Expression, purification, and refolding of biologically active Acinetobacter baumannii OmpA from Escherichia coli inclusion bodies.

Abstract:

:Infections caused by Acinetobacter baumannii have emerged as a significant clinical problem due to the increase in infections caused by antibiotic resistant strains. A. baumannii OmpA is a highly conserved membrane protein that has multiple roles in interacting with the host during infection, and thus represents an attractive target for the development of novel antibacterial therapies. In the present study, the coding sequence of the mature form of A. baumannii OmpA was cloned into the vector pET-15b and purified under denaturing conditions from Escherichia coli inclusion bodies using nickel affinity chromatography. A Triton X-114 wash step was incorporated into the purification method in order to remove endotoxin, resulting in endotoxin levels of <1.3 EU/mg of protein. A protocol was developed for refolding the purified protein by dilution into the non-ionic detergent n-octyl-β-D-glucopyranoside followed by dialysis to remove excess denaturant and detergent. Cytotoxicity assays demonstrated that refolded A. baumannii OmpA was able to induce cell death in A549 cells. In addition, a polyclonal antibody was raised against the refolded protein and used to assess extracellular secretion of OmpA by Western blot. This protein expression and purification system may be useful for further characterization of A. baumannii OmpA.

journal_name

Protein Expr Purif

authors

McConnell MJ,Pachón J

doi

10.1016/j.pep.2010.11.019

subject

Has Abstract

pub_date

2011-05-01 00:00:00

pages

98-103

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(10)00329-3

journal_volume

77

pub_type

杂志文章
  • Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris.

    abstract::This is the first report of an insect esterase efficiently expressed in the methylotrophic yeast Pichia pastoris (so far insect esterases have been produced only in the baculovirus system). Having isolated a Tribolium castaneum carboxylesterase cDNA (TCE), we were initially unable to express it in Escherichia coli or ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2005.04.011

    authors: Delroisse JM,Dannau M,Gilsoul JJ,El Mejdoub T,Destain J,Portetelle D,Thonart P,Haubruge E,Vandenbol M

    更新日期:2005-08-01 00:00:00

  • Expression and purification of the alpha-subunit of eukaryotic initiation factor eIF2: use as a kinase substrate.

    abstract::The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2alpha at Ser51. In the...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.1998.0863

    authors: Kimball SR,Horetsky RL,Jagus R,Jefferson LS

    更新日期:1998-04-01 00:00:00

  • Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

    abstract::Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with speci...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2014.12.003

    authors: Kumar S,Jain KK,Singh A,Panda AK,Kuhad RC

    更新日期:2015-06-01 00:00:00

  • Affinity purification of GST fusion proteins for immunohistochemical studies of gene expression.

    abstract::Fusion proteins expressed in bacteria are often insoluble or inefficiently purified by standard procedures previously reported to work well for the non-fused proteins. We report here a simple but general procedure that can be used to quickly customize and optimize the purification of milligram quantities of most GST f...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/s1046-5928(02)00524-7

    authors: Mercado-Pimentel ME,Jordan NC,Aisemberg GO

    更新日期:2002-11-01 00:00:00

  • Expression and purification of active recombinant human bikunin in Pichia pastoris.

    abstract::Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in the methylotrophic yeast Pichia pastoris and established the purification pr...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2008.03.025

    authors: Jian-qiu W,Feng-qin Y,Dou-dou W,Lei B,Nan S,Cong-yan L,Ting Z,Wei-qun Y

    更新日期:2008-08-01 00:00:00

  • Increasing the expression levels of papillomavirus major capsid protein in Escherichia coli by N-terminal deletion.

    abstract::The major capsid protein L1 of human papillomavirus (HPV) contains the immunodominant neutralization epitopes of the virus and can auto-assembles to form virus-like particles (VLPs). Therefore, HPV L1 capsid proteins have been well investigated as potential vaccine candidates. To express large quantities of human papi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2007.05.010

    authors: Ma Z,Chen B,Zhang F,Yu M,Liu T,Liu L

    更新日期:2007-11-01 00:00:00

  • Heterologous expression and characterization of CYP61A1 from dandruff-causing Malassezia globosa.

