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

Abstract:

: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.

journal_name

Protein Expr Purif

authors

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

doi

10.1016/j.pep.2018.10.002

subject

Has Abstract

pub_date

2019-02-01 00:00:00

pages

98-103

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(18)30369-3

journal_volume

154

pub_type

杂志文章
  • Facile production of Aspergillus niger α-N-acetylgalactosaminidase in yeast.

    abstract::α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) 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

    doi:10.1016/j.pep.2011.09.009

    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.

    abstract::We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella entericaprpBCDE promoter (P(prpB)) and compared it to that from the strongest IPTG-inducible promoter, P(T7). In contrast to our previous study showing slight...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2008.06.008

    authors: Lee SK,Keasling JD

    更新日期:2008-10-01 00:00:00

  • Functional expression, purification, and characterization of human Flt3 ligand in the Pichia pastoris system.

    abstract::Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30 mg...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2005.04.001

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

    更新日期:2005-08-01 00:00:00

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

    abstract::Huperzine A (HupA) is a drug used for the treatment of Alzheimer's disease. However, the biosynthesis of this medicinally important compound is not well understood. The HupA biosynthetic pathway is thought to be initiated by the decarboxylation of lysine to form cadaverine, which is then converted to 5-aminopentanal b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2016.07.013

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

    更新日期:2016-12-01 00:00:00

  • BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies.

    abstract::Baculovirus mediated gene transduction of mammalian cells (BacMam) is an emerging technique for rapid recombinant protein expression in mammalian cells. We constructed two baculovirus transfer vectors that incorporate several mammalian transcriptional regulatory elements necessary for high-level protein expression in ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2008.08.004

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

    更新日期:2008-12-01 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

    doi:10.1016/s1046-5928(02)00596-x

    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.

    abstract::Pullulanase is crucial to the specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in the starch-processing industry. Recombinant Bacillus subtilis that employs an inducible promoter would be a suitable candidate for pullulanase expression because of its safety and contr...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2018.03.012

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

    更新日期:2018-08-01 00:00:00

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

    abstract::The main principles of higher-order protein oligomerization are elucidated by many structural and biophysical studies. An astonishing number of proteins self-associate to form dimers or higher-order quaternary structures which further interact with other biomolecules to elicit complex cellular responses. In this study...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2018.08.010

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

    更新日期:2019-01-01 00:00:00

  • A simplified method for purification of recombinant soluble DnaA proteins.

    abstract::An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA p...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2006.01.010

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

    更新日期:2006-07-01 00:00:00

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

    abstract::NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein. This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1. Different heterologous expression sy...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.1999.1098

    authors: Gierlich A,van 't Slot KA,Li VM,Marie C,Hermann H,Knogge W

    更新日期:1999-10-01 00:00:00

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

    abstract:INTRODUCTION:Glycodelin is a glycoprotein with different oligosaccharides that are responsible for its diverse biological functions in contraception and immunosuppression. Therefore, it is necessary to have access to adequate amounts of glycodelin with retained carbohydrate structure for functional studies because the ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2016.09.020

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

    更新日期:2017-02-01 00:00:00

  • Expression and purification of milligram levels of inactive G-protein coupled receptors in E. coli.

    abstract::G-protein coupled receptors (GPCRs) are seven transmembrane helical proteins involved in cell signaling and response. They are targets for many existing therapeutic agents, and numerous drug discovery efforts. Production of large quantities of these receptors for drug screening and structural biology remains challengi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2006.10.017

    authors: Bane SE,Velasquez JE,Robinson AS

    更新日期:2007-04-01 00:00:00

  • Synthesis of a human lysosomal enzyme, beta-hexosaminidase B, using the baculovirus expression system.

    abstract::Human lysosomal beta-hexosaminidase exists in two major forms: the A isoform is composed of both alpha and beta chains, while the B form is a homopolymer of beta chains. Deficiency of beta-hexosaminidase underlies the GM2 gangliosidoses. We have produced active beta-hexosaminidase B in cultured insect (Sf9) cells by i...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/1046-5928(90)90003-h

    authors: Boose JA,Tifft CJ,Proia RL,Myerowitz R

    更新日期:1990-11-01 00:00:00

  • Purification and characterization of the hexokinase from Schistosoma mansoni, expressed in Escherichia coli.

