Large-scale protein expression for proteome research

Korf, Ulrike; Kohl, Thorsten; Zandt, Hans van der; Zahn, Regina; Schleeger, Simone; Ueberle, Barbara; Wandschneider, Silke; Bechtel, Stephanie; Schnölzer, Martina; Ottleben, Holger; Wiemann, Stefan; Poustka, Annemarie

doi:10.1002/pmic.200401195

Cited by 51 publications

(34 citation statements)

References 29 publications

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“…This probably stems from the intracellular nature of GST and the construction of secretion-based vectors in this system. Of fusion proteins commonly used in E. coli, GST is also one of the least effective, 11 although it has proven to be of some utility for the production of intracellular proteins in baculovirus. It is also a particularly effective purification tag.…”

Section: Discussionmentioning

confidence: 99%

Fusion partners can increase the expression of recombinant interleukins via transient transfection in 2936E cells

et al. 2010

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The expression levels of five secreted target interleukins 15, 17B, 32, and IL23 p19 subunit) were tested with three different fusion partners in 2936E cells. When fused to the N-terminus, human serum albumin (HSA) was found to enhance the expression of both IL-17B and IL-15, cytokines which did not express at measurable levels on their own. Although the crystallizable fragment of an antibody (Fc) was also an effective fusion partner for IL-17B, Fc did not increase expression of IL-15. Fc was superior to HSA for the expression of the p19 subunit of IL-23, but no partner led to measurable levels of IL-32c secretion. Glutathione S-transferase (GST) did not enhance the expression of any target and suppressed the production of IL-11, a cytokine which expressed robustly both on its own and when fused to HSA or Fc. Cleavage of the fusion partner was not always possible. The use of HSA or Fc as N-terminal fusions can be an effective technique to express difficult proteins, especially for applications in which the fusion partner need not be removed.

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Section: Discussionmentioning

confidence: 99%

Fusion partners can increase the expression of recombinant interleukins via transient transfection in 2936E cells

et al. 2010

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“…Bacterial transformations and protein inductions were performed as described in ref. 62. Bacterial BL21 (DE3) cells were grown overnight in 2ϫ YTA medium using ampicillin (Sigma) selection.…”

Section: Methodsmentioning

confidence: 99%

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Kabbani

Woll

Levenson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Nicotine acetylcholine receptors (nAChRs) comprise a family of ligand-gated channels widely expressed in the mammalian brain. The ␤2 subunit is an abundant protein subunit critically involved in the cognitive and behavioral properties of nicotine as well as in the mechanisms of nicotine addiction. In this work, we used matrixassisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS) to uncover protein interactions of the intracellular loop of the ␤2 subunit and components of immunoprecipitated ␤2-nAChR complexes from mouse brain. Using the ␤2-knockout mouse to exclude nonspecific binding to the ␤2 antibody, we identify 21 nAChR-interacting proteins (NIPs) expressed in brain. Western blot analysis confirmed the association between the ␤2 subunit and candidate NIPs. Based on their functional profiles, the hypothesis is suggested that the identified NIPs can regulate the trafficking and signaling of the ␤2-nAChR. Interactions of the ␤2 subunit with NIPs such as G protein ␣, G protein-regulated inducer of neurite outgrowth 1, and G proteinactivated K ؉ channel 1 suggest a link between nAChRs and cellular G protein pathways. These findings reveal intracellular interactions of the ␤2 subunit and may contribute to the understanding of the mechanisms of nAChR signaling and trafficking in neurons.mass spectrometry ͉ receptor complex N icotine is a common drug of addiction and a leading cause of preventable deaths in developed countries (1, 2). The target of nicotine is a class of nicotinic acetylcholine receptors (nAChRs) existing as pentameric channels made up of various receptor subunits and distributed throughout the brain (3). To date, 11 neuronal subunits have been identified: ␣2-␣9 and ␤2-␤4. The specific combination of these subunits confers the pharmacological and physiological properties of the nAChR (4). Studies show that Ϸ90% of the high-affinity nicotine receptor sites within the brain consist of the ␣4␤2-nAChR (5, 6). Topological analysis of nAChR subunits indicates the presence of a large, highly divergent intracellular loop (M3-M4 loop) that is implicated in trafficking and targeting properties of nAChRs (7,8). Recent analysis of a prokaryotic homolog of the nAChR family reveals that the M3-M4 loop is unique to the evolution of the nAChR in eukaryotes (9).The generation of subunit-specific knockout (KO) mice has proven valuable in defining the role of individual nAChR subunits in brain physiology and animal behavior (10). Experiments in mice lacking the ␤2 subunit (␤2 Ϫ/Ϫ ) demonstrate an important role for this receptor subunit in neuronal development and plasticity (10), protection from excitotoxicity (11), and the mechanisms underlying cognitive and social behaviors (12). ␤2 Ϫ/Ϫ mice also exhibit a loss of nicotine self-administration and nicotine-elicited firing and dopamine release from dopaminergic neurons of the ventral tegmental area (13,14). Because reexpression of the ␤2 subunit in ␤2 Ϫ/Ϫ mice was found sufficient to restore the electrophysiological and beh...

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“…Previous work has established that various protein fusion vectors featuring rTEV protease cleavage sites are viable for the purification of high levels of recombinant protein through highthroughput approaches (Dummler et al, 2005;Korf et al, 2005) or via ligation independent cloning (Cabrita et al, 2006). Here we report on the construction of two new series of vectors to the growing family of rTEV-cleavable protein fusion vectors that allow for efficient purification of recombinant bacterial proteins.…”

Section: Introductionmentioning

confidence: 99%

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

Rocco

Dennison

Klenchin

et al. 2008

Plasmid

119

121

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We describe the construction and use of two sets of vectors for the over-expression and purification of protein from E. coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His 6 ) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His 6 and maltose-binding protein tags in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.

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Large-scale protein expression for proteome research

Cited by 51 publications

References 29 publications

Fusion partners can increase the expression of recombinant interleukins via transient transfection in 2936E cells

Fusion partners can increase the expression of recombinant interleukins via transient transfection in 2936E cells

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

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