Database Study on the Expression and Purification of Membrane Proteins

: Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta-barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

Dysfunction of EAAT3 enhances LPS-induced Postoperative Cognitive Dysfunction

Background Studies have shown that excitatory amino acid transporter 3 (EAAT3) function inhibition is related to several neurodegenerative diseases. Our previous studies also found that the EAAT3 function is intimately linked to learning and memory. In this study, we examined the role of EAAT3 in postoperative cognitive dysfunction (POCD) and explored the potential benefit of riluzole against POCD. Methods We measured EAAT3 protein expression in hippocampus of male mice at different ages. Next, we established a recombinant adeno-associated viral (rAAV)-mediated shRNA to knockdown EAAT3 expression in the hippocampus of adult male mice. And then the mice received 2µg of lipopolysaccharide (LPS) intracerebroventricular microinjection to construct the POCD model. In addition, we intraperitoneally injected 4mg/kg of riluzole 2 days before LPS microinjection for consecutive 3 days in elderly male mice. Cognitive function was assessed using a Morris water maze 24h after LPS microinjection. Animal behavioral tests, as well as pathological and biochemical assays, were performed to clarify the role of EAAT3 function in POCD and evaluate the effect of activation of EAAT3 function by riluzole. Results We found that the expression of EAAT3 was significantly decreased in old mice and EAAT3 knockdown in hippocampus aggravated LPS-induced learning and memory deficits in adult male mice. LPS significantly inhibited hippocampal EAAT3 membrane protein expression and GluA1 protein phosphorylation level in adult male mice. Moreover, riluzole pretreatment significantly increased hippocampal EAAT3 membrane protein expression and ameliorated LPS-induced cognitive impairment in old male mice. Conclusions Our results demonstrated that the dysfunction of EAAT3 is an important risk factor for POCD susceptibility and riluzole may be a promising strategy for prevention and treating of POCD in the elderly people.

Dysfunction of EAAT3 Enhances LPS-induced Postoperative Cognitive Dysfunction

Background: Studies have shown that excitatory amino acid transporter 3 (EAAT3) function inhibition is related to several neurodegenerative diseases. Our previous studies also found that the EAAT3 function is intimately linked to learning and memory. In this study, we examined the role of EAAT3 in postoperative cognitive dysfunction (POCD) and explored the potential benefit of riluzole against POCD. Methods: We measured EAAT3 protein expression in hippocampus of male mice at different ages. Next, we established a recombinant adeno-associated viral (rAAV)-mediated shRNA to knockdown EAAT3 expression in the hippocampus of adult male mice. And then the mice received 2μg of lipopolysaccharide (LPS) intracerebroventricular microinjection to construct the POCD model. In addition, we intraperitoneally injected 4mg/kg of riluzole 2 days before LPS microinjection for consecutive 3 days in elderly male mice. Cognitive function was assessed using a Morris water maze 24h after LPS microinjection. Animal behavioral tests, as well as pathological and biochemical assays, were performed to clarify the role of EAAT3 function in POCD and evaluate the effect of activation of EAAT3 function by riluzole. Results: We found that the expression of EAAT3 was significantly decreased in old mice and EAAT3 knockdown in hippocampus aggravated LPS-induced learning and memory deficits in adult male mice. LPS significantly inhibited hippocampal EAAT3 membrane protein expression and GluA1 protein phosphorylation level in adult male mice. Moreover, riluzole pretreatment significantly increased hippocampal EAAT3 membrane protein expression and ameliorated LPS-induced cognitive impairment in old male mice. Conclusions: Our results demonstrated that the dysfunction of EAAT3 is an important risk factor for POCD susceptibility and riluzole may be a promising strategy for prevention and treating of POCD in the elderly people.