    abstract::The hexokinase (ATP;D-hexose 6-phosphotransferase, EC 2.7.1.1) of Schistosoma mansoni has been expressed in Escherichia coli as a fusion protein including an N-terminal polyhistidine tag and enterokinase cleavage site. The enzyme was purified by metal chelate chromatography to > 95% homogeneity, based on analysis by S...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.1996.0113

    authors: Armstrong RL,Wilson JE,Shoemaker CB

    更新日期:1996-11-01 00:00:00

  • Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta.

    abstract::Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2005.06.002

    authors: Ikehara T,Shinjo F,Ikehara S,Imamura S,Yasumoto T

    更新日期:2006-01-01 00:00:00

  • Kinetic characterization of recombinant human cystathionine beta-synthase purified from E. coli.

    abstract::Cystathionine beta-synthase (CBS) catalyzes the pyridoxal-50-phosphate-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chrom...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2008.10.012

    authors: Belew MS,Quazi FI,Willmore WG,Aitken SM

    更新日期:2009-04-01 00:00:00

  • High yield expression, refolding, and characterization of recombinant interferon alpha2/alpha8 hybrids in Escherichia coli.

    abstract::Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral infections and cancers. The low-cost production of IFNs with high biological value and the discovery of IFNs with improved properties are important for the treatment of these diseases as well as for understanding the physi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/s1046-5928(03)00187-6

    authors: Platis D,Foster GR

    更新日期:2003-10-01 00:00:00

  • 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)...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.1998.0929

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

    更新日期:1998-11-01 00:00:00

  • One-step isolation of alpha 1-acid glycoprotein.

    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

    doi:10.1016/1046-5928(91)90006-5

    authors: Chan J,Yu D

    更新日期:1991-02-01 00:00:00

  • 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...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2003.10.010

    authors: Mohanty AK,Wiener MC

    更新日期:2004-02-01 00:00:00

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

    abstract::Nedd4 is an E3 ubiquitin ligase that has received increased attention due to its role in the maintenance of proteostasis and in cellular stress responses. Investigation of Nedd4 enzymology has revealed a complex enzymatic mechanism that involves intermolecular interactions with upstream E2 conjugating enzymes and with...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2020.105649

    authors: Hatstat AK,McCafferty DG

    更新日期:2020-09-01 00:00:00

  • 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

    doi:10.1016/j.pep.2016.02.011

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

    更新日期:2016-06-01 00:00:00

  • 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

    doi:10.1016/j.pep.2007.05.014

    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

    doi:10.1016/1046-5928(90)90041-v

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

    更新日期:1990-09-01 00:00:00

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

    abstract::Myrosinases are thioglucosidases that hydrolyze the natural plant products glucosinolates. We have expressed the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae. The recombinant myrosinase was enzymatically active which shows that the MYR1, which in the plant is complex bound with myrosinase-binding pr...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.1999.1158

    authors: Chen S,Halkier BA

    更新日期:1999-12-01 00:00:00

  • 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

    doi:10.1016/j.pep.2005.07.002

    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

    doi:10.1016/j.pep.2009.05.013

    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.

    abstract::Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) prot...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2010.06.015

    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.

    abstract::Human prolactin was expressed in insect culture cells by recombinant baculoviruses carrying prolactin gene cDNA placed under the transcriptional control of polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus. Preliminary results of recombinant human prolactin expression as extracellular as we...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.2001.1419

    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.

    abstract::To obtain strong bacterial expression of proteins that seem to be hard to express in bacteria or are highly toxic for bacteria, it is possible to create a palette of similar constructs, differing only by several nucleotides, gradually deleted from the full-length clone by exonuclease III. When a construct is equipped ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2004.12.013

    authors: Pecenková T,Filigarová M,Cerovská N

    更新日期:2005-05-01 00:00:00