Purification and immunohistochemical analysis of calcium-binding proteins expressed in the chick pineal gland

ArticleinJournal of Pineal Research 34(3):208-16 · May 2003with5 Reads
DOI: 10.1034/j.1600-079X.2003.00031.x · Source: PubMed
The pineal gland is a site of melatonin production, of which intracellular calcium ions (Ca2+) are likely involved in various aspects. To investigate the identity of molecules responsible for the Ca2+-dependent processes in the pineal cells, we prepared a cellular extract from 2000 chick pineal glands and isolated a series of Ca2+-binding proteins by taking advantage of their Ca2+-dependent hydrophobic interaction with phenyl-Sepharose beads. The proteins identified by micro-sequencing analysis included calmodulin, neurocalcin, sorcin, annexin II and annexin V. Immunohistochemical analysis of the chick pineal sections revealed that both calmodulin and sorcin are expressed in the follicular and parafollicular pinealocytes. On the other hand, neurocalcin was expressed in a few neuron-like cells located predominantly in the parafollicular layer of the pineal follicle. These results suggest that calmodulin and sorcin may contribute to cellular functions in the chick pinealocytes.
    • "The opening of the Fh8's structure exposes a large hydrophobic surface that becomes available for interaction with its targets (Fraga et al., 2010). In this study, the Fh8 tag and Fh8-fused proteins presented a calcium-dependent interaction with a hydrophobic resin, and, as reported for other calcium-binding proteins (Rozanas, 1998; Shimizu et al., 2003), this interaction was still occurring even with low salt concentration in the mobile phase. The low salt concentration decreases the unspecific binding of other proteins from the E. coli extracts, thus promoting selectivity toward the purification of the fusion protein of interest (Costa et al., 2013b). "
    [Show abstract] [Hide abstract] ABSTRACT: Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide's immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system.
    Full-text · Article · Feb 2014
    • "The later was selected to perform the purification of Fh8 fusion proteins as it allowed a single-step and rapid elution of all bound proteins. The use of a calcium chelating agent has already been demonstrated to be effective for the elution of other calcium binding proteins [17,22,23]. In fact, this elution mechanism proved to be highly selective towards Fh8 tag since other proteins of superior molecular weight (between 20–25 and 50–75 kDa) were only observed in the elution with pH 10, after the first elution with EDTA. "
    [Show abstract] [Hide abstract] ABSTRACT: Downstream processing is still a major bottleneck in recombinant protein production representing most of its costs. Hence, there is a continuing demand of novel and cost-effective purification processes aiming at the recovery of pure and active target protein. In this work, a novel purification methodology is presented, using the Fh8 solubility enhancer tag as fusion handle. The binding properties of Fh8 tag to a hydrophobic matrix were first studied via hydrophobic interaction chromatography (HIC). The Fh8 tag was then evaluated as a purification handle by its fusion to green fluorescent protein and superoxide dismutase. The purification efficiency of the Fh8-HIC strategy was compared to the immobilized metal ion affinity chromatography (IMAC) using the His6 tag. Results showed that the Fh8-HIC binding mechanism is calcium-dependent in a low salt medium, making the purification process highly selective. Both target proteins were biologically active, even when fused to Fh8, and were successfully purified by HIC, achieving efficiencies identical to those of IMAC. Thus, the Fh8 acts as an effective affinity tag that, together with its previously reported solubility enhancer capability, allows for the design of inexpensive and successful recombinant protein production processes in Escherichia coli.
    Full-text · Article · Sep 2013
    • "Each pool of the frozen glands was homogenized separately with 200 lL of sodium dodecyl sulfate–polyacrylamide gel electrophoresis loading buffer containing 20 mmol/L NaH 2 PO 4 , 10 mmol/L Na 2 H 2 P 2 O 7 , 1.6 mmol/L EDTA, and 1.6 mmol/L EGTA, and the total protein concentration was estimated by the method of Bradford (1976) to normalize sample-to-sample variation. Equal amounts of proteins were loaded onto an 8.5% polyacrylamide gel and were subjected to immunoblotting as described previously (Shimizu et al. 2003). "
    [Show abstract] [Hide abstract] ABSTRACT: Stimulus-induced transcription of the Period gene is a critical step for phase-shift of vertebrate circadian systems. The promoter region of chicken Period2 contains a canonical calcium/cAMP-responsive element, but its functional relevance is not known. The present study shows that cAMP-responsive element-binding protein (CREB) and activating transcription factor-2 (ATF-2) bind to the promoter region of the Period2 gene in the chick pineal gland. In transient transfection assays, a reporter construct containing 0.7-kbp upstream region of chicken Period2 was transactivated by ATF-2, but it was poorly responsive to CREB. In the chick pineal gland, phosphorylation of CREB protein at the kinase-inducible domain was negatively regulated by light. On the other hand, phosphorylation of ATF-2 at the amino-terminal transactivation domain exhibited a circadian rhythm with a daytime peak, suggesting a role for ATF-2 in circadian rhythmicity in the chick pineal gland.
    Article · Jan 2008
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