Part:BBa_K4968020
MSmtA4-CBM-sfGFP
The component part consist of MSmtA4 (BBa_K4968000), CBM (BBa_K4968002), sfGFP (BBa_K4968003).
Metallothioneins are a class of low molecular weight proteins rich in cysteine residues that have the ability to adsorb heavy metals and chelate them in an inactive form (Carpenè et al., 2007). MSmtA4 (BBa_K4968000) was obtained by mutating four specific sites in the Cyanobacterial metallothionein SmtA from Synechococcus PCC 7942. It has a stronger surface affinity for heavy metal ions and greater chelation capacity for heavy metal ions near the protein's surface.
By introducing a carbohydrate-binding module (CBM) (BBa_K4968002), our goal is to tightly bind the protein to cellulose, creating a biocompatible adsorbent with potential applications. Recombinant fusion proteins containing superfolder Green Fluorescent Protein (sfGFP)(BBa_K4968003) are used to confirm protein expression, monitor the immobilization process, and study protein dynamics.
Usage & Biology
The Cyanobacterial metallothionein SmtA, originating from Synechococcus PCC 7942, belongs to the metallothionein protein family. It has the ability to sense and bind specific heavy metal ions such as cadmium, lead, zinc, and assists in regulating the tolerance and metabolism of these toxic metals in cells. It achieves this by forming complexes with these metal ions, preventing their entry into the cell or transferring them to cellular compartments, thereby protecting the cell from the toxicity of heavy metals.
To enhance the surface affinity for heavy metal ions and improve the metal ion chelation capacity near the protein surface, we designed a variant of Cyanobacterial metallothionein SmtA, named MSmtA4 (BBa_K4968000). Mutations were carefully chosen at positions that would not affect the protein's spatial structure and function. When selecting the mutated amino acids, we prioritized residues with larger accessible surface areas (ASA) and smaller root mean square fluctuations (RMSF), particularly those with positive charges, to enhance electrostatic interactions. Therefore, we selected Arg 26, Lys 8, and Lys 22 to be mutated into Cys. This mutation effectively enhanced the protein's adsorption capacity for heavy metal ions, especially cadmium ions.
Carbohydrate-binding modules (CBMs)(BBa_K4968002) are components of several enzymes and can bind specific carbohydrates. One of their primary functions is to be used in constructing specific bifunctional proteins (Oliveira et al., 2015). Cellulose is an environmentally friendly material commonly used as an immobilization matrix. CBMs (BBa_K4968002) can recognize and specifically bind to characteristics on the crystal surface, providing possibilities for modifying and enhancing cellulose. Kevin Aïssa and others have found that CBMs (BBa_K4968002) exhibit a strong affinity for crystalline cellulose. Introducing CBMs (BBa_K4968002) to the cellulose surface for modification can provide powerful non-covalent modification, increase the redispersibility of functionalized cellulose nanocrystals after drying, and improve suspension stability (Aïssa et al., 2019).
In the XJTLU-2023 project, we selected a family 2 CBM (BBa_K4968002) from C. fimi's endoglucanase A, commonly used for enzyme immobilization.
Superfolder Green Fluorescent Protein (sfGFP)(BBa_K4968003)was engineered by Pédelacq and others with the purpose of ensuring that proteins do not misfold when fused and expressed with other proteins. This means that sfGFP (BBa_K4968003) can maintain a well-folded state even when fused with poorly folded peptides, thereby enhancing the stability of the fusion protein. Compared to other variants of Green Fluorescent Protein (GFP), sfGFP (BBa_K4968003) exhibits superior performance in various aspects, such as tolerance to circular permutation, resistance to chemical denaturants, and folding kinetics. This makes sfGFP (BBa_K4968003) a more reliable label suitable for various protein expression and research contexts. Its outstanding stability and reliability have made it one of the preferred fluorescence labeling tools in biological research, especially in the study of protein interactions, localization, and expression. The unique properties of sfGFP (BBa_K4968003) have found extensive applications in the field of biological sciences, contributing to the advancement of scientific research.
This fusion protein offers several advantages. Firstly, MSmtA4 (BBa_K4968000) plays a crucial role in sensing and regulating heavy metal ions, significantly enhancing the protein's adsorption capacity for heavy metal ions. Additionally, the carbohydrate-binding module (CBM) (BBa_K4968002) can be used to modify the surface of cellulose, improving its redispersibility and suspension stability, thereby providing potential applications for environmentally friendly materials. Lastly, superfolder Green Fluorescent Protein (sfGFP) (BBa_K4968003) exhibits excellent folding performance, maintaining a stable folded state even when fused with other proteins, making it a reliable labeling tool widely used in protein research. This multifunctional fusion protein demonstrates flexibility, versatility, and convenience in various biological research and engineering applications.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 676
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 282
Illegal BsaI.rc site found at 275
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