Part:BBa_K4968021
SUMO-MSmtA4-CBM-sfGFP
The component part consist of MSmtA4 (BBa_K4968000), CBM (BBa_K4968002), sfGFP (BBa_K4968003), and SUMO (BBa_K4968007).
Also the improvement of the part MSmtA4-CBM-sfGFP (BBa_K4968020) by adding the optimized SUMO tag (BBa_K4968007).
MSmtA4 (BBa_K4968000) is a variant of the Cyanobacterial metallothionein SmtA from Synechococcus PCC 7942, created by introducing mutations at four specific sites. This variant exhibits a higher surface affinity for heavy metal ions and a stronger chelation capacity for heavy metal ions near the protein's surface. It can adsorb heavy metals and chelate them in an inactive form.
Carbohydrate-binding modules (CBMs) (BBa_K4968002) are components of several enzymes that can bind to specific carbohydrates. sfGFP (BBa_K4968003) ensures proper protein folding while producing green fluorescence when fused, preventing incorrect protein folding during fusion expression. Additionally, to enhance protein expression levels, facilitate proper folding of the target protein, and increase the solubility of recombinant proteins, a small ubiquitin-like modifier (SUMO) (BBa_K4968007) is used as a fusion tag and molecular chaperone.
These components together create a fusion protein with several advantages, including superior heavy metal adsorption capabilities, optimized protein expression, improved stability, enhanced functionality, and ease of purification.
Usage & Biology
SUMO (BBa_K4968007), which stands for Small Ubiquitin-like Modifier, is a type of ubiquitin-like protein found in eukaryotic organisms. It is a highly conserved large protein involved in protein post-translational modification (PTM) through a process called sumoylation. SUMO (BBa_K4968007) plays a crucial role in regulating protein stability and biological functions.
SUMO (BBa_K4968007) is also commonly used as an efficient and useful tag for protein expression and purification. In comparison to tags like GST (Glutathione S-transferase), MBP (Maltose-binding protein), or NusA, SUMO (BBa_K4968007) serves not only as a fusion tag for recombinant protein expression but also functions as a molecular chaperone. It can facilitate proper protein folding, exhibit tolerance to heat and proteases, and contribute to maintaining the stability of the target protein. Fusion expression of SUMO (BBa_K4968007) at the N-terminus of the target protein can improve protein folding, enhance solubility, and increase protein yield. Additionally, SUMO tags (BBa_K4968007) come with associated proteases (with high specificity) that recognize the tertiary structure of SUMO (BBa_K4968007) and cleave it with exceptional specificity, leaving no residual amino acids behind. This feature makes it suitable for recombinant protein expression and purification.
Metallothioneins (MTs) are a class of low molecular weight proteins rich in cysteine residues. They have the ability to adsorb heavy metals and chelate them in an inactive form (Carpenè et al., 2007). MSmtA4 (BBa_K4968000), derived from the Cyanobacterial metallothionein SmtA found in Synechococcus PCC. 7942, is a variant designed to enhance the surface affinity for heavy metal ions, particularly cadmium ions, and improve the chelation capacity of heavy metal ions near the protein's surface. This modification helps cells tolerate and metabolize toxic heavy metals.
Carbohydrate-binding modules (CBMs) (BBa_K4968002) are components of several enzymes that can bind to specific carbohydrates. One of their primary functions is to build specific bifunctional proteins (Oliveira et al., 2015). Cellulose, an environmentally friendly material, is commonly used as an immobilization matrix. CBM (BBa_K4968002) can recognize and specifically bind to characteristics on crystal surfaces, providing the potential for modifying and enhancing cellulose's properties. Studies by Kevin Aïssa and others have shown that CBM (BBa_K4968002) have a strong affinity for crystalline cellulose, and their introduction to cellulose surfaces for modification can provide powerful non-covalent modifications, increasing the redispersibility of functionalized cellulose nanocrystals and improving suspension stability (Aïssa et al., 2019). In the XJTLU-2023 project, a family 2 CBMs (BBa_K4968002) from C. fimi endoglucanase A was chosen for enzyme immobilization.
