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












  • Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa.

    abstract::Glucose-6-phosphate dehydrogenase (G6PDH) (EC plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Acero-Navarro KE,Jiménez-Ramírez M,Villalobos MA,Vargas-Martínez R,Perales-Vela HV,Velasco-García R

    更新日期:2018-02-01 00:00:00

  • Biotechnological applications of elastin-like polypeptides and the inverse transition cycle in the pharmaceutical industry.

    abstract::Proteins are essential throughout the biological and biomedical sciences and the purification strategies of proteins of interest have advanced over centuries. Elastin-like polypeptides (ELPs) are compound polymers that have recently been highlighted for their sharp and reversible phase transition property when heated ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章,评审


    authors: Fletcher EE,Yan D,Kosiba AA,Zhou Y,Shi H

    更新日期:2019-01-01 00:00:00

  • High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts.

    abstract::(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones. This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hasslacher M,Schall M,Hayn M,Bona R,Rumbold K,Lückl J,Griengl H,Kohlwein SD,Schwab H

    更新日期:1997-10-01 00:00:00

  • Purification of proteins with native terminal sequences using a Ni(II)-cleavable C-terminal hexahistidine affinity tag.

    abstract::The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a met...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Abd Elhameed HAH,Hajdu B,Balogh RK,Hermann E,Hunyadi-Gulyás É,Gyurcsik B

    更新日期:2019-07-01 00:00:00

  • Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli.

    abstract::We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of criti...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Joshi BH,Puri RK

    更新日期:2005-02-01 00:00:00

  • Generation and application of a 293 cell line stably expressing bovine interferon-gamma.

    abstract::A stable mammalian cell line expressing highly active bovine interferon-gamma (BoIFN-γ) was generated using Flp recombinase-mediated integration. This recombinant 293 cell line (B1) efficiently secreted FLAG-tagged BoIFN-γ protein into the culture supernatant, as determined by ELISA and Western blot. The recombinant B...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Xu Z,Shan F,Shan F,Meng C,Zhou X,Zhang X,Chen X,Jiao X

    更新日期:2014-07-01 00:00:00

  • Zebrafish Sp1-like protein is structurally and functionally comparable to human Sp1.

    abstract::The transcription factor Sp1 is a regulator of TATA-less genes. It belongs to the Cys₂-His₂ zinc finger domain-containing family. A zebrafish cDNA encoding a peptide homologous to mammalian Sp1 was cloned and inserted into a pET43.1a vector and expressed in Escherichia coli Rosetta (DE3) cells as a Nus-His-tag fusion ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lin CJ,Hsiao TH,Chung YS,Chang WN,Yeh TM,Chen BH,Fu TF

    更新日期:2011-03-01 00:00:00

  • Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase.

    abstract::A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: di Salvo ML,Delle Fratte S,De Biase D,Bossa F,Schirch V

    更新日期:1998-07-01 00:00:00

  • Expression and structural properties of a chimeric protein based on the ectodomains of E1 and E2 hepatitis C virus envelope glycoproteins.

    abstract::Hepatitis C virus encodes two enveloped glycoproteins, E1 and E2, which are involved in viral attachment and entry into target cells. We have obtained in insect cells infected by recombinant baculovirus a chimeric secreted recombinant protein, E1(341)E2(661,) containing the ectodomains of E1 and E2. The described proc...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Tello D,Rodríguez-Rodríguez M,Yélamos B,Gómez-Gutiérrez J,Ortega S,Pacheco B,Peterson DL,Gavilanes F

    更新日期:2010-06-01 00:00:00

  • A simple and efficient method for generating high-quality recombinant Mical enzyme for in vitro assays.

    abstract::We have recently identified a new family of multidomain oxidoreductase (redox) enzymes, the MICALs, that directly regulate the actin cytoskeletal elements necessary for the morphology, motility, and trajectory of cells. Our genetic assays reveal that Mical is both necessary and sufficient for actin organization and ce...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wu H,Hung RJ,Terman JR

    更新日期:2016-11-01 00:00:00

  • Heterologous expression and pro-peptide supported refolding of the high specific endopeptidase Lys-C.

