当前位置： SCI文献检索 > PROTEIN EXPRESSION AND PURIFICATION期刊下所有文献 > High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein.

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 of the induction protocol, we achieved a high-level LLO synthesis (about 10% of total cell proteins) in hemolytically active form. The expressed hemolysin was then purified to homogeneity, as revealed by SDS-PAGE and Western blot analysis, by a hydroxyapatite adsorption chromatography, followed by an SP Sepharose ion-exchange chromatography. The recombinant protein showed the same properties determined for LLO purified from L. monocytogenes cultures and the characteristics of the sulfhydryl-activated toxins such as inactivation by oxidation and by reaction with cholesterol. By a combination of the pET expression system and the simple purification method, we obtained a significant amount of toxin (4.5 mg/litre cell culture) in a hemolytically active form (1.25 x 10(6)HU/mg protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures, which is a difficult task, mainly owing to the low levels of toxin released in the culture media. The recombinant hemolysin, purified in sufficient quantities, could be very useful for structural studies and for diagnostic and pharmaceutical applications.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

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

doi

10.1016/s1046-5928(02)00682-4

keywords:

subject

Has Abstract

pub_date

2003-03-01 00:00:00

pages

78-85

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046592802006824

journal_volume

28

pub_type

杂志文章

相关文献

PROTEIN EXPRESSION AND PURIFICATION文献大全
  • Large-scale fractionation of molluscan shell matrix.

    abstract：:The numerous proteins occluded within the molluscan shell play a key role in the control of the mineralization process. Although extensively studied, these proteins are still poorly known, mainly because they are difficult to fractionate. In the present paper, we present, for the first time, a simple combined strategy...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.2001.1487

    authors： Marin F,Pereira L,Westbroek P

    更新日期：2001-10-01 00:00:00

  • Expression, purification and characterization of cold shock protein A of Corynebacterium pseudotuberculosis.

    abstract：:The gram-positive bacterium Corynebacterium pseudotuberculosis is the causative agent of different diseases that cause dramatically reduced yields of wool and milk, and results in weight loss, carcass condemnation and also death mainly in sheep, equids, cattle and goats and therefore globally results in considerable e...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2015.04.006

    authors： Lindae A,Eberle RJ,Caruso IP,Coronado MA,de Moraes FR,Azevedo V,Arni RK

    更新日期：2015-08-01 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

  • High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli.

    abstract：:In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with iso...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2004.04.024

    authors： Cipáková I,Hostinová E,Gasperík J,Velebný V

    更新日期：2004-09-01 00:00:00

  • Cloning, expression and functional characterization of two sesquiterpene synthase genes from moso bamboo (Phyllostachys edulis).

    abstract：:The purpose of this work was to characterize the functions of two sesquiterpene synthase genes from moso bamboo (Phyllostachys edulis). Two novel sesquiterpene synthase genes, belonging to the Tpsa subfamily, were isolated from moso bamboo. MoTPS2 was 1641 bp in length and encoded a protein of 63 kDa, whereas MoTPS6 w...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2015.11.019

    authors： Chen X,Wang Y,Sun J,Wang J,Xun H,Tang F

    更新日期：2016-04-01 00:00:00

  • Design and purification of active truncated phosphoinositide 3-kinase gamma protein constructs for structural studies.

    abstract：:Phosphoinositide 3-kinase gamma (PI3Kγ) is a lipid kinase that plays a crucial role in cell migration, chemotaxis, oxidative burst and myocardial contractility. It is activated downstream of G protein-coupled receptors (GPCRs) and small GTPases of Ras superfamily. PI3Kγ is a heterodimer composed of a catalytic and a r...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2017.04.011

    authors： Vujičić Žagar A,Scapozza L,Vadas O

    更新日期：2017-07-01 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

  • A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization.

    abstract：:Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in o...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.1999.1054

    authors： Maurice S,Hädge D,Dekel M,Friedman A,Gertler A,Shoseyov O

    更新日期：1999-08-01 00:00:00

  • Cloning and constitutive expression of His-tagged xylanase GH 11 from Penicillium occitanis Pol6 in Pichia pastoris X33: purification and characterization.

    abstract：:High-level constitutive expression of xylanase GH11 from Penicillium occitanis Pol6 termed PoXyn2 was achieved using the methylotrophic yeast Pichia pastoris. The PoXyn2 cDNA encoding for a mature xylanase of 320 amino acids was subcloned into the pGAPZαA vector, to construct recombinant xylanse with six histidine res...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2012.02.012

    authors： Driss D,Bhiri F,Ghorbel R,Chaabouni SE

    更新日期：2012-05-01 00:00:00

  • High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells.

    abstract：:We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhe...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.2000.1290

    authors： Das T,Johns PW,Goffin V,Kelly P,Kelder B,Kopchick J,Buxton K,Mukerji P

    更新日期：2000-11-01 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

    doi：10.1016/j.pep.2010.10.010

    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

  • Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I.

