Difference between revisions of "Part:BBa K4380000"

Latest revision as of 00:33, 14 October 2022

Cellulose Binding domain (CBD)

This part contains cellulose binding protein, which can be used as a novel way to immobilize proteins on cellulose. The part was used extensively in Vilnius-Lithuania iGEM 2022 team project "NanoFind".

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.

Sequence and features

Profile

Name: CBD (Cellulose binding domain)
Base Pairs: 489 bp
Origin: Clostridium Thermocellum, synthetic
Properties: Celulose binding domain, which can robustly attach to cellulose
Safety: Biosafety level 1 laboratory
Molecular weight (protein): 17899.45 Da
Instability Index (protein): 11.64 (stable)
Isoelectric Point (pI) (protein): 5.22

Introduction

The cellulose-binding domain (CBD) is a protein that can robustly bind cellulose. The protein coded from this sequence comes from a thermophilic, anaerobic bacterium Clostridium thermocellum [1]. The part can be successfully used in a safety level 1 laboratory for different, but useful reasons. The CBM3 is overexpressed in Escherichia coli and it is possible to take advantage of its affinity properties to purify recombinant proteins on cellulose fibers [2], (reducing significantly the costs of purification), to immobilize proteins on a cellulose membrane as well as a purification tag for antimicrobial peptides [3] (Read more Usage ). CBM3 proteins can not only bind to crystalline cellulose but can also interact with smaller affinity (~500 lower) to chitin and xyloglucan [4].

Biology

Figure 1: C. thermocellum cellulosome. C. thermocellum scaffoldin (CipA) contains nine type I cohesins and thus organizes a multienzyme complex that incorporates nine enzymes. (Brás et al., 2012)

Many bacterial and fungal enzymes that hydrolyze insoluble carbohydrates share a familiar structure composed of a catalytic domain linked to a carbohydrate-binding module (CBM). Carbohydrate-binding modules (CBMs) are non-catalytic domains that anchor glycoside hydrolases into complex carbohydrates. Clostridium thermocellum produces a multi-enzyme complex of cellulases and hemicellulases, termed the cellulosome, which is organized by the scaffoldin protein CipA. The binding of the cellulosome to the plant cell wall results from the action of CipA family 3 CBM (CBM3), which presents a high affinity for crystalline cellulose. CBMs that are specific for insoluble cellulose (cellulose binding domains – CBDs) represent the predominant category. The CBMs can be grouped into distinct families based on amino acid sequence similarities. CBM3 is a family of protein modules specific for Gram-positive bacterial families. The proteins comprise of around 150 amino acids. The family of proteins is divided into four subgroups: CBM3a, CBM3b, CBM3c, and CBM3d. The major ligand recognized by CBM3as and CBM3bs is crystalline cellulose with an affinity (Kd) of 0,4 uM determined by depletion isotherms [5]. The family 3a (scaffoldin) and 3b (mainly free enzymes) are closely similar in their primary structures and both types bind strongly to crystalline cellulose. Members of the family IIIc fail to bind crystalline cellulose but serve in a helper capacity by feeding a single incoming cellulose chain into the active site of the neighboring catalytic module pending hydrolysis [6]. (Figure 1)

Figure 2: 3D structure of CBM3a from Clostridium Thermocellum

Structure

The crystal structure of CBM3 has been solved. It has nine beta-strands, which form a compact domain. It is arranged in two antiparallel beta sheets. Two defined structures, located on opposite sides of the molecule, contain conserved polar and aromatic residues, which are presumably involved in the binding to the cellulose. (Figure 2)

Usage

The protein coded by this part can be used for several useful applications:

Immobilization of proteins

The ability of the cellulose-binding domain to bind to cellulose may form the basis of an immobilization platform, used for the display of highly specific binding reagents on cellulosic filters for sensing pathogens, biomarkers, or environmental pollutants. Cellulose is an attractive, easy-to-use support matrix for the development of novel biosensing surfaces because of its chemical and physical stability, low cost, low nonspecific affinity for proteins, and approval for human and therapeutic use. Paper-based microfluidic devices have been shown to perform well as low-cost analytical systems for colorimetric bioassays. Interestingly, CBMs have had a high specific affinity for a variety of soluble and insoluble celluloses, depending on their subfamily origin and in the last years, a fusion of CBMs to other proteins offers the possibility of targeted immobilization of antibodies, proteins, bacteriophages, and bacteria onto cellulose matrices to develop sensor, microarray, and protein purification applications. [7]

