Expression and reconstitution of NF-kappaB from insect cells using a baculovirus vector.

Abstract:

:NF-kappaB is a pleiotropic transcriptional activator originally identified by its ability to regulate immunoglogulin kappa light chain expression. Purification of this DNA-binding complex demonstrated that NF-kappaB is a heterodimer composed of two subunits, NFKB1 and RelA. Previous studies have shown that truncated versions of these proteins could be expressed and purified from bacterial cells. In the present study, we utilize a baculovirus expression vector system (BEVS) to overexpress each subunit independently to produce homodimers or together to reconstitute functional NF-kappaB. These proteins can be enriched to >70% homogeneity on a kappaB-agarose DNA- affinity column. The purified proteins are active in DNA binding as measured by electrophoretic mobility shift assays. Finally, transcriptional activation of these recombinant proteins can be measured by their ability to activate a kappaB-CAT reporter plasmid in transiently transfected/infected SF-9 cells. Thus, BEVS provides a method for production of full-length, transcriptionally active NF-kappaB proteins.

journal_name

Protein Expr Purif

authors

Coleman TA,Huddleston KA,Ruben SM,Rosen CA,Gentz R

doi

10.1006/prep.1996.0670

subject

Has Abstract

pub_date

1997-02-01 00:00:00

pages

40-8

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(96)90670-1

journal_volume

9

pub_type

杂志文章
  • Expression and purification of stable 33-kDa soluble human CD23 using the Drosophila S2 expression system.

    abstract::CD23, a 45-kDa type II membrane glycoprotein present on B cells, monocytes, and other human immune cells, is a low-affinity receptor for IgE. The extracellular region of the membrane-bound human CD23 is processed into at least four soluble (s) CD23 forms, with apparent molecular masses of 37, 33, 29, and 25 kDa. High ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.2001.1448

    authors: Khandekar SS,Mayer RJ,Cusimano DM,Katchur SR,Appelbaum ER

    更新日期:2001-07-01 00:00:00

  • Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli.

    abstract::Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/s1046-5928(03)00159-1

    authors: Gasparian ME,Ostapchenko VG,Schulga AA,Dolgikh DA,Kirpichnikov MP

    更新日期:2003-09-01 00:00:00

  • Protective immunity induced by the vaccination of recombinant Proteus mirabilis OmpA expressed in Pichia pastoris.

    abstract::Proteus mirabilis (P. mirabilis) is a zoonotic pathogen that has recently presented a rising infection rate in the poultry industry. To develop an effective vaccine to protect chickens against P. mirabilis infection, OmpA, one of the major outer membrane proteins of P. mirabilis, was expressed in Pichia pastoris. The ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2014.10.001

    authors: Zhang Y,Yang S,Dai X,Liu L,Jiang X,Shao M,Chi S,Wang C,Yu C,Wei K,Zhu R

    更新日期:2015-01-01 00:00:00

  • Requirement of continuous transcription for the synthesis of sufficient amounts of protein by a cell-free rapid translation system.

    abstract::To understand the key processes of cell-free protein synthesis, the synthesis of adipose-type fatty acid binding protein (A-FABP) by a rapid translation system was examined under various conditions. The synthesis of A-FABP was achieved by using an expression vector of A-FABP containing a T7 promoter. However, synthesi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.2001.1570

    authors: Hino M,Shinohara Y,Kajimoto K,Terada H,Baba Y

    更新日期:2002-03-01 00:00:00

  • Recombinant expression of biologically active rat leptin in Escherichia coli.

    abstract::Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.2001.1412

    authors: Park JH,Lee HH,Na SY,Ju SK,Lee YJ,Lee MK,Kim KL

    更新日期:2001-06-01 00:00:00

  • High-level bacterial expression and purification of human SirT2 protein for NMR studies.

