Difference between revisions of "Part:BBa K5374025"

Latest revision as of 12:16, 24 September 2024

FTD-BMP-4 (Fibrinogen-like Domain fused with Bone Morphogenetic Protein-4). A fusion protein combini

The Fibrinogen-like Domain (FTD) provides strong collagen-binding properties, enabling the fusion protein to localize in collagen-rich environments. BMP-4 is a key growth factor involved in the differentiation of mesenchymal stem cells into osteoblasts, thus promoting bone formation and regeneration. By fusing BMP-4 with the FTD, the protein can be directed specifically to collagen-based scaffolds, ensuring a localized and sustained release of BMP-4. This is particularly useful in bone repair applications, where BMP-4 needs to be concentrated at the site of injury to stimulate effective bone regeneration.

FTD-BMP-4 proved more challenging. The presence of multiple disulfide bonds in BMP-4 led to the formation of inclusion bodies when expressed in BL21, which made the protein insoluble and inactive. To overcome this, we explored various optimized bacterial strains designed to facilitate proper folding of disulfide bond-containing proteins.

Several strains were tested:

• E. coli Origami (DE3) improved disulfide bond formation but still resulted in considerable protein aggregation.
• E. coli Shuffle T7-B enhanced soluble expression, but its slow growth rate limited its practicality for large-scale production.
• Finally, ArcticExpress (DE3) pRARE was selected as the most suitable expression host, balancing both high protein yield and improved solubility of FTD-BMP-4.

The culmination of this stage was obtaining both fusion proteins in their active, soluble forms, laying the foundation for further testing of their controlled release and biological activity in subsequent phases. In Fig3.2, lanes 1 to 4, 5 to 8, and 9 to 12 are whole bacteria, supernatant after disruption, and precipitate after disruption, respectively. Each group consists of four lanes, and the order within each group is BL21, ArcticExpress (DE3) pRARE, Shuffle T7-B, Origami (DE3)

Fig1 Results of expression system optimization a) SDS-PAGE; b) semi-quantitative analysis (Lanes 1 to 4, 5 to 8, and 9 to 12 are whole bacteria, supernatant after disruption, and precipitate after disruption, respectively. Each group consists of four lanes, and the order within each group is BL21, ArcticExpress (DE3) pRARE, Shuffle T7-B, Origami (DE3))

According to Fig1, ArcticExpress (DE3) pRARE stands out for its high proportion of soluble protein expression.The reasons for ArcticExpress's success in producing a high level of soluble protein are as follows:

Chaperone Support:
ArcticExpress is engineered to co-express two cold-adapted chaperones, Cpn60 and Cpn10, which facilitate the proper folding of proteins, especially those with complex structures like FTD-BMP-4, which contains multiple disulfide bonds. These chaperones help reduce the formation of misfolded proteins and inclusion bodies, promoting correct folding at lower temperatures, which is ideal for proteins prone to aggregation.

pRARE Plasmid:
The pRARE plasmid in ArcticExpress provides additional tRNAs for rare codons that are underrepresented in standard E. coli strains. This allows for more efficient and accurate translation of eukaryotic or complex proteins like BMP-4, which may otherwise experience stalling during translation in standard hosts due to rare codon usage.

Cold-Adapted System:
ArcticExpress is designed to perform well at lower temperatures, which can slow down the protein synthesis process, giving more time for complex proteins with disulfide bonds to fold correctly and avoid aggregation.

In the gel (left) and the bar chart (right), ArcticExpress shows significantly improved soluble protein expression compared to other strains like BL21, which primarily produces inclusion bodies. This makes ArcticExpress the optimal choice for expressing soluble and active FTD-BMP-4, ensuring that a higher percentage of the total protein is in the desired soluble form.

Figure2 Effect of Gradual vs. Direct NaCl Reduction on FTD-BMP-4 Solubility During Dialysis

Figure2 showcases the solubility and precipitation behavior of FTD-BMP-4 during dialysis under different conditions of salt concentration reduction.

The protein FTD-BMP-4 tends to precipitate when dialyzed into low-salt conditions. However, some protein remains soluble in the supernatant, as seen in the SDS-PAGE gel. The key observations are:

Gradual Salt Reduction (20mM Tris, 150mM NaCl):

When the protein was initially dissolved in 500mM NaCl and then gradually dialyzed down to 150mM NaCl, some precipitation occurred, but most of the protein remained soluble.

This suggests that a gradual decrease in salt concentration only temporarily disrupts the protein's hydration layer, leading to reversible salting-out. Once equilibrated, a significant amount of protein stays soluble, as indicated by the prominent band in the supernatant lane (green arrow).

Direct Salt Reduction (20mM Tris, 50mM NaCl):

When the protein solution (initially at 500mM NaCl) was dialyzed directly down to 50mM NaCl, substantial precipitation was observed, with only a small amount of protein remaining in the supernatant.

This supports the hypothesis that rapid salt concentration changes cause irreversible protein denaturation and precipitation, as indicated by the smaller band in the supernatant lane (green arrow) and the visible precipitate (red arrow) in the solution.

So, this experiment highlights the importance of gradual salt reduction to avoid protein denaturation and precipitation. The sharp change in salt concentration causes irreversible aggregation, while gradual reduction merely disrupts the hydration layer temporarily, allowing the protein to remain soluble after equilibration. This insight is critical for optimizing the conditions for FTD-BMP-4’s stability during downstream processing.

We characterized the sustained-release activity and biological activity of this part.

