Difference between revisions of "Part:BBa K4380000"

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Cellulose Binding domain (CBD)

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

Sequence and features

Profile

Name: CBD (Cellulose binding domain)
Base Pairs: 489 bp
Origin: Clostridium Thermocellum, synthetic
Properties: Celulose binding domain, which has the abillity to robustly attach cellulose
Safety: Biosafety level 1 laboratory

Introduction

Cellulose binding domain (CBD) is a protein that has the ability to robustly bind cellulose. The protein coded from this sequence comes from a thermophilic, anaerobic bacterium Clostridium thermocellum . The part can be succesfully used in 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, (reducing significantly the costs of purification), to imobilize proteins on a cellulose membrane as well as a purification tag for antimicrobial peptides (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.

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 hydrolyse insoluble carbohydrates share a familiar structure composed of a catalytic domain linked to 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. 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 domain – CBD) represent the predominant category. The CBMs can be grouped into distinctive families on the basis of 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, CBM3d. The major ligand recognised by CBM3as and CBM3bs is crystalline cellulose with an affinity (Kd) of 0,4 uM determined by depletion isotherms. 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, fails to bind crystalline cellulose, but serves in a 'helper' capacity by feeding a single incoming cellulose chain into the active site of the neighbouring catalytic module pending hydrolysis.

Structure

Figure 1:

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 moleclue, contain conserved polar and aromatic residues, which are presumably involved in the binding to the cellulose.

Usage

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

Immobilization of proteins

The ability of cellulose binding domain to bind to cellulose may form the basis of immobilization platform, used for display of highly specific binding reagents on cellulosic filters for sensing pathogens, biomarkers or enviromental 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 colourimetric bioassays. Interestingly, CBMs have have a high specific affinity for a a variety of soluble and insoluble celluloses, depending on their subfamily origin and in the last years, fusion of CBMs to other proteins offers the possibility of targeted immobilization of antibodies, proteins, bacteriophages and bacteria onto cellulose matrices with the goal of developint sensor, microarray, and protein purification applications.

Protein purrification

Although affinity tags are a convenient and easy way to purify target recombinant proteins, the presense of a tag in in a purified recombinant protein is undersirable 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.

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 remarkalbe resistance to microorganism adaptation. CBM has the ability to 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.

Experimental characterisation

Vilnius-Lithuania Igem 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 peptide bound to a fluorescent protein, other peptide immobilized on cellulose membrane by cellulose binding domain.

Cultivation and purification

Figure 1: Binding assay perfomed with CBD SDS-PAGE analysis

• 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 4-8 p.m. 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.

To check the functionality of CBD, cellulose binding test was performed.

Figure 4: CBD SDS PAGE analysis

Binding assays

The following experiment was performed:
1. Finely trim grade Whatman paper (20 mg) into a glass test tube.
2. Pour 200ul 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, centrifuge at 3200 g 1min through the membrane into a glass 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 Whatman paper, only this band disappeared from the supernatant and was recovered after elution, indicating the preservation of its cellulose-binding functionality. After addition of high pH Tris-NaOH solution, unbound protein also appeared on SDS-PAGE analysis.

Antimicrobial peptide immobilization on cellulose

One of the goals of Vilnius-Lithuania iGEM team was to immobilize two peptides (namely TA2 (Tachystatin 2) and LCI (Liquid chromatography peak 1) on cellulose membrane in order to create a fusion protein, capable of interacting with plastic nano particles. These two peptides were chosen as predominant in this project due to the fact that it has been prooven, that these peptides and their mutated variants have the ability to attach to plastic. TA2 mutated variant has been prooven to bind to polystyrene microplates and LCI was prooven to bind to polypropylene microplates. For this reason, Vilnius-Lithuania iGEM team decided to add these peptides to cellulose by creating two fusion protein: BBa_K4380015 and BBa_K4380017.