    abstract::Silent information regulator 2 (Sir2) proteins are a class of protein deacetylase enzymes that play key roles in transcriptional gene silencing, DNA repair, and aging. Here, we describe the high-level bacterial expression and purification of a human SirT2 construct that yields high resolution NMR spectra. By removing ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2005.12.006

    authors: Sun C,Song D,Marcotte PA,Richardson PL,Hajduk PJ

    更新日期:2006-07-01 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

  • Increased peptide deformylase activity for N-formylmethionine processing of proteins overexpressed in Escherichia coli: application to homogeneous rubredoxin production.

    abstract::Deformylation of the initiator N-formylmethionine does not always proceed to completion for proteins overexpressed in Escherichia coli. To overcome this limitation, the def gene encoding the Escherichia coli peptide deformylase was cloned into the plysS plasmid under the tetracycline (Tc) promoter control. The efficie...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2004.03.007

    authors: Tang J,Hernández G,LeMaster DM

    更新日期:2004-07-01 00:00:00

  • Improvements to the throughput of recombinant protein expression in the baculovirus/insect cell system.

    abstract::Recombinant baculoviruses have proved to be a very useful means to express many proteins over the last 20 years. Since their introduction, there have been a number of significant improvements that have simplified and speeded up the construction of baculoviruses. One of the most commonly used methods relies upon recomb...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2005.03.021

    authors: McCall EJ,Danielsson A,Hardern IM,Dartsch C,Hicks R,Wahlberg JM,Abbott WM

    更新日期:2005-07-01 00:00:00

  • One-step extraction of functional recombinant aquaporin Z from inclusion bodies with optimal detergent.

    abstract::Aquaporins are integral membrane channel proteins found in all kingdoms of life. The Escherichia coli aquaporin Z (AqpZ) has been shown to solely conduct water at high permeability. Functional AqpZ is generally purified from the membrane fraction. However, the quantity of the purified protein is limited. In this study...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2015.08.014

    authors: Wang L,Zhou H,Li Z,Lim TK,Lim XS,Lin Q

    更新日期:2015-11-01 00:00:00

  • Use of nonreducing SDS-PAGE for monitoring renaturation of recombinant protein synthesis initiation factor, eIF-4 alpha.

    abstract::The purification of biologically active human protein synthesis initiation factor 4 alpha, eIF-4 alpha, overexpressed in Escherichia coli, is complicated by its localization in insoluble inclusion bodies, as well as its possession of four cysteines. Two of these cysteines have been reported to be reduced in the native...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.1993.1041

    authors: Stern BD,Wilson M,Jagus R

    更新日期:1993-08-01 00:00:00

  • Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase.

    abstract::Escherichia coli tyrosine kinase (Etk) is a membrane bound kinase in gram-negative bacteria that regulates the export of capsular polysaccharides (CPS). The molecular mechanism behind CPS regulation remains unclear, despite access to a crystal structure of the cytoplasmic kinase domain of Etk. In this study, an effici...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2015.08.029

    authors: Chesterman C,Jia Z

    更新日期:2016-09-01 00:00:00

  • Design and preparation of non-tagged Yersinia pestis LcrV antigen in Escherichia coli and its immunogenicity in BALB/c mice.

    abstract::The whole encoding sequence for Yersinia pestis LcrV antigen was cloned into pET-32a(+) and expressed in Escherichia coli BL21 (DE3). The LcrV was high level expressed in the E. coli cytoplasm in a completely soluble form. Recombinant LcrV could be purified from the supernatant of the bacteria lysate after chromatogra...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2007.10.011

    authors: li J,Li B,Li G,Ren J,Zhang J,Xu C,Yang X,Liu S,Fu L,Chen W

    更新日期:2008-02-01 00:00:00

  • Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli.

    abstract::Cyclohexanone monooxygenase (CMO) is a soluble flavoenzyme originally isolated from Acinetobacter spp. which carries out Baeyer-Villiger reactions with cyclic ketone substrates. In the present study we cloned the Acinetobacter CMO gene and modified it for facile purification from heterologous expression systems by inc...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.2000.1340

    authors: Cheesman MJ,Kneller MB,Kelly EJ,Thompson SJ,Yeung CK,Eaton DL,Rettie AE

    更新日期:2001-02-01 00:00:00

  • Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR.