Protein purification

Although affinity tags are a convenient and easy way to purify target recombinant proteins, the presence of a tag in a purified recombinant protein is undesirable in many applications. Different CBMs offer a cost-effective method of purification. For example, CBM3, which binds to microcrystalline cellulose, has been used as the affinity tag to purify human-interleukin-6 with a high yield from leaf extracts of Nicotiana benthamiana by Agrobacterium-mediated transient expression. This approach can be used as a powerful method to produce and purify recombinant proteins in plants for specific approaches. [2]

Expression and purification of antimicrobial peptides

Antimicrobial peptides (AMPs) are molecules that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, parasites, viruses, and fungi. These molecules now are getting more attention and importance as a consequence of their remarkable resistance to microorganism adaptation. CBM can be fused to different AMPs using recombinant DNA technology and the fusion recombinant proteins were expressed at high levels in Escherichia coli cells. CBM3 does not present antibacterial activity and does not bind to the bacterial surface. However, the four recombinant proteins retained the ability to bind cellulose, suggesting that CBM3 is a good candidate polypeptide to direct the binding of AMPs into cellulosic supports. [3]

Experimental characterization and design

Vilnius-Lithuania Igem's 2022 team used this part as a novel way for peptide immobilization. The team 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 another peptide is immobilized on a cellulose membrane by a cellulose binding domain.

Figure 3: CBD SDS-PAGE analysis

Cultivation and purification

• medium: Luria Bertani (LB) medium
• strain: E.coli BL21(DE3)
• antibiotics: 30 µg mL^-1 Kanamycin
• temperature: 37 °C
• cultivation time: 16 h

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 at 16 °C for 16 hours at 37 °C. Cells are collected by centrifugation for 5 min. at 7000 rpm at 4 °C. (Figure 3)

Figure 4: Binding assay perfomed with CBD SDS-PAGE analysis

Binding assays

To check the functionality of CBD, a cellulose-binding test was performed.
For this, the following experiment was performed:
1. Finely trim grade Whatman paper (20 mg) into a test tube.
2. Pour 200 µl CBD cell lysate and incubate for 30 min at room temperature shaking at 600 rpm.
3. Centrifuge at 3200g for 1 min through the 0.45 µm pore size, hydrophilic PVDF into a tube and collect as an unbound portion.
4. Resuspend with 200 µl of 50 mM Tris-HCl pH 7.4 buffer and centrifuge at 3200 g 1 min through the membrane into a tube.
5. Resuspend with 200 µl Tris–NaOH (pH 11), incubate at RT, 600 rpm for 20 min, and centrifuge at 3200 g 1 min through the membrane into a tube.

A specific band corresponding to CBD at 18 kDa was detected in whole-cell lysates, demonstrating the successful expression of the designed biomolecule. Upon addition of the Whatman paper, only this band disappeared from the supernatant and was recovered after elution, indicating the preservation of its cellulose-binding functionality. After the addition of high pH Tris-NaOH solution, unbound protein also appeared on SDS-PAGE analysis.

Peptide immobilization on cellulose

One of the goals of the Vilnius-Lithuania iGEM team was to immobilize two peptides (namely TA2 (Tachystatin 2) and LCI (Liquid chromatography peak 1) on cellulose membrane to create a fusion protein, capable of interacting with plastic nanoparticles. These two peptides were chosen as predominant in this project since it has been proven, that these peptides and their mutated variants can attach to the plastic. TA2 mutated variant has been proven to bind to polystyrene microplates and LCI was proven to bind to polypropylene microplates. For this reason, the Vilnius-Lithuania iGEM 2022 team decided to add these peptides to cellulose by designing two fusion proteins: BBa_K4380015 and BBa_K4380017. We have successfully proven, that our fusion CBD-peptide proteins are expressed and CBD can be successfully used as an immobilization unit. See more on NanoFind.

Saturation measurement

For immobilization application, it is important to fully saturate cellulose as unspecific binding for substrate and cellulose membrane might occur. We designed an experiment based on Miller et al. 2018 [9], 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. The concentrations of all purified proteins were assessed using a bicinchoninic acid (BCA) assay and were tested three times to obtain greater accuracy. Protein purity was defined via an SDS-PAGE gel image. Unmodified Whatman grade 1 chromatography paper was used as an immobilization platform for our CBD proteins immobilization assay. After immobilization, absorbance at 562 nm was measured to evaluate protein binding efficiency onto the cellulose membranes.

CBD protein, merged with peptides was tested for its saturation (parts: BBa_K4380015,BBa_K4380017).
Therefore, we have shown, that peptides, attached to cellulose membrane, can be fully saturated by around 3 nmol, or 80 µg of pure protein. (Figure 5)

Figure 5: CBD binding to cellulose membrane measurement, parts used for this assay were BBa_K4380015 and BBa_K4380017. The curves were fitted by applying local polynomial regression (R function loess)

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-TA2 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.

