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Celluose binding domain - mutated LCI peptide fusion protein

Introduction

Vilnius-Lithuania Igem 2022 project NanoFind was working to create an easily accessible nanoplastic detection tool, using peptides, whose interaction with nanoplastic particles would lead to an easily interpretable response. The system itself focused on smaller protein molecules, peptides, which are modified to acquire the ability to connect to the surface of synthetic polymers – plastics. The detection system works when peptides and nanoplastic particles combine and form a sandwich complex - one nanoplastic particle is surrounded by two peptides, attached to their respective protein. The sandwich complex consisted of two main parts – one is a peptide bound to a fluorescent protein, and the other peptide is immobilized on a cellulose membrane by a cellulose binding domain.
This specific part is a bound cellulose binding domain bound to a specific plastic binding LCI mutated peptide fused by a linker.

Experimental characterisation

During NanoFindproject this part was tested very throughly in order to be a part of the NanoFind nanoplastic detection system. The part was used for immobilization of LCI peptide on a cellulose membrane in order to catch specific polypropylene nanoplastic particles in a sample.

Purification

Figure 1: CBD-LCI SDS-PAGE analysis. L - protein ladder (PageRuler™ Plus Prestained Protein Ladder #26619), C - cell-free extract of no-plasmid control, S - cell-free extract, P - purified and peptide.

Cultivation and purification

• medium: Luria Bertani (LB) medium
• strain: E.coli BL21(DE3)
• antibiotics: 30 µg mL^-1 Kanamycin
• temperature: 37 °C
• cultivation time: 2 h 37 °C
• pH of lysis buffer : 7.4

E.coli strain was transformed with plasmid expression vector pet29b(+) containing the desired CBD protein. Bacteria night culture is grown, which after 16 h 1/30 dilution is sown into a larger volume of liquid LB medium with appropriate antibiotic (Kanamycin). Cell culture was grown at 37 ℃ at 200 rpm until OD600 value suitable for induction (0.5-0.6) is achieved. IPTG is then added to the growth medium to a concentration of 0,5 mM and the bacteria are further grown for 2 h at 37 °C. Cells are collected by centrifugation for 5 min. at 7000 rpm at 4 °C. (Figure 1)

Figure 2: CBD-LCI binding to Whatman cellulose paper. L - protein ladder (PageRuler™ Plus Prestained Protein Ladder #26619), S - cell free extract of CBD-LCI, U - unbound fraction, W - washed fraction, B - bound fraction of peptides.

Binding to plastics and cellulose

Binding to cellulose paper

To be able to immobilize CBD-LCI peptides on cellulose, we first checked whether it binds to it. Cell free extracts that contain the peptides were incubated with Whatman paper which was cut into small pieces. After washing the unbound fraction, bound peptides were eluted. A faint band can be seen in the bound fraction for both CBD-LCI (Figure 2). Furthermore, the amount of peptide that was removed during the washing step was comparably lower than in the bound fraction. This showed that both peptides could successfully be immobilized on Whatman paper.

Binding to plastics

Figure 3: x CBD-LCI binding to macroplastics. A, peptide binding to PS; B, binding to PP; C, binding to PE. L - protein ladder (PageRuler™ Plus Prestained Protein Ladder #26619), S - cell-free extract of CBD-LCI, U - unbound fraction, W - washed fraction, B - a bound fraction of peptides.

To prove that the peptides bind to plastics, a binding experiment was performed. Cell-free extracts were incubated with macro-sized pieces of plastics. After washing the unbound fraction, bound peptides were eluted. A very faint band in the B line could be seen for all peptide and plastic combinations, confirming that the peptide binds to PS, PP, and PE plastics (Figure 3).

