Based on the human NMDAR1 gene sequence, Phyre 2 software was used to predict the tertiary structure of the protein and analyze its domains. Primers were designed to amplify nucleic acid fragments encoding different domains of NMDAR1 outer membrane protein using RT-PCR method, and inserted into the prokaryotic expression vector pCold-SUMO to construct a recombinant plasmid. The correct recombinant was identified to transform E. coli BL21(DE3), and IPTG induced the expression and purification of the target protein. Protein purity was determined using SDS-PAGE, and immunoreactivity was identified using Western blotting.
Three DNA sequences of the extramembrane part of NMDAR1 were cloned, namely NR1-M1, NR1-S1 and NR1-S2. The NR1-S1 and NR1-S2 fragments are connected using G (glycine) and T (threonine) as linkers to form a composite fragment. After colony PCR screening and sequencing identification, the recombinant plasmids pCold-SUMO-M1 and pCold-SUMO-S1-GT-S2 were successfully constructed. SDS-PAGE identification results showed that the recombinant plasmid could express soluble NR1-M1 and NR1-S1-GT-S2 proteins in E. coli. The expression product was subjected to affinity chromatography and gel filtration chromatography to obtain high-purity target protein. Western blotting confirmed that the purified target protein can specifically bind to the corresponding antibody.
Figure 1. Analysis of the purified protein by Western blotting. M1-1: 0.5 μg NR1-M1 purified protein, incubated with mouse anti-NMDAR1 monoclonal antibody; M1-2: pCold-SUMO vector control, incubated with mouse anti-NMDAR1 monoclonal antibody; M1-3: 0.5 μg NR1-M1 purified protein incubated with mouse IgG; S1-GT-S2-1: 0.5 μg NR1-S1-GT-S2 purified protein, incubated with rabbit anti-NMDAR mAb; S1-GT-S2-2: pCold-SUMO vector control, incubated with rabbit anti-NMDAR mAB; S1-GT-S2-3: 0.5 μg S1-GT-S2 purified protein incubated with rabbit IgG; M: protein marker. (Bao, Ding, et al., 2017)
A: The prokaryotic expression vector pCold-SUMO is modified from the pCold vector, whose promoter (CS Promoter) originates from Antarctic psychophilic bacteria to initiate protein expression at low temperatures (15 degrees).
A: Under the low temperature, it slows down the protein synthesis speed, which increases the probability of correct protein folding, improves the soluble expression of protein, and enhances the expression ratio of active protein
A: Cold promoter;Induced expression at low temperature;Limit the protein solubility.
A: The pCold-SUMO vector is reformed based on the pCold carrier vector.
A: The CS Promoter is derived from Antarctic eosinophilic bacteria.
A: Protein expression can be activated at low temperature (15 degrees).
A: The SUMO tag greatly improves the expression of small molecular weight proteins and increases the soluble expression probability of the protein.
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The SUMO tag contained in this expression system can greatly increase the expression amount of small molecular weight protein, and further improve the soluble expression probability of the protein.
The TEE signal peptide of this vector can enhance the high expression of the target protein under the regulation of the cold promoter.
The vector can greatly increase the protein expression, and its molecular chaperone can assist the correct folding of the protein, which is recommended.
The proteins we studied are difficult to induce expression, and the protein expression was greatly increased after using this vector.
I have been using the pCold-SUMO vector for my protein expression experiments, and I am extremely satisfied with the results. The design of this vector, incorporating the cold shock gene CSPA promoter and the lac operon, allows for efficient and controllable expression of proteins in E.coli.
The pCold-SUMO vector has revolutionized our protein expression experiments. Its innovative design, utilizing the cold shock gene CSPA promoter, has significantly improved the efficiency and yield of protein expression in our lab.
I've recently purchased and used the pCold-sumo vector for my protein research and have found it to be an indispensable tool in my lab work. This product stands out because of its significant efficiency and high performance.
The pCold-sumo vector is designed for high-level protein expression and its SUMO (Small Ubiquitin-like Modifier) fusion system enhances the solubility of proteins, which is beneficial to the function and structure of some proteins.
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