    abstract::The high specific lysyl endopeptidase (Lys-C; EC is often used for the initial fragmentation of polypeptide chains during protein sequence analysis. However, due to its specificity it could be a useful tool for the production of tailor-made protein hydrolysates with for example bioactive or techno functiona...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Stressler T,Eisele T,Meyer S,Wangler J,Hug T,Lutz-Wahl S,Fischer L

    更新日期:2016-02-01 00:00:00

  • Xenopus phospho-CDK7/cyclin H expressed in baculoviral-infected insect cells.

    abstract::The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. Th...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lawrie AM,Tito P,Hernandez H,Brown NR,Robinson CV,Endicott JA,Noble ME,Johnson LN

    更新日期:2001-11-01 00:00:00

  • Production and characterization of a bacterial single-chain antibody fragment specific to B-cell-activating factor of the TNF family.

    abstract::An active form of a single-chain antibody fragment (scFv) from the murine monoclonal antibody ABL-1, which is specific for B-cell-activating factor of the TNF family, was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Cao P,Tang XM,Guan ZB,Diao ZY,Zhang SQ

    更新日期:2005-10-01 00:00:00

  • Remedial strategies in structural proteomics: expression, purification, and crystallization of the Vav1/Rac1 complex.

    abstract::The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 bindi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Brooun A,Foster SA,Chrencik JE,Chien EY,Kolatkar AR,Streiff M,Ramage P,Widmer H,Weckbecker G,Kuhn P

    更新日期:2007-05-01 00:00:00

  • Rapid generation of stable cell lines expressing corticotropin-releasing hormone receptor for drug discovery.

    abstract::Human HEK293 cells that stably express the Epstein Barr nuclear antigen 1 (EBNA1) support the episomal replication of plasmids containing the Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E) cell line expressing the human corticotropin-releasing hormone receptor subtype I (CRHR1) from an episomal ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Horlick RA,Sperle K,Breth LA,Reid CC,Shen ES,Robbins AK,Cooke GM,Largent BL

    更新日期:1997-04-01 00:00:00

  • Optimized bacterial expression of human apolipoprotein A-I.

    abstract::Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structur...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ryan RO,Forte TM,Oda MN

    更新日期:2003-01-01 00:00:00

  • Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1.

    abstract::Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble re...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Khine AA,Lu PC,Ko TP,Huang KF,Chen HP

    更新日期:2020-11-01 00:00:00

  • Effects of co-expressing chaperone BiP on functional antibody production in the baculovirus system.

    abstract::The assembly pathway of the insect cell Spodoptera frugiperda (Sf-9) was engineered to include expression of the murine chaperone immunoglobulin heavy chain binding protein (BiP) using the baculovirus vector. The impact of BiP coexpression on the production and secretion of functional and soluble recombinant immunoglo...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hsu TA,Eiden JJ,Bourgarel P,Meo T,Betenbaugh MJ

    更新日期:1994-12-01 00:00:00

  • Purification and characterization of a long-acting ciliary neurotrophic factor via genetically fused with an albumin-binding domain.

    abstract::Ciliary neurotrophic factor (CNTF) is a promising candidate for the treatment of neurodegenerative or metabolic diseases, but suffers rapid clearance in body. Herein we constructed a new long-acting recombinant human CNTF (rhCNTF) by genetic fusion with an albumin-binding domain (ABD) through a flexible peptide linker...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Xu L,Zhang C,Liu L,Zhang Y,Wang Q,Wang J,Liu Y,Su Z

    更新日期:2017-11-01 00:00:00

  • Expression and purification of amyloid-beta peptides from Escherichia coli.

    abstract::Soluble oligomers and fibrillar deposits of amyloid beta (Abeta) are key agents of Alzheimer's disease pathogenesis. However, the mechanism of amyloid aggregation and its interaction with live cells still remain unclear requiring the preparation of large amounts of pure and different Abeta peptides. Here we describe a...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Garai K,Crick SL,Mustafi SM,Frieden C

    更新日期:2009-07-01 00:00:00

  • The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.

    abstract::Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer po...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Nespovitaya N,Barylyuk K,Eichmann C,Zenobi R,Riek R