Binding assays - protocol

Peptide binding to plastics or cellulose was tested using 3 ml glass bottles.
1. 20 mg of PS, PP, PE, or cellulose was added to a glass bottle.
2. 300 µl of a cell-free extract was added into the bottle and the mixture was incubated for 30 min. at room temperature, shaking at 300 rpm.
3. After incubation, a sample containing unbound proteins was taken for SDS-PAGE.
4. The leftover liquid was centrifuged through a hydrophilic PVDF membrane at 3200 rcf for 1 min.
300 µl of 50 mM Tris-HCl, pH 7,4 was added and a sample containing washed proteins was taken for SDS-PAGE.
5. The leftover liquid was centrifuged through a hydrophilic PVDF membrane at 3200 rcf for 1 min. 100 µl of 50 mM Tris-HCl, pH 7,4, 300 mM NaCl, 0,1 % Triton X-100 was added to elute peptides. The mixture was incubated for 30 min. at room temperature, shaking at 300 rpm.
6. After incubation, a sample containing bound peptides was taken for SDS-PAGE.
In the case of the low solubility or concentration of proteins in the cell-free extract, we recommend, that plastic binding might need to be proved by the Western blot experiment additionally.

Optimization of saturation

Figure 4: CBD-LCI saturation test. Each amount of added CBD-LCI was tested in triplicate. The curves were fitted by applying local polynomial regression (R function loess).

We designed an experiment based on Miller et al. 2018 [1], to see whether our CBD can bind cellulose and measure the sufficient protein amount needed to fully saturate cellulose. For this test, purified proteins were needed. For this assay, CBD-LCI composite part was used on a glass-coated microplate to identify the saturation of CBD on Whatman grade 1 chromatography paper. The concentrations of all purified proteins were assessed using a bicinchoninic acid (BCA) assay, and all standards and purified samples were tested three times to obtain greater accuracy. Protein purity was defined via SDS-PAGE gel image. Unmodified Whatman grade 1 chromatography paper was used as an immobilization platform for our CBD fusion proteins immobilization assays. After immobilization, absorbance at 562 nm was measured to evaluate protein binding efficiency onto the cellulose membranes. The constants showed that the protein of interest can bind to the cellulose with high efficiency, thus allowing us to move further with the experiments . We performed cellulose binding domain saturation experiments and found out that our cellulose can be fully saturated by CBDs. We tested 8 different protein concentrations, ranging from 100µg to 10µg of total purified protein, and selected the most effective one.
Therefore, we have shown, that the peptide, attached to the cellulose membrane, can be fully saturated by around 3 nmol, or 80 µg of pure protein. (See Figure 4)

Cellulose saturation test - protocol

1. At the bottom of the well, a round piece of Whatman paper (grade 1; with a diameter of 5.5 ± 0.5 mm) is placed.
200 µl of mQ water is poured inside the well and cellulose paper is incubated for 10 min.
2. Water is collected and 200 µl 50 mM Tris-HCl, pH 7,4, 100 mM NaCl, and 3 mM CaCl2 are poured and incubated for 10 min. Buffer is collected and cellulose binding protein is poured onto the Watman paper with ranging concentrations.
3.Cellulose binding proteins are incubated for 30 min 300 rpm RT.
4.Cellulose with attached cellulose binding domains is washed 3 times with 200 µl 50 mM Tris-HCl, pH 7,4, 100 mM NaCl 3 mM CaCl2 buffer.
5. 150 µl of 40 mM sodium acetate pH 5.5 is added onto the cellulose paper and incubated for 5 min. with shaking at 600 rpm.
6. Additional protein samples (100 µl of known concentration) were mixed with 50 µl of 120 mM sodium acetate, pH 5.5 for a calibration curve, to find out the bound portion of the CBD-LCI peptide.
7. 150 µl of BCA Working Reagent (Sigma Aldrich) is poured onto the paper sheets or into calibration wells and the microplate is incubated for 2 h at 300 rpm at 37°C.
8. The solution was collected to a new clear bottom microplate and absorbance was measured at 562 nm.

References

[1] EMiller, E. A., Baniya, S., Osorio, D., Maalouf, Y. J. A., & Sikes, H. D. (2018). Paper-based diagnostics in the antigen-depletion regime: High-density immobilization of rcSso7d-cellulose-binding domain fusion proteins for efficient target capture. Biosensors & bioelectronics, 102, 456-463. https://doi.org/10.1016/j.bios.2017.11.050