Testing the presence of CBD on cellulose with FITC

Another test that was performed to prove that this part can successfully attach to cellulose was using a FITC antibody. For this, a part BBa_K4380002, encoding an antibody-recognized sequence, was added to the composition of this part. The composed part BBa_K4380021 and empty BL21(DE3) bacterial strain were used for this assay alongside CBD. The presence of CBD on the cellulose membrane was evaluated using a conjugated FITC antibody recognition sequence. The following experiment was performed:
1. Incubate bacterial cell-free extract on cellulose membrane for 30 min.
2. After incubation for 30 min., wash the membrane with 1x PBS buffer 3 times.
3. After washing steps, measure fluorescence at excitation 485 nm / emission 525 nm.
To evaluate results, quantitative and qualitative data was gathered.

Qualitative measurement

To evaluate whether our CBD has successfully attached to the cellulose membrane, we used a blue light to evaluate the fluorescence of the Whatman paper. (Figure 7)

Quantitative measurement

To evaluate whether our CBD has successfully been attached to the cellulose membrane, we measured fluorescence at excitation 485 nm / emission 525 nm with a microplate reader. (Figure 8)

Figure 8: CBD fluorescence measurements. Quantitative evaluation.

Figure 7: CBD antibody test. Qualitative evaluation.

References

[1] Morag, E., Lapidot, A., Govorko, D., Lamed, R., Wilchek, M., Bayer, E. A., & Shoham, Y. (1995). Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum. Applied and Environmental Microbiology, 61(5), 1980–1986. https://doi.org/10.1128/aem.61.5.1980-1986.1995
[2]Islam, M. R., Kwak, J., Lee, J., Hong, S., Khan, M. R. I., Lee, Y., Lee, Y., Lee, S., & Hwang, I. (2019). Cost‐effective production of tag‐less recombinant protein in Nicotiana benthamiana. Plant Biotechnology Journal, 17(6), 1094–1105. https://doi.org/10.1111/pbi.13040
[3]Guerreiro, C. I. P. D., Fontes, C. M. G. A., Gama, M., & Domingues, L. (2008). Escherichia coli expression and purification of four antimicrobial peptides fused to a family 3 carbohydrate-binding module (Cbm) from Clostridium thermocellum. Protein Expression and Purification, 59(1), 161–168. https://doi.org/10.1016/j.pep.2008.01.018
[4]Hernandez-Gomez, M. C., Rydahl, M. G., Rogowski, A., Morland, C., Cartmell, A., Crouch, L., Labourel, A., Fontes, C. M. G. A., Willats, W. G. T., Gilbert, H. J., & Knox, J. P. (2015). Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate-binding module. FEBS Letters, 589(18), 2297–2303. https://doi.org/10.1016/j.febslet.2015.07.009
[5]Pasari, N., Adlakha, N., Gupta, M., Bashir, Z., Rajacharya, G. H., Verma, G., Munde, M., Bhatnagar, R., & Yazdani, S. S. (2017). Impact of Module-X2 and Carbohydrate Binding Module-3 on the catalytic activity of associated glycoside hydrolases towards plant biomass. Scientific Reports, 7(1), 3700. https://doi.org/10.1038/s41598-017-03927-y
[6] Brás, J. L. A., Alves, V. D., Carvalho, A. L., Najmudin, S., Prates, J. A. M., Ferreira, L. M. A., Bolam, D. N., Romão, M. J., Gilbert, H. J., & Fontes, C. M. G. A. (2012). Novel clostridium thermocellum type I cohesin-dockerin complexes reveal a single binding mode. Journal of Biological Chemistry, 287(53), 44394–44405. https://doi.org/10.1074/jbc.M112.407700
[7] Ong, E., Gilkes, N. R., Miller, R. C., Warren, A. J., & Kilburn, D. G. (1991). Enzyme immobilization using a cellulose-binding domain: Properties of a beta-glucosidase fusion protein. Enzyme and Microbial Technology, 13(1), 59–65. https://doi.org/10.1016/0141-0229(91)90189-h
[8] Yang, J. M., Kim, K. R., Jeon, S., Cha, H. J., & Kim, C. S. (2021). A sensitive paper-based lateral flow immunoassay platform using engineered cellulose-binding protein linker fused with antibody-binding domains. Sensors and Actuators B: Chemical, 329, 129099. https://doi.org/10.1016/j.snb.2020.129099
[9] Miller, 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