Part:BBa_K4968019
SUMO-SmtA-CBM-sfGFP
The Composite part consist of SmtA (BBa_K4968001), CBM (BBa_K4968002), sfGFP (BBa_K4968003), and SUMO (BBa_K4968007).
Also is designed for the improvement of the part SmtA-CBM-sfGFP (BBa_K4968018) by adding the optimized SUMO tag (BBa_K4968003).
SmtA (BBa_K4968001), CBM (BBa_K4968002) is a protein belonging to the metallothionein family, and it has the ability to adsorb heavy metals and chelate these heavy metals in an inactive form. Carbohydrate-binding modules (CBMs) (BBa_K4968002) are components of several enzymes and can bind specific carbohydrates. sfGFP (BBa_K4968003) can produce green fluorescence while ensuring that there is no misfolding of the fused proteins during expression. Additionally, to enhance the expression levels of proteins, promote proper folding of the target protein, and increase the solubility of recombinant proteins, we have employed a Small Ubiquitin-like Modifier (SUMO) (BBa_K4968003) as a fusion tag and molecular chaperone.
Usage & Biology
SUMO (Small Ubiquitin-like Modifier)(BBa_K4968003) is a ubiquitin-like protein found in eukaryotic organisms and is highly conserved (Vertegaal, 2022). It belongs to a class of large proteins involved in protein-small ubiquitin-like modification (SUMOylation), a common post-translational modification (PTM). SUMO (BBa_K4968003) plays a significant role in regulating the stability and biological functions of proteins.
SUMO (BBa_K4968003) is also frequently used as an efficient and valuable tag for protein expression and purification. In comparison to tags like GST, MBP, or NusA, SUMO (BBa_K4968003) not only serves as a fusion tag for recombinant protein expression but also functions as a molecular chaperone. It promotes proper protein folding, exhibits tolerance to heat and proteases, and enhances the stability of the target protein. When SUMO (BBa_K4968003) is fused to the N-terminus of the target protein as a tag, it can improve protein folding, solubility, and yield. Additionally, SUMO tags (BBa_K4968003) are recognized by highly specific proteases that cleave SUMO (BBa_K4968003) at its tertiary structure, leaving no residual amino acids behind, making it suitable for recombinant protein expression.
Metallothionein (MT), is a class of metal-binding proteins widely found in living organisms. It is a low molecular weight protein (2-7 kD) rich in cysteine (20%-30%) and devoid of histidine and aromatic amino acids (Feng et al., 2017). MT can be induced by metals, cytokines, hormones, cytotoxic drugs, organic chemicals, and stress. MT plays a crucial role in regulating the concentration of trace elements within the organism and detoxifying heavy metals. Additionally, it is involved in the regulation of hormones, cellular metabolism, control of cell differentiation and proliferation, and participates in UV-induced reactions and free radical scavenging (Haq, 2003).
Cyanobacterial metallothionein (BBa_K4968001), originating from Synechococcus PCC 7942, is a protein belonging to the metallothionein family. It can sense and bind specific heavy metal ions such as cadmium, lead, zinc, and others, thereby assisting in regulating the cell's tolerance and metabolism of these toxic metals. It prevents these metal ions from entering the cell or transferring them to cellular compartments by forming complexes with them, thus protecting the cell from the toxicity of heavy metals (Blindauer, 2011; Blindauer, Razi, Campopiano, & Sadler, 2007). 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). CBMs (BBa_K4968002) can recognize and selectively bind to characteristics on the crystal surface, providing possibilities for modifying cellulose. Research by Kevin Aïssa and others has found that CBMs (BBa_K4968002) have a strong affinity for microcrystalline cellulose, and the addition of this module assists in protein immobilization.
sfGFP (superfolder green fluorescent protein) (BBa_K4968003) is a fluorescent tagging tool designed to ensure that it does not induce misfolding of fused proteins when expressed alongside other proteins. This means that even when fused with poorly folded peptides (Pédelacq et al., 2005), sfGFP (BBa_K4968003) can maintain a well-folded state, thus improving the stability of fused proteins.
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 |These figures (from A-E) indicated that the Scanning Electron Microscopy (SEM) observation of the fusion protein SUMO-SmtA-CBM-sfGFP. From picture A to E, the magnifications are 1.00KX, 2.00KX, 5.00KX, 10.00KX, 20.00KX(done by Yuantest Laboratory).
Figure 3 |Comparison of microcrystalline cellulose and microcrystalline cellulose bound with fusion protein containing CBM part.
Figure 4 |The figure shows the ICP-MS results of the fusion protein SUMO-SmtA-CBM-sfGFP adsorbing cadmium ions, copper ions, and lead ions at a time of 4 hours, a temperature of 25°C, and a pH of 6. The concentrations of the three ions are all 20 mg/L. It can be seen from the figure that the adsorption efficiency for cadmium ions is 52.8%, the adsorption efficiency for copper ions is 62.2%, and the adsorption efficiency for lead ions is 71.5%.
The composite forms of these components have diverse applications: SUMO-fusion tags enhance the stability and solubility of target proteins, Metallothionein composite components are used to regulate cell tolerance and metabolism of heavy metals, Carbohydrate-Binding Modules (CBMs) can be employed for specific enzyme engineering and protein immobilization, and sfGFP, as a fluorescent tag, is useful for studying protein localization and interactions. The combination of these components forms multifunctional tools that contribute to protein engineering, heavy metal tolerance research, and various applications in molecular biology 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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