Expression and Biological Activity Analysis of Recombinant Fibronectin3 Protein in Bacillus subtilis

Abstract

Fibronectin (FN), a primary component of the extracellular matrix (ECM), features multiple structural domains closely linked to various cellular behaviors, including migration, spreading, adhesion, and proliferation. The FN3 domain, which contains the RGD sequence, is critical in tissue repair because it enables interaction with integrin receptors on the cell surface. However, the large molecular weight of wild-type FN presents challenges for its large-scale production through heterologous expression. Therefore, this study focused on cloning the FN3 functional domain of full-length FN for expression and validation. This study selected Bacillus subtilis as the expression host due to its prominent advantages, including efficient protein secretion, absence of endotoxins, and minimal codon bias. The recombinant vector pHT43-FN3 was successfully constructed through homologous recombination technology and transformed into Bacillus subtilis WB800N. The FN3 protein was successfully expressed after induction with IPTG. Following purification of the recombinant FN protein using a His-tag nickel column, SDS-PAGE analysis showed that the molecular weight of FN3 was approximately 27.3 kDa. Western blot analysis confirmed the correct expression of FN3, and the BCA protein assay kit determined a protein yield of 5.4 mg/L. CCK8 testing demonstrated the good biocompatibility of FN3. In vitro cell experiments showed that FN3 significantly promoted cell migration at a 20 μg/mL concentration and enhanced cell adhesion at 10 μg/mL. In summary, this study successfully utilized Bacillus subtilis to express the FN3 functional domain peptide from FN protein and has validated its ability to promote cell migration and adhesion. These findings not only provide a strategy for the expression of FN protein in B. subtilis, but also establish an experimental foundation for the potential application of FN3 protein in tissue repair fields such as cutaneous wound healing and cartilage regeneration.

Keywords: Bacillus subtilis; cell adhesion; cell migration; recombinant fibronectin3 protein.

Figure 1

Construction process and PCR verification of pHT43-FN3. (A) Preparation of the recombinant plasmid pHT43-FN3. (B) PCR amplification of the FN3 fragment. (C) Single-enzyme digestion of the pHT43 vector. (D) Colony PCR fragments of the pHT43-FN3 recombinant strain.

Figure 2

Bioinformatics prediction of FN3 and optimization of fermentation conditions. (A) Functional domain prediction of recombinant FN3. (B) Structural prediction of recombinant FN3. (C) SDS-PAGE electrophoresis of pellet and supernatant from recombinant B. subtilis before and after induction. M: protein marker; 1: bacterial culture before induction; 2: bacterial cells after induction; 3–5: fermentation supernatants at 28 °C with IPTG induction concentrations of 0.1, 0.5, and 1 mM in sequence; 6–8: fermentation supernatants at 37 °C with IPTG induction concentrations of 0.1, 0.5, and 1 mM in sequence.

Figure 3

SDS-PAGE electrophoretogram of recombinant FN3 after ammonium sulfate precipitation and purification. (A) The fermentation supernatant was subjected to ammonium sulfate precipitation. M: protein marker; lanes 1–6: fermentation supernatants precipitated with 20%, 30%, 40%, 50%, 60%, and 70% ammonium sulfate, respectively. (B) The target protein was purified by a His-tag nickel column. M: protein marker; 1: flow-through fraction; 2–4: the 1st–3rd tubes of the eluent containing impurity proteins eluted with 3 mM imidazole in sequence; 5–12: The 1st–8th tubes of the eluent containing the target protein eluted with 50 mM imidazole in sequence.

Figure 4

Western blot of recombinant FN3 protein. 1: empty vector negative control; 2–4: purified FN3 protein.

Figure 5

FN3 induces concentration-dependent effects on the viability of L-929 cells. ns p > 0.05 denotes a non-significant difference between the experimental and control groups, n = 6.

Figure 6

Effect of FN3 on the migration of L-929 cells. (A) The migration area of L-929 cells at different time points (×200). (B) The relative quantitative assessment of the migration area in L-929. * p < 0.05 shows a significant difference between the experimental and control groups; ** p < 0.01, *** p < 0.001, **** p <0.0001 show a highly significant difference between the experimental and control groups, n = 3.

Figure 7

Detection of the adhesion effect of recombinant FN protein on L-929 cells by crystal violet staining. (A) cell images stained with crystal violet (×200). (B) Quantification of adhered cells. *** p < 0.001, **** p < 0.0001 indicate a highly significant difference between the experimental and control groups, n = 3.