    更新日期:2014-07-01 00:00:00

  • Identification, expression, and purification of a unique stable domain from human HSPC144 protein.

    abstract::HSPC144 is a newly identified gene in human CD34(+) hematopoietic stem/progenitor cells. In this work, we have expressed and purified the 225-residue protein from Escherichia coli BL21 (DE3) and identified a stable fragment HSPC144-P (residues 44-225) by limited proteolysis method. The HSPC144-P fragment exhibits high...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Song AX,Chang YG,Gao YG,Lin XJ,Shi YH,Lin DH,Hang QH,Hu HY

    更新日期:2005-07-01 00:00:00

  • Analysis of human alpha-thrombin by hydrophobic interaction high-performance liquid chromatography.

    abstract::The major active form of human thrombin, alpha-thrombin, was analyzed by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). The chromatographic system included a polymeric phenyl column and elution was performed by a gradient, 2-0M sodium chloride (5-20 min). Total analysis time was 30 min per ...

    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

  • Expression, proteolytic analysis, reconstitution, and crystallization of the tau60/tau91 subcomplex of yeast TFIIIC.

    abstract::The transcription factor IIIC (TFIIIC) is a multisubunit DNA-binding factor required for promoter recognition and TFIIIB assembly on tRNA genes transcribed by RNA polymerase III. Yeast TFIIIC consists of six subunits, organized in the two globular subcomplexes tauA and tauB, which recognize two internal tDNA promoter ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Mylona A,Acker J,Fernández-Tornero C,Sentenac A,Müller CW

    更新日期:2006-02-01 00:00:00

  • Purification of granulin-like polypeptide from the blood-sucking leech, Hirudo nipponia.

    abstract::A cysteine-rich (approximately 20%), low molecular weight (MW 6 kDa) polypeptide has been isolated from the Korean blood-sucking leech, Hirudo nipponia. From its amino acid composition and N-terminal amino acid sequence analysis, the new protein is similar to granulin (or epithelin), and so it has been named leech gra...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hong SJ,Kang KW

    更新日期:1999-07-01 00:00:00

  • Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones.

    abstract::The production of a single-chain variable fragment (scFv) antibody against bovine ribonuclease A in the cytoplasm of Escherichia coli trxB/gor double mutant was investigated. Previous reports have shown that the thioredoxin (Trx) protein fusion strategy is useful for the correct folding of scFvs and that the expressio...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sonoda H,Kumada Y,Katsuda T,Yamaji H

    更新日期:2010-04-01 00:00:00

  • Heterologous expression of 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability.

    abstract::The gene for the extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed structural and functional studies of the enzyme. The recombinant enzyme was purified to homogenei...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Cacciapuoti G,Fusco S,Caiazzo N,Zappia V,Porcelli M

    更新日期:1999-06-01 00:00:00

  • Cloning, expression, purification, and properties of a putative plasma membrane hexokinase from Solanum chacoense.

    abstract::A full-length hexokinase cDNA was cloned from Solanum chacoense, a wild relative of the cultivated potato. Analysis of the predicted primary sequence suggested that the protein product, ScHK2, may be targeted to the secretory pathway and inserted in the plant plasma membrane, facing the cytosol. ScHK2 was expressed as...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Claeyssen E,Wally O,Matton DP,Morse D,Rivoal J

    更新日期:2006-05-01 00:00:00

  • Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells.

    abstract::Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Cadel S,Gouzy-Darmon C,Petres S,Piesse C,Pham VL,Beinfeld MC,Cohen P,Foulon T

    更新日期:2004-07-01 00:00:00

  • Purification of major lignin peroxidase isoenzymes from Phanerochaete chrysosporium by chromatofocusing.

    abstract::The basidiomycete Phanerochaete chrysosporium produces several isoforms of lignin peroxidase, which catalyzes the oxidative depolymerization of lignin To date, ion-exchange chromatography and preparative isoelectric focusing (IEF) have been commonly used for isolation of lignin peroxidase isoenzymes. In this work we h...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ollikka P,Leppänen VM,Anttila T,Suominen I

    更新日期:1995-06-01 00:00:00