Latest revision as of 21:10, 1 October 2024

BaCBM2_RFP_3xMad10

This CBM2 protein is fused with the red fluorescent protein (miRFP670). This fusion enhances the visualization of CBM2. The protein also has three Mad10 peptides [ ], which serve as a magnetic tag that facilitates the purification of the protein through magnetic separation techniques.

This part was used as template to construct BBa_K5396003

Usage and Biology

BaCBM2

This CBM2, or Carbohydrate-Binding Module 2, is a protein sourced from Bacillus anthracis. It belongs to a broader family of carbohydrate-binding modules that are crucial for the degradation of polysaccharides. These modules are important to break down complex carbohydrates, enabling microorganisms to convert them into usable energy sources.

A recent study has shown that CBM2 has the ability to bind to certain types of plastics, especially those derived exhibiting similar structural features of polysaccharides. This binding ability is largely due to the protein's carbohydrate-binding properties, which facilitate interactions with specific functional groups found on plastic surfaces. [1]

Figure 1. 3D simulation of BaCBM2-RFP-3xMad10

miRFP670

miRFP670 is a monomeric near-infrared fluorescent protein (NIR FP) derived from the bacterial photoreceptor Rhodopseudomonas palustris. It exhibits excitation and emission maxima at 643 nm and 670 nm, respectively, making it suitable for applications in multicolor microscopy. With a molecular weight of approximately 34.5 kDa, miRFP670 is characterized by its high brightness and photostability in various cells, outperforming many other NIR FPs in terms of fluorescence intensity. The protein utilizes biliverdin as its chromophore, which enhances its effective brightness and allows for efficient labeling in live-cell imaging. The miRFP670 demonstrates low cytotoxicity and stability across a range of pH levels. Its structural properties enable the development of biosensors and facilitate protein-protein interaction studies.

Mad10 peptide

Mad10 (Magnetosome-associated deep-branching 10) is a protein derived from the magnetotactic bacterium Desulfamplus magnetovallimortis BW-1, known for its ability to synthesize magnetite crystals, which are essential for its navigation in aquatic environments. This protein is important for the formation and stabilization of magnetosomes (organelles that contain magnetic particles). [2]

The Mad10 tag refers to a peptide derived from Mad10, designed to include the most conserved amino acids within the protein sequence. The Mad10-derived peptide exhibits a strong affinity for magnetite, allowing it to effectively bind to magnetic columns during purification processes.

Protein Expression and Purification

The sequence for this part was chemically synthesized by Genscript and arrived pre-cloned in the pET-15b(+) vector. This vector includes a T7 promoter, lacI, a ribosome binding site (RBS), a 6xHis tag, and a T7 terminator. After the arrival, we proceeded with the transformation of the plasmid into the E. coli BL21(DE3) strain.

Optimization of Protein Expression

We initiated the process by growing a pre-inoculum of E. coli BL21(DE3) expressing BaCBM2-RFP-Mad10 in LB medium with ampicillin overnight at 37ºC. The next day, the pre-inoculum was transferred to a larger culture and incubated until it reached the desired cell density. Protein expression was induced by adding IPTG, followed by shaking at 37ºC for 3 hours.

The harvested culture was frozen, and the pellets were later resuspended in a lysis buffer containing Tris-HCl, NaCl, imidazole, lysozyme, and a protease inhibitor cocktail. The cells were lysed through sonication and the debris was removed by centrifugation. The protein was then purified using Immobilized Metal Affinity Chromatography (IMAC) on an ÄKTA system with a Ni-NTA column.

This protocol resulted in the successful purification of BaCBM2-RFP-Mad10, highlighting the efficiency of the combined expression and purification methods.

Figure 2. Chromatogram of BaCBM2-RFP-Mad10 purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The peak corresponding to the BaCBM2-RFP-Mad10 protein is highlighted.

To confirm the identity of the protein, an SDS-PAGE analysis was performed, revealing a band consistent with the expected size of BaCBM2-RFP-Mad10 (Figure 3).

Figure 3. Analysis of BaCBM2-RFP-3xMad10 expression and purification by SDS-PAGE.

Despite successfully purifying a significant amount of BaCBM2-RFP-Mad10, SDS-PAGE analysis revealed that a large portion of the protein remained in the insoluble fraction, which was prepared from the pellet formed during the final centrifugation step before loading the sample onto the ÄKTA system. A similar result was observed for Barbie1-RFP-3xMad10 (BBa_K5396001). Consequently, we decided to optimize the extraction process by testing different detergents to improve solubility and protein recovery.

Expression and Purification of BaCBM2-RFP-Mad10 with Triton X-100

To improve protein solubility, we resuspended the E. coli pellet in 40 mL of Buffer A with the addition of 1% Triton X-100, along with lysozyme and a protease inhibitor cocktail. Triton X-100 played a crucial role in ensuring effective cell lysis and preventing aggregation during extraction. The mixture was incubated on ice for 30 minutes. After incubation, the sample was centrifuged at 14,000 rpm for 1 hour at 4°C, and the supernatant was filtered using a 0.45 µm filter.

The supernatant was loaded onto a HisTrap column, equilibrated with Buffer A using the ÄKTA system. After loading, the column was washed with 10 column volumes of Buffer A, and protein elution was performed using 5-10 column volumes of Buffer B. Fractions were collected in glass tubes to avoid protein adhesion to plastic surfaces.

Figure 4. Chromatogram of BaCBM2-RFP-Mad10 purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The peak corresponding to theBaCBM2-RFP-Mad10 protein is highlighted.