Superfolder Green Fluorescent Protein (sfGFP) (BBa_K4968003) was engineered by Pédelacq and others with the aim of ensuring proper protein folding when fused with other proteins. This means that sfGFP (BBa_K4968003) maintains a well-folded state even when fused with poorly folding peptides, thus enhancing the stability of fusion proteins. Compared to other Green Fluorescent Protein (GFP) variants, 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 tag suitable for various protein expression and research scenarios. Its outstanding stability and reliability have made it one of the preferred fluorescence labeling tools in biological research, particularly in the study of protein interactions, localization, and expression. Its unique properties have contributed to advancing scientific research.
Figure 1 |(A) The color of E. coli Bl21 (DE3) without inducement is grey, while that after IPTG inducement is fluorescent green. (B) The supernatant and precipitate are fluorescent green after centrifugation. (C) The supernatant is fluorescent green after moving the precipitate.
Figure 2 |The binding test of microcrystalline cellulose by fusion protein containing CBM module. CBM module can bind to microcrystalline cellulose, thus the total protein concentration decreases.
Figure 3 |Scanning Electron Microscopy (SEM) analysis. A represents Microcrystalline cellulose that is not bound to any protein, and B represents Microcrystalline cellulose bound to proteins containing CBM (done by Yuantest Laboratory).
Figure 4 |These figures (from A-E) indicated that the Scanning Electron Microscopy (SEM) observation of the fusion protein SUMO-MSmtA4-CBM-sfGFP. From picture A to E, the magnifications are 1.00KX, 2.00KX, 5.00KX, 10.00KX, 20.00KX (done by Yuantest Laboratory).
These recombinant fusion proteins offer multiple advantages: firstly, the SUMO tag (BBa_K4968007) not only improves the solubility and yield of the target protein but also positively affects folding and stability, while providing efficient purification. Secondly, metallothionein SmtA plays a crucial role in sensing and regulating heavy metal ions, significantly enhancing the protein's adsorption capacity for heavy metal ions and contributing to the management of heavy metal pollution in biotechnology and environmental applications. Thirdly, the carbohydrate-binding module (CBM) (BBa_K4968002) can be used to modify cellulose surfaces, enhancing the stability and redispersibility of cellulose nanocrystals and expanding the applications of cellulose-based materials. Lastly, superfolder Green Fluorescent Protein (sfGFP) (BBa_K4968003) possesses excellent folding performance and stability, serving as a reliable tag for protein research and expression. These fusion proteins leverage the strengths of each component and provide powerful tools and flexibility in biological research.
Figure 5 |The adsorption efficiency of the biosorbent (SmtA) and biosorbent (MSmtA) in the mixed system. For all the three metal ions, the adsorption efficiency of the biosorbent (MSmtA4) is higher than that of the biosorbent (SmtA). The initial concentrations of cadmium, copper, and lead ions are 20 mg/L, 20 mg/L, and 20 mg/L, respectively.
Figure 6 |The adsorption efficiency of the biosorbent (SmtA) and biosorbent (MSmtA) in the mixed system. For all the three metal ions, the adsorption efficiency of the biosorbent (MSmtA4) is higher than that of the biosorbent (SmtA). The initial concentrations of cadmium, copper, and lead ions are 50 mg/L, 20 mg/L, and 20 mg/L, respectively.
This composite component, comprised of MSmtA4 for heavy metal adsorption, Carbohydrate-Binding Modules (CBMs) for carbohydrate binding and modification, Superfolder Green Fluorescent Protein (sfGFP) for precise protein localization and stable folding, and Small Ubiquitin-like Modifier (SUMO) for efficient protein engineering and purification, serves as a versatile toolkit with applications spanning from enhanced heavy metal pollution management to improved cellulose-based material functionality. It enables researchers to conduct efficient protein expression, enhance stability, and gain visual insights into protein dynamics, offering multifaceted benefits in biological research and biotechnology.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 206
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 206
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 206
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 206
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 206
Illegal AgeI site found at 988 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 81
Illegal BsaI site found at 594
Illegal BsaI.rc site found at 587
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