    abstract::Malassezia globosa is pathogenic fungus that causes skin disorders including dandruff in humans. Many yeast cytochrome CYP enzymes are involved in the biosynthesis of sterols and are considered major targets of azole antifungal agents. Here, we report on the expression and characterization of the MGL_0310 gene product...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2015.07.002

    authors: Ohk SO,Park HG,Lee H,Kwon YJ,Kim BJ,Kim D,Chun YJ

    更新日期:2015-10-01 00:00:00

  • Cloning, expression, and characterization of single-chain variable fragment antibody against mycotoxin deoxynivalenol in recombinant Escherichia coli.

    abstract::Deoxynivalenol (DON), a mycotoxin produced by several Fusarium species, is a worldwide contaminant of food and feedstuffs. The DON-specific single-chain variable fragment (scFv) antibody was produced in recombinant Escherichia coli. The variable regions of the heavy chain (V(H)) and light chain (V(L)) cloned from the ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2003.12.008

    authors: Choi GH,Lee DH,Min WK,Cho YJ,Kweon DH,Son DH,Park K,Seo JH

    更新日期:2004-05-01 00:00:00

  • Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr-Abl oncoprotein fused to the cytoplasmic transduction peptide.

    abstract::Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2008.10.023

    authors: Huang SF,Liu DB,Zeng JM,Xiao Q,Luo M,Zhang WP,Tao K,Wen JP,Huang ZG,Feng WL

    更新日期:2009-04-01 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

    doi:10.1016/j.pep.2020.105715

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

    更新日期:2020-11-01 00:00:00

  • Soluble expression and purification of a full-length asparaginyl tRNA synthetase from Fasciola gigantica.

    abstract::We report the molecular cloning, expression, and single-step homogeneous purification of a full-length asparaginyl tRNA synthetase (NRS) from Fasciola gigantica (FgNRS). Fasciola gigantica is a parasitic liver fluke of the class Trematoda. It causes fascioliasis that infects the liver of various mammals, including hum...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2017.10.009

    authors: Vijayakumar R,Tripathi T

    更新日期:2018-03-01 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

    doi:10.1016/j.pep.2014.04.012

    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

  • 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

  • Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus.

    abstract::BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome b(5) fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2009.10.017

    authors: Kumar GS,Sarma SP

    更新日期:2010-04-01 00:00:00

  • Heterologous expression and in vitro assembly of the transmembrane cytochrome b6.

    abstract::Folding and assembly studies with alpha-helical membrane proteins are often hampered by the absence of high-level expression systems as well as by missing suitable in vitro refolding procedures. Experimental constraints and requirements for heterologous expression and in vitro assembly of cytochrome b6 have been exami...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2007.08.007

    authors: Prodöhl A,Dreher C,Hielscher R,Hellwig P,Schneider D

    更新日期:2007-12-01 00:00:00

  • Purification methods for recombinant Lactobacillus casei thymidylate synthase and mutants: a general, automated procedure.

    abstract::General procedures for the rapid, large-scale purification of recombinant Lactobacillus casei thymidylate synthase and its mutants have been established. An effective method employs sequential phosphocellulose and hydroxylapatite chromatography. Crude cell extracts are directly applied to phosphocellulose, and the enz...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/s1046-5928(05)80039-7

    authors: Kealey JT,Santi DV

    更新日期:1992-10-01 00:00:00

  • Enhancing the soluble expression of an amylase in Escherichia coli by the mutations related to its domain interactions.

    abstract::The sequence and structure of the target protein exert a marked effect on its soluble expression in Escherichia coli. The effects of the mutation of an amylase isolated from Bacillus licheniformis (BLA) on its soluble expression in E. coli were investigated. A random mutation library of BLA was constructed to screen f...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2015.12.010

    authors: Wang P,Qin W,Xu J,Yan Y,Tian J,Wu N,Yao B

    更新日期:2016-04-01 00:00:00

  • Expression, purification, and molecular characterization of Plasmodium falciparum FK506-binding protein 35 (PfFKBP35).