1 sustained-release activity

Fig3 Time-Dependent Release Profiles of CBD MMPs-VEGF and FTD-BMP-4 from Collagen Hydrogel

FTD-BMP-4 exhibited a much slower and more controlled release. During the first 10 days, less than 20% of BMP-4 was released, indicating a more gradual release process. The release steadily increased over time, with the curve following a linear trend. By day 20, around 30% of BMP-4 had been released, and by day 30, approximately 50% was released. The release continued at a steady pace, reaching close to 100% around day 40. This extended release profile reflects the higher binding affinity of the FTD domain to collagen, which ensures a slower and sustained release of BMP-4 over the course of the experiment.

These contrasting release profiles demonstrate the effectiveness of using different collagen-binding domains to control the timing and rate of cell factor release, with CBD MMPs facilitating a faster release for VEGF and FTD ensuring a prolonged release for BMP-4.These results confirm that the controlled, sequential release of the two cell factors can be achieved by leveraging the different binding affinities of the CBDs, with CBD MMPs-VEGF releasing faster to initiate angiogenesis, followed by the slower release of FTD-BMP-4 to promote osteogenesis.

2 biological activity

In our osteogenic differentiation experiments, MC3T3-E1 cells, a well-established pre-osteoblast cell line derived from mouse calvaria, were used. These cells are commonly employed in bone research because they have the inherent capacity to differentiate into mature osteoblasts when cultured under osteogenic conditions. During the early stages of osteogenic differentiation, MC3T3-E1 cells express alkaline phosphatase (ALP), which is critical for mineralization. ALP staining can be used to measure the early osteogenic activity in these cells. As differentiation progresses, MC3T3-E1 cells begin to deposit calcium in the extracellular matrix, which is a key indicator of bone formation. This can be detected using Alizarin Red staining, which binds specifically to the calcium deposits and allows for the visualization of mineralization. Thus, by using both ALP staining and Alizarin Red staining, the osteogenic differentiation process of MC3T3-E1 cells can be effectively tracked from early osteoblast activity (ALP expression) to late-stage bone formation (calcium deposition).
So, ALP (Alkaline Phosphatase) Staining and Alizarin Red Staining are two key methods used to assess different stages of osteogenic differentiation in cells, providing insights into the bone formation process.

ALP Staining Mechanism:
ALP is an early marker of osteogenic differentiation, highly expressed by pre-osteoblasts and osteoblasts during the early stages of bone formation. ALP plays a crucial role in the mineralization process by hydrolyzing phosphate-containing compounds, and releasing inorganic phosphate, which contributes to the formation of hydroxyapatite crystals. When cells are induced toward osteogenic differentiation, an increase in ALP activity is observed. During ALP staining, a substrate (e.g., BCIP/NBT) reacts with ALP to produce a blue-purple color, indicating active ALP expression. By measuring this staining, you can assess how efficiently the cells are entering the osteogenic pathway and preparing for mineral deposition.

Alizarin Red Staining Mechanism:
Alizarin Red S staining detects calcium deposits, a hallmark of late-stage osteogenic differentiation. As osteoblasts mature, they begin to produce and deposit extracellular matrix, which undergoes mineralization as calcium and phosphate ions crystallize into hydroxyapatite, the main mineral component of bone. Alizarin Red S binds specifically to calcium ions, forming an orange-red complex that can be observed microscopically. By assessing the intensity and extent of red staining, you can determine the extent of mineralization, indicating that the cells have progressed to the later stages of osteoblast differentiation and are actively contributing to bone matrix formation.

Together, ALP staining and Alizarin Red staining offer a comprehensive view of osteogenic differentiation. ALP staining identifies early differentiation events, while Alizarin Red staining confirms matrix mineralization, showing that the cells are functionally mature osteoblasts capable of bone formation.

For the ALP staining, cells were cultured for 7 and 14 days, then washed with PBS and fixed with 4% paraformaldehyde for 10-15 minutes at room temperature. After washing with PBS, the cells were incubated with ALP staining solution (e.g., BCIP/NBT) for 30-60 minutes at 37°C. ALP activity, indicating early osteogenic differentiation, was visualized as blue-purple staining under a microscope. For the Alizarin Red staining, cells were cultured for 21 and 28 days, washed with PBS, and fixed with 4% paraformaldehyde for 10-15 minutes. After washing with distilled water, Alizarin Red S solution (pH 4.1-4.3) was applied for 20-30 minutes to detect calcium deposits. The cells were then rinsed with distilled water, and red staining was observed to indicate matrix mineralization, a marker of mature osteoblast activity.

Fig4 ALP and Alizarin Red Staining of MC3T3-E1 Cells at Different Time Points During Osteogenic Differentiation

ALP Staining Results (Day 7 and Day 14): From the images provided, on Day 7 (top-left images), the ALP staining shows minimal blue-purple staining, suggesting that osteogenic differentiation had only just begun. By Day 14 (bottom-left images), there is a noticeable increase in blue-purple staining, indicating elevated ALP activity, which correlates with increased osteoblast differentiation. The stronger ALP activity seen on Day 14 demonstrates that the cells are undergoing early osteogenic differentiation, which is being effectively stimulated by the BMP-4 cell factor, confirming its biological activity in promoting bone formation.

Alizarin Red Staining Results (Day 21 and Day 28): In the Day 21 images (top-right), there are initial signs of mineralization with scattered red deposits, indicating the onset of calcium deposition. By Day 28 (bottom-right), the staining intensity has significantly increased, with larger and more densely stained red areas, showing substantial mineralization of the extracellular matrix. This confirms the later-stage osteogenic activity, as BMP-4 continues to drive the maturation of osteoblasts and the deposition of calcium, which is a hallmark of bone tissue formation.

These results confirm that the BMP-4 produced in our system retains its biological activity, as it successfully stimulates both early (ALP activity) and late (mineralization) osteogenic differentiation.

Sequence and Features