PelB-EGF-R

This part generates epidermal growth factor (EGF). And it is a small protein involved in epidermal cell growth, oncogenesis, and wound healing. R is the DNA sequence of a repetitive region of Pyriform Spidroin1 (PySp1) from the piriform glandular filament of Arachnid. The function of the repeat area is mainly related to the mechanical properties of spider silk fiber. In the N-terminal is a pelB signal sequence to send the protein to periplasmic space on and then be cleaved the 5' end this also allows for potential increased stability of the folded protein.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 54
Illegal AgeI site found at 328
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 208

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

[1].Perry D J, Bittencourt D, Siltberg-liberles J, et al. (2010) Piriform spider silk sequences reveal unique repetitive elements. Biomacromolecules, 11(11): 3000-3006.

[2].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

[3].Tuo Yi (2019) Research on effects of repeat modules on properties of recombinant spidroins. Master's thesis, Donghua University.

Contribution From OTIA-Hangzhou 2024

Overview

This year, our team has significantly improved the performance of OTIA-Hangzhou 2023 hydrogel made of sodium alginate as a raw material, increasing its antioxidant properties and enhancing its ability to remove excess ROS from skin wounds when used as a wound dressing. This hydrogel further promotes wound healing and has been found to provide some meaningful data and conclusions in the process. The hydrogels were prepared using sodium alginate, neoagaroligosaccharides(NAOS), agarose, and enzymes, and their antioxidant properties were tested extensively. The resulting hydrogels are now more effective at removing harmful free radicals from wounds, reducing inflammation and promoting faster healing times.

Experience and Results

Hydrogels made with NAOS added to sodium alginate or AgaA added to agagose have excellent antioxidant properties.

Obtain plasmid

We obtained the EGF-R_pET-21a plasmid from OTIA-Hangzhou 2023 team. The plasmid was transferred to receptive E. coli (Rosetta) by heat shock method. Fig. 1A shows the growth of the expression strain after transformation. There were obvious single colonies in LB solid medium, and appropriate amount of single colonies could be selected to further verify whether the plasmid was successfully transferred to the expression strain. The selected single bacterial colonies were cultured under appropriate conditions (37℃, 220 rpm) for about 4 h, and then the cultured strains were used as templates for PCR verification. The verification results were shown in Fig. 1B. The eight selected single bacterial colonies all showed the same bands as the positive control, indicating that the plasmid was successfully transferred to the expressing strain. One of these strains can be selected for culture to harvest the target protein EGF-R.

Fig. 1 Plasmid transformation. (A) E. coli Rosetta Transformation Results. (B) E. coli Rosetta PCR Results. M: DNA marker; 1: positive control; 2 to 9: PCR results of positive clones.

EGF-R protein induced expression

4 mL of overnight cultured seed solution (E. coli Rosetta EGF-R_pET-21a) was added to 200 mL of LB liquid medium containing ampicillin and cultured at 37℃ and 200 rpm until OD600 was about 0.5. 160 µL of 1 M IPTG (final concentration: 0.8 mM) was added and continued to be cultured at 37℃ and 200 rpm for 18 h. The cultured strains were collected and crushed by ultrasound, and the soluble proteins and inclusion bodies were collected after centrifugation. 10% SDS-PAGE (Fig. 2) showed that EGF-R protein was successfully expressed and mainly existed in the form of inclusion bodies. Collecting the crude enzyme solution of EGF-R could prepare for the next step of making hydrogels.

Fig. 2: The induced expression of protein EGF-R. M: Protein marker; 1: Before IPTG induction; 2: The supernatant from ultrasonication after IPTG induction; 3: The precipitate from ultrasonication after IPTG induction.

To make a hydrogel

Weigh 0.2 g of sodium alginate and agarose and 0.5 g of CaCl2, respectively, into 15 mL centrifuge tubes, add an appropriate amount of 20 mM of Tris HCl (pH 7.0) solution in turn, and fully dissolve (agarose solution needs to be heated at high temperature to dissolve), then set the volume to 10 mL respectively. Sodium alginate solution and calcium chloride solution can be temporarily stored at room temperature, agarose solution needs to be kept in a 55℃ water bath to prevent early solidification. Different types of hydrogels are prepared according to the following systems: Group 1: 200 µL agarose +300 µL Tris HCl buffer Group 2: 200 µL agarose +90 µL AgaA+210 µL Tris HCl buffer Group 3: 200 µL sodium alginate +200 µL NAOS+10 µL CaCl2+90 µL EGF-R Group 4: 200 µL sodium alginate +90 µL EGF-R+10 µL CaCl2+200 µL Tris HCl buffer The prepared reaction system was mixed and incubated at room temperature for 30 minutes to form different kinds of hydrogels (Fig. 3).

Fig. 3: Different types of hydrogels

Oxidation resistance test

1/10 of each hydrogel was purified and placed in 2 mL EP tube (the control group was 50 µL Tris HCl buffer), and 950 µL antioxidant detection reagent (ABTS detection kit, Shanghai) was added successively. After incubation at room temperature for 20 minutes, compared with the control group, experimental groups have obvious color fading reaction (Fig. 4), which can be tested for antioxidant capacity.

Fig. 4: The antioxidant properties of different hydrogels were detected by ABTS method

200 µL was taken from each group and added to the 96-well plate, and the absorbance value of each group of samples at the wavelength of 734 nm was detected by enzyme-labeled instrument(Fig. 5), and the results were shown in Tab. 1.

Fig. 5: The antioxidant capacity of different hydrogels was detected by enzymolysis

The ABTS removal capacity of each hydrogel group can be calculated by the following formula:

ABTS free radical clearance rate (%) = (Control group A - Experimental group A) ÷ Control group A × 100%

The calculation results(Fig. 6) show that the hydrogel of OTIA-Hangzhou 2023 team has almost no antioxidant capacity, but after adding NAOS, a product of our project, the ABTS free radical clearance rate has increased by 31.17%. In addition, the hydrogel prepared directly from agarose was also increased to 10%, and with the addition of AgaA, it was increased to 28%, possibly due to AgaA hydrolyzing the NAOS produced by agarose. The experiment can further improve the performance of hydrogels.

Tab.1 Absorption values of different reaction groups at wavelength 734 nm

Fig. 6: ABTS free radical scavenging efficiency

Part:BBa_K4865002