    abstract：:The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass o...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2004.02.008

    authors： He A,Simpson DR,Daniels L,Rosazza JP

    更新日期：2004-06-01 00:00:00

  • Expression, purification, and initial structural characterization of nonstructural protein 2B, an integral membrane protein of Dengue-2 virus, in detergent micelles.

    abstract：:Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2011.08.008

    authors： Huang Q,Chen AS,Li Q,Kang C

    更新日期：2011-12-01 00:00:00

  • Domain isolation, expression, purification and proteolytic activity of the metalloprotease PrtV from Vibrio cholerae.

    abstract：:The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-prote...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2014.01.012

    authors： Edwin A,Grundström C,Wai SN,Ohman A,Stier G,Sauer-Eriksson AE

    更新日期：2014-04-01 00:00:00

  • Heterologous expression of Class IIb bacteriocin Plantaricin JK in Lactococcus Lactis.

    abstract：:Plantaricin JK (PlnJK) is a Class IIb LAB bacteriocin that includes two peptides; i.e., PlnJ and PlnK, which can synergistically halt many types of gram-positive bacteria, including food spoilage organisms. Purification of these peptides from natural lactic acid bacteria is difficult therefore, their application remai...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2019.02.013

    authors： Xu Y,Yang L,Li P,Gu Q

    更新日期：2019-07-01 00:00:00

  • Expression in E. coli and purification of the nucleoside diphosphate kinase b from Leishmania major.

    abstract：:Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2006.05.010

    authors： de Oliveira AH,Ruiz JC,Cruz AK,Greene LJ,Rosa JC,Ward RJ

    更新日期：2006-10-01 00:00:00

  • Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein αi1.

    abstract：:Molecular-level investigation of proteins is increasingly important to researchers trying to understand the mechanisms of signal transmission. Heterotrimeric G proteins control the activation of many critical signal transmission cascades and are also implicated in numerous diseases. As part of a longer-term investigat...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2012.06.003

    authors： Maly J,Crowhurst KA

    更新日期：2012-08-01 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

  • Strategy for improvement of enteropeptidase efficiency in tag removal processes.

    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. It has also great biotechnological interest because of the unique subs...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2011.04.005

    authors： Gasparian ME,Bychkov ML,Dolgikh DA,Kirpichnikov MP

    更新日期：2011-10-01 00:00:00

  • Purification of recombinant chimeric B72.3 Fab' and F(ab')2 using streptococcal protein G.

    abstract：:Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab'...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(05)80037-3

    authors： Proudfoot KA,Torrance C,Lawson AD,King DJ

    更新日期：1992-10-01 00:00:00

  • Production of recombinant Conkunitzin-S1 in Escherichia coli.

    abstract：:Conkunitzin-S1 from the cone snail Conus striatus is the first member of a new neurotoxin family with a canonical Kunitz domain fold. Conk-S1 is 60 amino acids long and lacks one of the three conserved disulfide bonds typically found in Kunitz domain modules. It binds specifically to voltage activated potassium channe...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2006.01.019

    authors： Bayrhuber M,Graf R,Ferber M,Zweckstetter M,Imperial J,Garrett JE,Olivera BM,Terlau H,Becker S

    更新日期：2006-06-01 00:00:00

  • Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium.

    abstract：:Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. O...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2008.01.015

    authors： Hanna ES,Roque-Barreira MC,Mendes GM,Soares SG,Brocchi M

    更新日期：2008-06-01 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

    doi：10.1016/j.pep.2018.09.006

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

    更新日期：2019-01-01 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

  • First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase.

    abstract：:2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia col...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2016.05.009

    authors： Ohshida T,Hayashi J,Satomura T,Kawakami R,Ohshima T,Sakuraba H

    更新日期：2016-10-01 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

  • 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 at...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2010.11.019

    authors： McConnell MJ,Pachón J

    更新日期：2011-05-01 00:00:00

  • Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells.

    abstract：:L1 is a human cell adhesion glycoprotein involved in the development of the central nervous system that comprises six immunoglobulin-like domains (Ig1-Ig6), five fibronectin-type III (FN1-FN5) domains, a single transmembrane region and a cytoplasmic domain. It contains 20 potential N-glycosylation sites and is heavily...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2006.10.008

    authors： Gouveia RM,Morais VA,Peixoto C,Sousa M,Regalla M,Alves PM,Costa J

    更新日期：2007-03-01 00:00:00

  • The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.

    abstract：:We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2003.08.014

    authors： Lamla T,Erdmann VA

    更新日期：2004-01-01 00:00:00

  • Expression and purification of functional, recombinant Trypanosoma cruzi complement regulatory protein.

    abstract：:The complement regulatory protein (CRP) of Trypanosoma cruzi is a developmentally regulated glycosylphosphatidylinositol (GPI)-anchored membrane protein that protects the parasite from complement-mediated killing, and is an important virulence determinant of the microorganism. CRP binds human complement components C3b...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(02)00562-4

    authors： Beucher M,Meira WS,Zegarra V,Galvão LM,Chiari E,Norris KA

    更新日期：2003-01-01 00:00:00