    abstract::Using a codon-optimized gene fragment, we report remarkable yields for extracellular domain of human NK cell receptor (NKp30ex) when produced on M9 minimal medium, even with low (2g/L) glucose concentration. The yields were identical using media containing (15)NH(4)Cl or (15)NH(4)Cl in combination with all-(13)C-d-glu...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2012.09.016

    authors: Grave L,Tůmová L,Mrázek H,Kavan D,Chmelík J,Vaněk O,Novák P,Bezouška K

    更新日期:2012-12-01 00:00:00

  • Kinetic characterization and Mg2+ enhancement of Streptomyces griseocarneus sphingomyelinase C produced by recombinant Streptomyces lividans.

    abstract::Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550-950 U/mg) and was obtained in h...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2011.10.004

    authors: Sugimori D,Matsumoto Y,Tomita Y,Murayama K,Ogino C

    更新日期:2012-02-01 00:00:00

  • Comparative analysis of native and cysteine-deficient HIV-1 reverse transcriptase.

    abstract::To study the subunit structure and the active site of human immunodeficiency virus reverse transcriptase (RT), the enzyme was expressed in E. coli and purified to homogeneity in large quantities. The recombinant enzyme consists of two major polypeptides of 66,000 and 53,000 Da in equimolar amounts and a minor species ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/1046-5928(92)90005-h

    authors: Fischer M,Lifshitz R,Katz T,Liefer I,Ben-Artzi H,Gorecki M,Panet A,Zeelon E

    更新日期:1992-08-01 00:00:00

  • Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli.

    abstract::Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively hig...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/s1046-5928(02)00573-9

    authors: Seddi R,Chaix JC,Puigserver A,Guo XJ

    更新日期:2003-02-01 00:00:00

  • Expression of Sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications.

    abstract::We have investigated the suitability of Pichia pastoris as an expression system for the candidate therapeutic protein, Sonic hedgehog fused to an immunoglobulin Fc domain (Shh-Fc). Sonic hedgehog is a morphogen protein involved in the patterning of a wide range of tissues during animal embryogenesis. The presence of S...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/s1046-5928(03)00062-7

    authors: Shapiro RI,Wen D,Levesque M,Hronowski X,Gill A,Garber EA,Galdes A,Strauch KL,Taylor FR

    更新日期:2003-06-01 00:00:00

  • Purification of the medium-chain/long-chain (COT/CPT) carnitine acyltransferase of rat liver microsomes.

    abstract::A procedure for the purification of the rat liver microsomal carnitine octanoyltransferase (COT) that catalyzes the reversible formation of medium-chain and long-chain acylcarnitines from acyl-coenzyme A is described. The K0.5 for L-carnitine is 0.6 mM and the K0.5 for both decanoyl-CoA and palmitoyl-CoA is 0.6 microM...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/1046-5928(92)90059-6

    authors: Murthy MS,Bieber LL

    更新日期:1992-02-01 00:00:00

  • Improved soluble expression and characterization of the Hc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli by using a PCR-synthesized gene and a Trx co-expression strain.

    abstract::Botulinum neurotoxin serotype A (BoNT/A) is an extremely potent bacterial protein toxin. The Hc fragment of BoNT/A (AHc) was shown to be non-toxic, antigenic, and capable of eliciting a protective immunity in animals challenged with homologous BoNT. In this study, we synthesized AHc gene by using T4 DNA ligase and PCR...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2009.11.007

    authors: Chen R,Shi J,Cai K,Tu W,Hou X,Liu H,Xiao L,Wang Q,Tang Y,Wang H

    更新日期:2010-05-01 00:00:00

  • High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein.

