Production of Phosphorylated Ric-8A proteins using protein kinase CK2.


:Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activities of Ric-8A are regulated by phosphorylation of five serine and threonine residues within protein kinase CK2 consensus sites. The traditional way that Ric-8A proteins have been purified is from Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells. Endogenous insect cell kinases do phosphorylate the critical regulatory sites of recombinant Ric-8A reasonably well, but there is batch-to-batch variability among recombinant Ric-8A preparations. Additionally, insect cell-production of some Ric-8 proteins with phosphosite alanine substitution mutations is proscribed as there seems to be interdependency of multi-site phosphorylation for functional protein production. Here, we present a method to produce wild type and phosphosite mutant Ric-8A proteins that are fully occupied with bound phosphate at each of the regulatory positions. Ric-8A proteins were expressed and purified from E. coli. Purified Ric-8A was phosphorylated in vitro with protein kinase CK2 and then re-isolated to remove kinase. The phosphorylated Ric-8A proteins were ∼99% pure and the completeness of phosphorylation was verified by chromatography, phos-tag SDS-PAGE mobility shifts, immunoblotting using phospho-site specific antibodies, and mass spectrometry analysis. E. coli-produced Ric-8A that was phosphorylated using this method promoted a faster rate of Gα subunit guanine nucleotide exchange than Ric-8A that was variably phosphorylated during production in insect cells.


Protein Expr Purif


Yu W,Yu M,Papasergi-Scott MM,Tall GG




Has Abstract


2019-02-01 00:00:00












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    abstract::α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC. is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae....

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Mrázek H,Benada O,Man P,Vaněk O,Křen V,Bezouška K,Weignerová L

    更新日期:2012-01-01 00:00:00

  • Heterologous protein production in Escherichia coli using the propionate-inducible pPro system by conventional and auto-induction methods.

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lee SK,Keasling JD

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zhang YL,Chen SS,Yang KG,Su L,Deng YC,Liu CZ

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  • Molecular cloning, expression, and functional analysis of the copper amine oxidase gene in the endophytic fungus Shiraia sp. Slf14 from Huperzia serrata.

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Yang H,Peng S,Zhang Z,Yan R,Wang Y,Zhan J,Zhu D

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Dukkipati A,Park HH,Waghray D,Fischer S,Garcia KC

    更新日期:2008-12-01 00:00:00

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Karlsson G

    更新日期:2003-01-01 00:00:00

  • Improved inducible expression of Bacillus naganoensis pullulanase from recombinant Bacillus subtilis by enhancer regulation.

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    pub_type: 杂志文章


    authors: Deng Y,Nie Y,Zhang Y,Wang Y,Xu Y

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  • A modified clear-native polyacrylamide gel electrophoresis technique to investigate the oligomeric state of MBP-5-HT3A-intracellular domain chimeras.

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    pub_type: 杂志文章


    authors: Pandhare A,Stuebler AG,Pirayesh E,Jansen M

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zawilak-Pawlik AM,Kois A,Zakrzewska-Czerwinska J

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  • Heterologous expression of the avirulence gene product, NIP1, from the barley pathogen Rhynchosporium secalis.

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    pub_type: 杂志文章


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  • Affinity purification of native glycodelin from amniotic fluid for biological investigations and development of a glycodelin ELISA for clinical studies.

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    pub_type: 杂志文章


    authors: Sørensen S,Myrhøj V,Nguyen TH,Aaslo P,Hansen YB

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    pub_type: 杂志文章


    authors: Bane SE,Velasquez JE,Robinson AS

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    pub_type: 杂志文章


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    pub_type: 杂志文章


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    pub_type: 杂志文章


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    pub_type: 杂志文章


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    pub_type: 杂志文章


    authors: Platis D,Foster GR

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  • Production and purification of a recombinant human hsp60 epitope using the cellulose-binding domain in Escherichia coli.

    abstract::The heat shock protein hsp60 plays a functional role in insulin-dependent diabetes mellitus. The hsp60 epitope p277 (aa 437-aa 460) is effective in vaccinating mice against diabetes. A synthetic peptide gene (p277) that encodes the human hsp60 epitope was cloned to the 3' end of the cellulose-binding domain gene (cbd)...

