Expression of Water-soluble, Ligand-binding Concatameric Extracellular Domains of the Human Neuronal Nicotinic Receptor α4 and β2 Subunits in the Yeast Pichia pastoris

Stergiou, Christos; Zisimopoulou, Paraskevi; Tzartos, Socrates J.

doi:10.1074/jbc.m110.171645

Cited by 9 publications

(5 citation statements)

References 42 publications

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“…Notably, the presence of a major band at (30 kDa) is well correlated with the size of the monomeric α-7 nAChR ECD. It should be noted that these results were in agreement with the expression of human neuronal nicotinic receptor α4 and β2 subunits in P. pastoris, which were also expressed in glycosylated forms [20]. Besides P. pastoris, the glycosylation of recombinant α-7 nAChR was also detected in the proteins expressed in Xenopus oocytes [21].…”

Section: Purification Of Recombinant Human α-7 Nachr Ecdsupporting

confidence: 84%

Expression and purification of human alpha-7 nicotinic acetylcholine receptor extracellular domain in Pichia pastoris

Chusuth¹,

Raksat²,

Hannongbua³

et al. 2023

ScienceAsia

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Human α-7 nicotinic acetylcholine receptor (α-7 nAChR) is an ion channel that plays a significant function in neuronal communication and development. Defects of α-7 nAChR could cause plethora of neurologic and psychiatric impairments such as schizophrenia, Alzheimer's, and inflammatory disorders. Hence, α-7 nAChR is an interesting candidate for drug development targeting neurological and inflammatory diseases. Therefore, the ability to obtain significant amount of the α-7 nAChR extracellular domain is crucial for further biochemical and structural investigations. We constructed a Pichia pastoris yeast strain that secretes the α-7 nAChR extracellular domain. The recombinant protein was hyperglycosylated and could be deglycosylated with Endoglycosidase H treatment. Crosslinking experiments suggested that the recombinant protein could form oligomeric states, consistent with the known property of the native α-7 nAChR. The optimized expression yield was 4.9 mg/l.

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Section: Purification Of Recombinant Human α-7 Nachr Ecdsupporting

confidence: 84%

Expression and purification of human alpha-7 nicotinic acetylcholine receptor extracellular domain in Pichia pastoris

Chusuth¹,

Raksat²,

Hannongbua³

et al. 2023

ScienceAsia

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“…At present, most studies about the relationship between nicotine administration and cannabinoid system are related to the addiction of nicotine [ 33 – 35 ]. Although nAchRs, including α 3-, α 4-, and α 7-AchRs, are believed to be involved in nicotine-induced bioactivities [ 36 – 38 ], and a study reported that α 3-nAchR and CB2 receptor were expressed in the same neural cells of brain tissue in mice [ 39 ], additional studies are needed to explore the mechanism that nicotine stimulates CB2 receptor and increases its expression.…”

Section: Discussionmentioning

confidence: 99%

Cannabinoid CB2 Receptor Mediates Nicotine-Induced Anti-Inflammation in N9 Microglial Cells Exposed toβAmyloid via Protein Kinase C

Jia

Peng

et al. 2016

Mediators of Inflammation

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Background. Reducing β amyloid- (Aβ-) induced microglial activation is considered to be effective in treating Alzheimer's disease (AD). Nicotine attenuates Aβ-induced microglial activation; the mechanism, however, is still elusive. Microglia could be activated into classic activated state (M1 state) or alternative activated state (M2 state); the former is cytotoxic and the latter is neurotrophic. In this investigation, we hypothesized that nicotine attenuates Aβ-induced microglial activation by shifting microglial M1 to M2 state, and cannabinoid CB2 receptor and protein kinase C mediate the process. Methods. We used Aβ1–42 to activate N9 microglial cells and observed nicotine-induced effects on microglial M1 and M2 biomarkers by using western blot, immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA). Results. We found that nicotine reduced the levels of M1 state markers, including inducible nitric oxide synthase (iNOS) expression and tumor necrosis factor α (TNF-α) and interleukin- (IL-) 6 releases; meanwhile, it increased the levels of M2 state markers, including arginase-1 (Arg-1) expression and brain-derived neurotrophic factor (BDNF) release, in the Aβ-stimulated microglia. Coadministration of cannabinoid CB2 receptor antagonist or protein kinase C (PKC) inhibitor partially abolished the nicotine-induced effects. Conclusion. These findings indicated that cannabinoid CB2 receptor mediates nicotine-induced anti-inflammation in microglia exposed to Aβ via PKC.

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“…We found a strategy to clone large fragments of DNA by dividing them to short PCR fragment cloning, followed by restriction digestion and ligation cloning 9 . This strategy shows dominance in amplifying efficiency, it helps overcome PCR problem, and constructs very big clones without turning to complex reagents and technologies, like Red/ET system 10 or TAR system 11 , 12 .…”

Section: Introductionmentioning

confidence: 99%

Restriction-based Multiple-fragment Assembly Strategy to Avoid Random Mutation during Long cDNA Cloning

Wang¹,

Chen²,

Zhang³

et al. 2015

J. Cancer

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Long fragment cloning is a challenge for its difficulty in accurate amplifying and tendency to get unwanted mutation. Here we discuss Restriction-based Multiple-fragment Assembly Strategy's advantages and limitations. In this strategy, rather than PCR amplifying the entire coding sequence (CDS) at one time, we amplified and sequenced smaller fragments which are shorter than 1.5kb spanning the CDS. After that, the sequence-proved fragments were assembled by digestion-ligation cloning to the target vector. We test its universality in our script programmed in Python. Our data shows that, among the entire human and mouse CDS, at least 70% of long CDS cloning will benefit from this strategy.

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Expression of Water-soluble, Ligand-binding Concatameric Extracellular Domains of the Human Neuronal Nicotinic Receptor α4 and β2 Subunits in the Yeast Pichia pastoris

Cited by 9 publications

References 42 publications

Expression and purification of human alpha-7 nicotinic acetylcholine receptor extracellular domain in Pichia pastoris

Expression and purification of human alpha-7 nicotinic acetylcholine receptor extracellular domain in Pichia pastoris

Cannabinoid CB2 Receptor Mediates Nicotine-Induced Anti-Inflammation in N9 Microglial Cells Exposed toβAmyloid via Protein Kinase C

Restriction-based Multiple-fragment Assembly Strategy to Avoid Random Mutation during Long cDNA Cloning

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