Difference between revisions of "Part:BBa K5283022"

Latest revision as of 07:05, 2 October 2024

pGroESL-SPusp45-LEISSTCDA-Linker-LMWP-FGF21-histag Plasmid Detailed Description1.

FGF21(Fibroblast growth factor 21), as a newly discovered endocrine hormone that can regulate carbohydrate and lipid metabolism, can lower blood sugar levels by improving insulin resistance, increasing gluconeogenesis, and promoting ketone production.Our plasmid can effectively respond to changes in cholate after food intake, promptly secreting FGF21 to promote blood glucose reduction, thereby achieving the goal of foodborne-stress induced secretion.

Fragment function

1. GroESL Inducible Promoter Function: The GroESL promoter is regulated by heat shock proteins and is typically activated under stress conditions, such as heat shock or chemical induction. Source: Derived from Escherichia coli heat shock protein genes, commonly utilized in industrial microbiology or synthetic biology. Application: Its inducible nature makes it suitable for controlled protein expression, particularly when expression needs to be initiated under specific conditions, aiding in maximizing protein yield and stability.

2. SPusp45 Signal Peptide Function: SPusp45 is a signal peptide sequence that directs the secretion of proteins out of the cell. Source: Typically sourced from the secretion system of Lactococcus lactis. Applicatio*: Used for secretory expression of recombinant proteins, enhancing solubility and purity, facilitating subsequent purification and application.

3. LEISSTCDA Sequence Function: LEISSTCDA is a peptide sequence used for protein modification or tagging. Source: This sequence may be obtained through in vitro selection or design, with specific functions depending on experimental requirements. Application: Employed to enhance protein functionality or facilitate detection and purification.

4. Linker (Peptide Linker) Function: Peptide linkers provide flexibility between protein domains, preventing structural interference. Source: Common linker sequences include (Gly_4Ser)_n, typically obtained through design. Application: Used to connect different functional domains, ensuring each domain can correctly fold and function independently.

5. LMWP (Low Molecular Weight Poly-Lysine) Function: LMWP is a cell-penetrating peptide sequence that aids in translocating proteins across cell membranes. Source: Generally, it is artificially designed or selected through screening. Application: Used in drug delivery systems to enhance intracellular delivery efficiency of genes or drugs.

6. FGF21 (Fibroblast Growth Factor 21) Function: FGF21 is a metabolic regulatory protein with roles in regulating glucose and lipid metabolism.**Source**: Naturally occurring in the human body and produced via gene cloning and expression systems. Application: Important in the research and treatment of metabolic diseases such as diabetes and obesity, potentially serving as a therapeutic protein drug.

7. His Tag Function: The His tag is a sequence of six histidines used for protein purification. Source: Widely utilized in molecular biology tools, added to recombinant proteins via genetic engineering. Application: Facilitates purification using nickel affinity chromatography (Ni-NTA), enabling high-purity target protein recovery.

Potential Applications

1. Metabolic Research: By inducing FGF21 expression, researchers can investigate its mechanisms in metabolic diseases.

2. Protein Drug Development: Production and purification of FGF21 for preclinical research and drug development for metabolic disorders.

3. Gene Therapy: Utilizing the cell-penetrating properties of LMWP to explore the feasibility of intracellular delivery of FGF21 genes or proteins.

4. Industrial Production: Using the GroESL inducible promoter to mass-produce FGF21 in industrial microorganisms, reducing production costs

Summary This plasmid combines multiple functional sequences to efficiently express and secrete FGF21 protein under specific conditions, facilitating subsequent purification and application. It holds significant potential for research and preclinical development in various fields.

Sequence and Features

Plasmid Construction

We chose pNZ8148 as our plasmid scaffold, modified into an inducible promoter pGroESL.

In the construction of engineered *L. lactis strain NZ9000* for therapy for diabetes, FGF21 is our key functional molecule. To achieve a long-lasting and non-invasive drug delivery method for diabetes,Due to the rapid onset of action of FGF21 and the wide range of receptor cells, we designed a cholate-induced secretion of FGF21. However, FGF21 needs to enter the circulatory system to function. The intestinal epithelium, composed of tightly connected intestinal columnar cells, restricts the passage of typical large molecular proteins. Additionally, the reliable, safe, and efficient delivery of proteins in the intestinal tract is challenging due to various factors, such as intestinal proteases(e.g., pancreatic proteases) and pH. By integrating FGF21 and LMWP (low molecular weight protamine) (BBa_K5283017) , which mediates the endocytosis of intestinal epithelial cells, we not only enhance the overall stability of the fusion protein but also ensure a safer and more reliable targeted cellular uptake compared to previous "intestinal penetration" strategies that compromised the integrity of the intestinal mucosal barrier. Regarding the LMWP connection issue, in order not to affect the binding of FGF21 to its receptor, we found by modeling molecular docking that LMWP attachment to the N-terminus of FGF21 did not affect its biological activity (plus linkage to the model), so we constructed LMWP-FGF21 fusion protein. In order to improve the secretion efficiency of lactic acid bacteria, we also designed an enhancer peptide LEISSTCDA (BBa_K5283014) matched with promoter USP45.

Figure 1. Changes in postprandial cholate in normal people.

Figure 2. The design of FGF21 plasmid.

Modeling and Simulation

Protein Active Site Prediction There is stability concern regarding the fusion protein of FGF21 with LMWP, as the fusion may impact the three-dimensional structure of FGF21, particularly in its receptor interactions, thereby reducing its functional efficiency. To investigate this, we performed a protein-protein interaction (PPI) network analysis using the STRING database (illustrated in the figure 3).