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    pub_type: 杂志文章


    authors: Shpigel E,Elias D,Cohen IR,Shoseyov O

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    abstract::alpha 1-Acid glycoprotein could be isolated by a one-step extraction method from human sera and plasma. Protein recovered in the water phase after extraction with phenol at 70 degrees C for 20 min was verified as human alpha 1-acid glycoprotein when it was compared with the reference standard human alpha 1-acid glycop...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


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  • Membrane protein expression and production: effects of polyhistidine tag length and position.

    abstract::Polyhistidine tags enable the facile purification of proteins by immobilized metal affinity chromatography (IMAC). Both the type and position of purification tags can affect significantly properties of a protein such as its expression level, behavior in solution, and its ability to form suitable samples (esp. suitable...

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    pub_type: 杂志文章


    authors: Mohanty AK,Wiener MC

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  • Robust and facile purification of full-length, untagged human Nedd4 as a recombinant protein from Escherichia coli.

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    pub_type: 杂志文章


    authors: Hatstat AK,McCafferty DG

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  • Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins.

    abstract::The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zheng Y,Xie J,Huang X,Dong J,Park MS,Chan WK

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  • Confronting high-throughput protein refolding using high pressure and solution screens.

    abstract::Over-expression of heterologous proteins in Escherichia coli is commonly hindered by the formation of inclusion bodies. Nevertheless, refolding of proteins in vitro has become an essential requirement in the development of structural genomics (proteomics) and as a means of recovering functional proteins from inclusion...

    journal_title:Protein expression and purification

    pub_type: 杂志文章,评审


    authors: Qoronfleh MW,Hesterberg LK,Seefeldt MB

    更新日期:2007-10-01 00:00:00

  • Purification of rat pro-atrial natriuretic factor: a simplified scheme using reversed-phase high-performance liquid chromatography.

    abstract::A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-s...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Belcourt D,Varma DR,Toney K,Bennett HP

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  • Functional expression and characterization of the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae.

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chen S,Halkier BA

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  • Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli.

    abstract::Human dermcidin, an anionic antimicrobial peptide expressed in the pons of the brain and the sweat glands, displays antimicrobial activity against pathogenic microorganisms such as Staphylococcus aureus and Candida albicans. Here, we describe the recombinant production of a 48 amino acid dermcidin variant with C-termi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Cipáková I,Gasperík J,Hostinová E

    更新日期:2006-02-01 00:00:00

  • Optimization of human D-amino acid oxidase expression in Escherichia coli.

    abstract::Human D-amino acid oxidase (hDAAO) is a flavoprotein that plays a key role in the pathophysiology of schizophrenia. So far, the biochemical characterization of this enzyme has been hampered by the difficulty of expressing it in a common heterologous host such as Escherichia coli. Increasing amounts of recombinant hDAA...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Romano D,Molla G,Pollegioni L,Marinelli F

    更新日期:2009-11-01 00:00:00

  • Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography.

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Tan LC,Chua AJ,Goh LS,Pua SM,Cheong YK,Ng ML

    更新日期:2010-11-01 00:00:00

  • Expression and one-step purification of intracellular human prolactin in insect cells.

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Strokovskaya L,Bartoszewicz Z,Szolajska E,Kikhno I,Solomko A,Michalik J

    更新日期:2001-07-01 00:00:00

  • Efficient bacterial expression of recombinant potato mop-top virus non-structural triple gene block protein 1 modified by progressive deletion of its N-terminus.

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    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Pecenková T,Filigarová M,Cerovská N

    更新日期:2005-05-01 00:00:00