    abstract::The immunosuppressive drug FK506 binds its targets FK506-binding protein (FKBP) family and modulates cellular processes. Recent studies demonstrated that FK506 shows anti-malaria effects. Newly identified FK506-binding protein 35 from Plasmodium falciparum (PfFKBP35) is assumed to be the molecular target of FK506 in t...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2006.12.019

    authors: Yoon HR,Kang CB,Chia J,Tang K,Yoon HS

    更新日期:2007-05-01 00:00:00

  • Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.

    abstract::Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-la...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2004.08.002

    authors: Kang TJ,Han SC,Kim MY,Kim YS,Yang MS

    更新日期:2004-11-01 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

  • Analysis of the tyrosine protein kinase p56lck expressed as a glutathione S-transferase fusion protein in Spodoptera frugiperda cells.

    abstract::A baculovirus vector system that expresses cloned DNA sequences as glutathione S-transferase fusion proteins was developed. This system was used to express and purify the lymphocyte-specific tyrosine kinase p56lck. This recombinant p56lck was purified to homogeneity in a single chromatography step using glutathione re...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1006/prep.1993.1051

    authors: Spana C,O'Rourke EC,Bolen JB,Fargnoli J

    更新日期:1993-10-01 00:00:00

  • A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2.

    abstract::Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signal...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2020.105628

    authors: Ådén J,Mushtaq AU,Dingeldein A,Wallgren M,Gröbner G

    更新日期:2020-08-01 00:00:00

  • Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse.

    abstract::Acyl coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-t...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2007.11.010

    authors: Petrescu AD,Huang H,Hostetler HA,Schroeder F,Kier AB

    更新日期:2008-04-01 00:00:00

  • High yield soluble bacterial expression and streamlined purification of recombinant human interferon α-2a.

    abstract::Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is cl...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2014.04.010

    authors: Bis RL,Stauffer TM,Singh SM,Lavoie TB,Mallela KM

    更新日期:2014-07-01 00:00:00

  • Expression and purification of a putative H-NS nucleoid-associated protein from the phytopathogen Xylella fastidiosa.

    abstract::The H-NS protein is one of the major constituents of the nucleoid structure that has been implicated in the DNA packaging and in the global regulation of gene expression. The study of this transcriptional regulator is an effort to fight Xylella fastidiosa, a citrus pathogen responsible for a range of economically impo...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/S1046-5928(03)00193-1

    authors: Paula DP,Azzoni AR,Siqueira SF,Catani CF,Rosselli LK,de Souza AP

    更新日期:2003-11-01 00:00:00

  • A screening strategy for heterologous protein expression in Escherichia coli with the highest return of investment.

    abstract::Heterologous protein expression in Escherichia coli is commonly used to obtain recombinant proteins for a variety of downstream applications. However, many proteins are not, or are only poorly, expressed in soluble form. High level expression often leads to the formation of inclusion bodies and an inactive product tha...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2011.08.030

    authors: Pacheco B,Crombet L,Loppnau P,Cossar D

    更新日期:2012-01-01 00:00:00

  • Reprint of: Immobilized-Metal Affinity Chromatography (IMAC): A Review.

    abstract::This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of im...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2011.08.021

    authors: Block H,Maertens B,Spriestersbach A,Brinker N,Kubicek J,Fabis R,Labahn J,Schäfer F

    更新日期:2011-09-03 00:00:00

  • New high fidelity polymerases from Thermococcus species.

    abstract::Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. P...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2006.07.022

    authors: Griffiths K,Nayak S,Park K,Mandelman D,Modrell B,Lee J,Ng B,Gibbs MD,Bergquist PL

    更新日期:2007-03-01 00:00:00

  • Production and single-step purification of EGFP and a biotinylated version of the Human Rhinovirus 14 3C protease.

    abstract::The fluorescent reporter enhanced Green Fluorescent Protein (EGFP) has been used for assaying a wide range of biological activities ranging from gene expression, or localization of target proteins through to intermolecular interactions. However, over-production of this protein in Escherichia coli has resulted in the p...

    journal_title:Protein expression and purification

    pub_type: 杂志文章

    doi:10.1016/j.pep.2011.05.003

    authors: Youell J,Fordham D,Firman K

    更新日期:2011-10-01 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

    doi:10.1016/j.pep.2014.03.011

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

    更新日期:2014-07-01 00:00:00