The receptor affinity score ranges from 0 to 1 and is calculated by integrating probabilities from various evidence channels, adjusted for the likelihood of random interactions (von Mering et al., 2005).To compute the combined score, we first remove a 'prior' probability (p=0.041) from each channel's score, then combine the scores as follows:

Using this formula, we calculated and compared the receptor affinity scores of highly homologous derivatives of FGF21 and constructed a PPI network for the top ten receptors. The core receptors with scores above 0.99 included KLB (0.999), FGFR1 (0.998), and FGFR4 (0.992), closely aligning with the receptors of unmodified native FGF21. This validation suggests that the FGF21-LMWP fusion protein does not introduce new active sites or alter existing ones.

Figure 3. The PPI network of potential receptors of FGF21-LMWP fusion protein.

Protein Stability Prediction A fusion protein combining fibroblast growth factor 21 (FGF21) with a transmembrane peptide, low molecular weight protamine (LMWP), was designed to enhance the transport and circulation of FGF21 during the protein engineering phase. We assessed the stability of this fusion protein. Using PyMOL for visualization, we examined the three-dimensional conformation of the FGF21-LMWP complex. As shown in Figure 2, the active sites of FGF21 are highlighted in magenta, while the LMWP segment is depicted in yellow. The LMWP peptide displays a relatively loose conformation with an undefined spatial structure and low polarity. These characteristics suggest minimal risk of detrimental intramolecular interactions between LMWP and FGF21, which could otherwise obstruct FGF21's binding sites to its receptor, FGFR.

Figure 4. The FGF21-LMWP fusion protein with its active site.

Figure 5. The hydrophobicity analysis of FGF21-LMWP fusion protein.

Construction of EFGF21 Previous research has reported low stability for native FGF21 (Onuma Y et al., 2015). To address this, we designed a more stable version, termed enhanced FGF21 (EFGF21). We employed SWISS-MODEL for homology modeling, using FGF21 with the protein ID 6m6e as the template based on both sequence coverage and identity. The sequence identity after alignment exceeds 40% (some suggest 35%), indicating that they are homologous proteins belonging to the same family.

We predicted the three-dimensional structure and physicochemical properties using appropriate methods for homologous protein modeling. Initially, we generated Ramachandran plots (Fig. 5, Fig 6), which illustrate the two dihedral angles of the α-carbon: φ (phi) for the C-N bond and ψ (psi) for the C-C bond. While these bonds can theoretically rotate freely, real molecular structures are constrained by steric hindrance and atomic interactions, leading to allowed and disallowed regions in the Ramachandran plot. This plot assesses the quality of homology modeling.

When comparing the Ramachandran plot of EFGF21 to that of FGF21, we observed that the amino acids in EFGF21, designed for enhanced stability, are more concentrated in the green allowed region. This indicates that EFGF21 adheres more closely to stereochemical rules.

Figure 6. The Ramachandran plot of FGF21.

Figure 7. The Ramachandran plot of EFGF21.

Expression and Verification

First, we set 15 ng/ml cholate as the low concentration stimulation condition according to the bile acid concentrations provided in the literature. However, since this concentration is relatively low, we established 100 ng/ml as the high concentration group to better simulate the bile acid levels in the human body. The duration was set to 30 minutes to explore the level of the bacteria's responses over a short period.

Figure 8-9. The ELISA result under different concentration condition.

Compared to the wild-type bacteria, the transformed bacteria produced a smaller amount of FGF21, with only 193 pg/ml secretion under the stimulation condition of 100 ng/ml cholate. Based on our analysis, we believe that this may be attributed to the lower bile acid concentration and shorter stimulation time. We have further designed the following experiments to investigate this.

Figure 10-11. The ELISA result with different stimulation time.

We found that by extending the stimulation time, the concentration of FGF21 in the supernatant was significantly increased. The FGF21 concentrations at stimulation times of 30 minutes, 60 minutes, and 120 minutes were 206, 663, and 1689 micrograms per μm, respectively, reaching the microgram level. This provides data support for our next experimental plan.

Function Verification

To verify the biological function of FGF21 (BBa_K5283016) in improving insulin resistance, we established an adipocyte model of insulin resistance. First, we treated this insulin resistance using FGF21 secreted by engineered *L. lactis strain NZ9000*. After that we measured the activation levels of downstream pathways after insulin stimulation to reflect the biological function of FGF21.

For the collection of media conditioned by RAW264.7 macrophages (RAW-CM), RAW264.7 macrophages were grown to 90% confluency in DMEM containing 10% FBS. Then, the cells were stimulated with 100 ng/ml lipopolysaccharide (LPS) for 3 h. After stimulation, the cells were cultured in new serum-free DMEM for 24 h. The media was collected, filtrated through a 0.22 μm filter, and used as RAW-CM. Insulin resistance of 3T3-L1 adipocytes could be induced by incubation with RAW-CM. To assess the insulin resistance improvement effects of FGF21, RAW-CM and 200 μg/ml FGF21 purified from engineered *L. lactis strain NZ9000* were added into 3T3-L1 adipocytes. After treatment for 24 h, 100 nM insulin was added to activate the insulin-induced PI3K/Akt/mTOR signaling pathway . The insulin sensitivity was detected by the AKT (Ser473) phosphorylation, which was qualified using Western Blot.

Figure 12. The overall process of verification.

Figure 12. The differentiation result.

The Western blot results showed that after FGF21 treatment, the proportion of phosphorylated Akt relative to total Akt significantly increased, indicating enhanced activation of the PI3K/Akt/mTOR signaling pathway. This demonstrated that FGF21 could improve the sensitivity of adipocytes to insulin, reflecting its favorable biological function.

Figure 12. The WB result.