SDS-PAGE analysis confirmed the presence of BaCBM2-RFP-3xMad10 in the collected fractions. Despite the UV curve from the ÄKTA system not following a typical pattern, fractions containing the BaCBM2-RFP-3xMad10 protein were successfully identified and collected, highlighting the effectiveness of Triton X-100 in the purification process.

Figure 5. Analysis of BaCBM2-RFP-3xMad10 expression and purification by SDS-PAGE.

Characterization

SEC-MALS

Knowing that the protein 6His-CBM-RFP-3xMAD10 has a 44.86 kDa molecular mass (MM), it is possible to observe three different oligomeric states in the SEC-MALS result shown in Figure 2. With a smaller normalized signal (dRI), it was possible to observe a MM of 180±2 kDa around 32 minutes of elution, which is close to the molecular mass of a 4 units oligomer.

Following on, a second elution was made around 34 minutes with 86.2±0.5 kDa. According to this molecular mass, it is possible to observe a dimer structure of the protein with a higher dRI. Finally, with the highest dRI and a molecular mass of 46.1±0.2 kDa at 37 minutes of elution, it was possible to identify the protein monomer state.

When compared to the other oligomer states, it was notable that this last elution had the lowest standard deviation when compared to the others. As a conclusion, it is possible to confirm that the BaCBM2-RFP-3xMAD10 construction is most likely to be a single unit protein.

Figure 6. SEC-MALS result of BaCBM2-RFP-3xMad10.

Circular Dichroism (CD)

CD can provide crucial insights due to the presence of beta sheets in their structures. Typically, β-sheets exhibit a characteristic negative band around 218 nanometers (nm) and a positive band near 195 nm, while α-helices show a negative band around 222 nm and a positive band around 190 nm. However, a limitation of the equipment used restricts the analyzed wavelength range to between 200 and 250 nm.

The pipeline acquisition of the spectrum was followed by a 2 seconds of integration for each point in each 6 spectrum calculated at room temperature (20 ºC). From the collected data, it is calculated its average, subtracted from the water absorbance, normalized, and smoothed using a Savitzky-Golay filter.

Figure 7. CD result for BaCBM2-RFP-3xMad10.

If you want to check the comparison between the BaCBM2 and Barbie1, see our Resultspage.

BaCBM2-RFP-3xMad10 Heating Ramp

An essential evaluation for the protein is the heating ramp, in which we can evaluate how the protein acts in different temperatures and their melting point. For doing so, the same procedure was followed, in which each protein was heated from 20 to 95ºC, as shown in Figure P. The data acquisition was made in steps of 5ºC.

The CD absorbance is shown as the figure’s color map. It is evident that the protein show absorbance in the secondary structure regions, indicating a thermal resistivity.

Figure 8. Heating ramp for BaCBM2-RFP-3xMad10.

BaCBM2-RFP-3xMad10 Pontual Spectra

With this result, it is interesting to observe that the protein could still maintain great results for tests like the described above. To further explore it, we also ran a protein pontual spectra, in which we measured CD absorbance for only 215 nanometers, but each measure made at 1ºC increase.

Figure 9. Pontual spectra for BaCBM2-RFP-3xMad10 at 215 nm.

By calculating the pontual spectra it was notable a constant absorbance for BaCBM2-RFP-3xMad10 through all temperatures (Figure 9).

Plastic Interaction Analysis

Through secondary structures absorbance analysis, it was determined that the BaCBM2 protein structure remains unchanged in the presence of plastic nanoparticles.

The final experimental evaluation using circular dichroism (CD) involved polystyrene nanoplastics. A 100-nanometer particle was introduced into a solution containing BaCBM2. However, as indicated by computational simulations, no conformational change is anticipated. Since CD primarily assesses absorbance related to secondary structures, significant differences are unlikely to occur during the experiment.

Consequently, as illustrated in Figure DDD, when the spectrum of BaCBM2 is plotted alongside the nanoparticle solution spectrum, only minor changes are observed. This contrast may be attributed to the scattering effects of the polystyrene nanoparticles, which can introduce noise into the measurements.

Figure 10. Plastic binding protein BaCBM2 spectra acquisition with 100 nm polystyrene nanoparticles.

Protein Corona Formation

We developed a very simple experiment of protein titration and particle size measurement in the Dynamic Light Scattering (DLS).

The experiment result is shown in Figure 11. Although we used a 100 nanometer polystyrene particle, it is important to note that the equipment has a 30 nm standard deviation, which reflects in the plotted result. In an initial moment shown on Subfigure 11(a), the BaCBM2 is added and starts to aggregate in the plastic particle, increasing the particle size.

On the other hand, in the final titrations, the protein has aggregated in the whole nanoplastic surface, which makes it stabilize its size, as represented on Subfigure 11(b). This effect can also be seen in the following points, since there is no size increase.

It is particularly interesting to note the theoretical and experimental comparison. Knowing that the protein has a 2.5 nm width, the expected theoretical particle size was a 5% increase (from 100 nm to 105 nm). On the other hand, the experimental value found for the experiment was an increase tax of 5.05% (from 122.7 to 128.9 nm), showing a great proximity between them.

Figure 11. Dynamic Light Scattering of the average particle size in function of the protein titration. On Subfigure (a), it is shown the initial titration state representation and the final state on Subfigure (b).

Dissociation Constant Calculation

From the protein-corona formation found in the previous section, it is now possible to fit the saturation bind curve, which is very used in the context of studying the affinity between ligands and receptors. In special, it measures the binding capacity.

In the context of studying the plastic binding affinity, uncovering the experimental protein dissociation constant is fundamental. Therefore, fitting the average value of each point as shown in Figure 12, it was possible to uncover BaCBM2-RFP-3xMad10 dissociation constant as 1.59 M.

Figure 12. Saturation binding curve fit in the DLS resulted data.

Sequence and Features