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

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.

Figure 1:

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.