Dysfunction of EAAT3 Enhances LPS-induced Postoperative Cognitive Dysfunction

Background: Studies have shown that excitatory amino acid transporter 3 (EAAT3) function inhibition is related to several neurodegenerative diseases. Our previous studies also found that the EAAT3 function is intimately linked to learning and memory. In this study, we examined the role of EAAT3 in postoperative cognitive dysfunction (POCD) and explored the potential benefit of riluzole against POCD. Methods: We measured EAAT3 protein expression in hippocampus of male mice at different ages. Next, we established a recombinant adeno-associated viral (rAAV)-mediated shRNA to knockdown EAAT3 expression in the hippocampus of adult male mice. And then the mice received 2μg of lipopolysaccharide (LPS) intracerebroventricular microinjection to construct the POCD model. In addition, we intraperitoneally injected 4mg/kg of riluzole 2 days before LPS microinjection for consecutive 3 days in elderly male mice. Cognitive function was assessed using a Morris water maze 24h after LPS microinjection. Animal behavioral tests, as well as pathological and biochemical assays, were performed to clarify the role of EAAT3 function in POCD and evaluate the effect of activation of EAAT3 function by riluzole. Results: We found that the expression of EAAT3 was significantly decreased in old mice and EAAT3 knockdown in hippocampus aggravated LPS-induced learning and memory deficits in adult male mice. LPS significantly inhibited hippocampal EAAT3 membrane protein expression and GluA1 protein phosphorylation level in adult male mice. Moreover, riluzole pretreatment significantly increased hippocampal EAAT3 membrane protein expression and ameliorated LPS-induced cognitive impairment in old male mice. Conclusions: Our results demonstrated that the dysfunction of EAAT3 is an important risk factor for POCD susceptibility and riluzole may be a promising strategy for prevention and treating of POCD in the elderly people.

Dysfunction of EAAT3 Enhances LPS-Induced Postoperative Cognitive Dysfunction

Background: Studies have shown that excitatory amino acid transporter 3 (EAAT3) function inhibition is related to several neurodegenerative diseases. Our previous studies also found that the EAAT3 function is intimately linked to learning and memory. In this study, we examined the role of EAAT3 in postoperative cognitive dysfunction (POCD) and explored the potential benefit of riluzole against POCD. Methods: We performed mutation analysis of SLC1A1 (encoding EAAT3) gene exons in patients of different age groups and measured EAAT3 protein expression in hippocampus of mice at different ages. Next, we established a recombinant adeno-associated viral (rAAV)-mediated shRNA to knockdown EAAT3 expression in the hippocampus of adult male mice. And then the mice received 2μg of lipopolysaccharide (LPS) intracerebroventricular microinjection to construct the POCD model. In addition, we intraperitoneally injected 4mg/kg of riluzole 2 days before LPS microinjection for consecutive 3 days in elderly male mice. Cognitive function was assessed using a Morris water maze 24h after LPS microinjection. Animal behavioral tests, as well as pathological and biochemical assays, were performed to clarify the role of EAAT3 function in POCD and evaluate the effect of activation of EAAT3 function by riluzole. Results: We found that point mutation of SLC1A1 gene exon in elderly patients was significantly different from children and adult people, and expression of EAAT3 was significantly decreased in old mice. And EAAT3 knockdown in hippocampus aggravated LPS-induced learning and memory deficits in adult male mice, and LPS significantly inhibited hippocampal EAAT3 membrane protein expression and GluA1 protein phosphorylation level in adult male mice. Moreover, riluzole pretreatment significantly increased hippocampal EAAT3 membrane protein expression and ameliorated LPS-induced cognitive impairment in old male mice. Conclusions: Our results demonstrated that the dysfunction of EAAT3 is an important risk factor for POCD susceptibility and riluzole may be a promising strategy for prevention and treating of POCD in the elderly people.

An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification

Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies.

Chemiluminescence imaging of cells (CLIC)

Here we describe a simple, sensitive and robust chemiluminescence-based immunoassay (chemiluminescence imaging of cells; CLIC) for relative quantification of proteins in cells. We first employed this method to quantify complement activation in cultured mammalian cells, and to quantify membrane protein expression, shedding, binding and internalization. Moreover, through specific membrane permeabilization we were able to quantify both cytosolic and nuclear proteins, and their translocation. We validated the CLIC quantification method by performing parallel experiments with other quantification methods like ELISA, qPCR, and immunofluorescence microscopy. The workflow of the immunoassay was found to be advantageous in certain instances when compared to these quantification methods. Since the reagents used for CLIC are common to other immunoassays with no need for specialized equipment, and due to the good linearity, dynamic range and signal stability inherent to chemiluminescence, we suggest that this assay is suitable for both small scale and high throughput relative protein quantification studies in whole cells