    abstract::Listeriolysin O (LLO) is a cholesterol-binding sulfhydryl-activated hemolysin encoded by Listeria monocytogenes hlyA gene. After analyzing the nucleotide coding sequence of this gene from the ATCC 9525 L. monocytogenes strain, we cloned it in a pET vector for expression in Escherichia coli. Thanks to the optimization ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/s1046-5928(02)00682-4

    authors: Giammarini C,Andreoni F,Amagliani G,Casiere A,Barocci S,Magnani M

    更新日期:2003-03-01 00:00:00

  • Improvements of a high-throughput protein purification process using a calcium-dependent setup.

    abstract::The Human Secretome Project aims to produce and purify all human secreted proteins as full-length. In order to enable this, a robust, gentle and effective purification process is needed, where multiple proteins can be purified in parallel. For this reason, a purification system based on a Protein C-tag and the HPC4 an...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2020.105698

    authors: Kanje S,Enstedt H,Dannemeyer M,Uhlén M,Hober S,Tegel H

    更新日期:2020-11-01 00:00:00

  • Expression and purification of a cold-adapted group III trypsin in Escherichia coli.

    abstract::The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from t...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2006.06.008

    authors: Pálsdóttir HM,Gudmundsdóttir A

    更新日期:2007-02-01 00:00:00

  • Generation and expression of a minimal hybrid Ig-receptor formed between single domains from proteins L and G.

    abstract::The Ig-binding properties of protein L from Peptostreptococcus magnus and protein G from Streptococcus have been successfully combined through the construction of a novel hybrid protein, consisting of a single Ig-binding domain from each protein. The biophysical and biochemical properties of this construct have been c...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2007.11.007

    authors: Harrison SL,Housden NG,Bottomley SP,Cossins AJ,Gore MG

    更新日期:2008-03-01 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 purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli.

    abstract::Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson's disease. In...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2012.05.007

    authors: Higgins CA,Vermeer LM,Doorn JA,Roman DL

    更新日期:2012-08-01 00:00:00

  • Small structural differences of targeted anti-tumor toxins result in strong variation of protein expression.

    abstract::Targeted anti-tumor toxins consist of a toxic functional moiety that is chemically linked or recombinantly fused to a cell-directing ligand. Ribosome-inactivating proteins (RIPs), especially type I RIPs such as saporin or dianthin, are commonly used as toxin components. Although expression of type I RIP-based fusion p...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2013.07.004

    authors: Gilabert-Oriol R,Thakur M,Weise C,Dernedde J,von Mallinckrodt B,Fuchs H,Weng A

    更新日期:2013-09-01 00:00:00

  • Characterization of ubiquitin C-terminal hydrolase 1 (YUH1) from Saccharomyces cerevisiae expressed in recombinant Escherichia coli.

    abstract::The YUH1 gene coding for ubiquitin C-terminal hydrolase 1, a deubiquitinating enzyme, was cloned from the Saccharomyces cerevisiae genomic DNA and expressed in Escherichia coli. YUH1 was fused with the 6 histidine tag at the N-terminus (H6YUH1) or C-terminus (YUH1H6) and purified by an immobilized metal affinity chrom...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2007.07.005

    authors: Yu HA,Kim SG,Kim EJ,Lee WJ,Kim DO,Park K,Park YC,Seo JH

    更新日期:2007-11-01 00:00:00

  • Delandin, a chitinase-like protein with antifungal, HIV-1 reverse transcriptase inhibitory and mitogenic activities from the rice bean Delandia umbellata.

    abstract::An antifungal protein with a chitinase-like N-terminal sequence, designated delandin, was isolated from the rice bean. The protein exhibited a molecular weight of 28 kDa and was adsorbed on both blue Affi-Gel and SP-Toyopearl. It exerted antifungal action toward Mycosphaerella arachidicola, Botrytis cinerea, Fu- sariu...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.2001.1596

    authors: Ye XY,Ng TB

    更新日期:2002-04-01 00:00:00