pressed into a relatively thin hydrogel form (Fig. 8).
Fig. 7 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.
Fig. 8 Pressed to thin hydrogel. (A) 2% Sodium alginate. (B) 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 (Fig. 9). A gel-like morphology is formed when the protein concentration is 7% (Fig. 10).
Fig. 9 The process of making biomaterial by formic acid. (A) and (B) Formic acid dissolves the protein. (C) Stirred the samples in an ice bath. (D) Dried naturally in a fume hood.
Fig. 10 Formic acid dissolves fusion proteins to produce biomaterials. (A) Stirred samples. (B) A jelly-like sample. (C-E) The morphology and structure of the sample were observed by inverted biological microscope (ECLIPSE TS100-F, Nikon; Magnification, 40 x)
Learn
We constructed the prokaryotic expression system of EGF-R fusion protein. The fusion protein was expressed after IPTG induced and mainly existed in the form of inclusion bodies. 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 dissolving proteins with formic acid presents the gel-like morphology and structure. However, since formic acid will break the peptide bond of proteins during the process of dissolving proteins, which may affect the function of proteins, we prefer to use sodium alginate hydrogel with EGF-R protein for further experiments in the future.
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.
Tuo Yi (2019) Research on effects of repeat modules on properties of recombinant spidroins. Master's thesis, Donghua University.