Abstract:
:The entire stx2 region from Escherichia coli O157:H7, containing two open reading frames (stx2a and stx2b), was cloned into pET-32a with a single promoter, and transformed into E. coli BL21 (DE3) pLysS. We used two methods of IPTG induction using LB medium and auto-induction using ZYM-5052 medium to express recombinant Shiga toxin 2 (rStx2). rStx2 was expressed in the E. coli periplasm in a completely soluble, biologically active form. The final yield of purified rStx2 from each liter of culture in LB medium and ZYM-5052 medium was approximately 2.3mg and 3.5mg, respectively. The highest amount of rStx2 accounted for 27.8% of total bacteria protein in ZYM-5052 medium. rStx2A and rStx2B isolated from rStx2 by electroelution were, respectively, identified by N-terminal protein sequencing. Signal peptides with the sequence MKCILFKWVLCLLLGFSSVSYS and MKKMFMAVLFALASVNAMA were identified at the N terminus of rStx2A and rStx2B, respectively. Our rStx2 possessed Vero cell CD(50) value about 500pg and LD(50) value approximately 6ng. rStx2 can be substitute for natural toxin Stx2, which can be used for animal models, drug screening, vaccine research, and so on.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Tu W,Cai K,Gao X,Xiao L,Chen R,Shi J,Liu H,Hou X,Wang Q,Wang Hdoi
10.1016/j.pep.2009.05.005subject
Has Abstractpub_date
2009-10-01 00:00:00pages
169-74issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(09)00118-1journal_volume
67pub_type
杂志文章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
更新日期:2010-11-01 00:00:00
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
更新日期:2005-07-01 00:00:00
abstract::Streptavidin is a versatile molecule for many in vitro and in vivo applications. To optimize the production of the full-length streptavidin in a soluble and functional form via secretion using Bacillus subtilis as the expression host, three different strategies were used. These strategies include the construction of a...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1582
更新日期:2002-04-01 00:00:00
abstract::p53 protein is an important regulation factor that can bind to p53 mRNA to regulate its translation in human and murine. To determine if a similar interaction exists in zebrafish and if the interaction affects zebrafish development, we cloned and expressed p53 protein from zebrafish in Escherichia coli. Soluble p53 pr...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2010.03.028
更新日期:2010-08-01 00:00:00
abstract::Methionine aminopeptidases (MetAPs), ubiquitous enzymes that play an important role in nascent protein maturation, have been recognized as attractive targets for the development of drugs against pathogenic protozoa including Plasmodium spp. Here, we characterized partial biochemical properties of a type I MetAP of Pla...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.01.003
更新日期:2015-04-01 00:00:00
abstract::Optimization of coding sequences to maximize protein expression yield is often outsourced to external service providers during commercial gene synthesis and thus unfortunately remains a black box for many researchers. The presented software program "CodonWizard" offers scientists a powerful but easy-to-use tool for cu...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2019.03.018
更新日期:2019-08-01 00:00:00
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
doi:10.1006/prep.1999.1077
更新日期:1999-07-01 00:00:00
abstract::κ-Carrageenase (EC3.2.1.83) is a class of glycoside hydrolase, which can be used for hydrolysis of κ-carrageenan to κ-carrageenan oligosaccharides. In this study, a bacterium, identified as Pseudoalteromonas sp. ZDY3 isolated from rotten algae, was capable to degrade κ-carrageenan. The κ-carrageenase produced by Pseud...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105768
更新日期:2021-02-01 00:00:00
abstract::Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously do...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.03.010
更新日期:2012-06-01 00:00:00
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
更新日期:2012-02-01 00:00:00
abstract::Glucagon is a pancreatic hormone that plays pivotal roles in regulating glucose homeostasis and metabolism. Glucagon exerts its action by binding to its receptor, glucagon receptor (GCGR), one of class B G-protein coupled receptors (GPCRs). Diabetes is a bihormonal disease in which excessive glucagon secretion is a ma...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2013.04.004
更新日期:2013-06-01 00:00:00
abstract::Heparinase I (HepA) was originally isolated from Flavobacterium heparinum (F. heparinum) and specifically cleaves heparin/heparan sulfate in a site-dependent manner, showing great promise for producing low molecular weight heparin (LMWH). However, expressing recombinant HepA is extremely difficult in Escherichia coli ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.03.016
更新日期:2012-06-01 00:00:00
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
更新日期:2005-08-01 00:00:00
abstract::Escherichia coli glutamic acid decarboxylase is a pyridoxal phosphate-dependent enzyme that catalyzes the alpha-decarboxylation of glutamate to yield 4-amino-butyrate and CO2. The E. coli chromosome contains two genes encoding for this enzyme, gadA and gadB, which map at distinct loci. Their protein products differ in...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0121
更新日期:1996-12-01 00:00:00
abstract::The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containin...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.08.020
更新日期:2012-11-01 00:00:00
abstract::An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open re...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1456
更新日期:2001-08-01 00:00:00
abstract::The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2011.04.009
更新日期:2011-10-01 00:00:00
abstract::Wild-type and deglycosylated forms of human prostate-specific antigen were expressed in Chinese hamster ovary (CHO) cells as zymogens. ProPSA was collected from conditioned medium and purified using a single cation-exchange chromatographic step for the deglycosylated form and cation-exchange followed by gel filtration...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2000.1342
更新日期:2000-12-01 00:00:00
abstract::A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpresse...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.05.016
更新日期:2006-01-01 00:00:00
abstract::Bovine follicle-stimulating hormone (bFSH) is a pituitary gonadotropin composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Low-level expression of the recombinant bFSH is the factor that limits its usefulness as a...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.03.016
更新日期:2007-08-01 00:00:00
abstract::The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L-1. Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2017.04.002
更新日期:2017-06-01 00:00:00
abstract::Receptor activator of NF-kappaB ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprot...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2000.1278
更新日期:2000-10-01 00:00:00
abstract::Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) 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
doi:10.1016/j.pep.2017.10.004
更新日期:2018-02-01 00:00:00
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
更新日期:2006-02-01 00:00:00
abstract::Peptide-based affinity tags are commonly used in recombinant production/purification of proteins, and are often preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal antibody 2H5 recognizing an undecapeptide tag called "eTev", which contains a recognition sequence f...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2018.03.004
更新日期:2018-07-01 00:00:00
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 v...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0670
更新日期:1997-02-01 00:00:00
abstract::Previously, the lysozyme gene of the Klebsiella phage K11 was partially sequenced in our lab. Using the sequence information the lysozyme gene of the Klebsiella phage K11 was amplified and cloned using the polymerase chain reaction of the pfu DNA polymerase. The nucleotide sequence of phage K11 lysozyme gene was deter...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.03.026
更新日期:2005-07-01 00:00:00
abstract::We have constructed a plasmid, pAHOB1, with a 482-b AluI fragment containing the Escherichia coli glutaredoxin gene (grx) cloned under lambda PL promoter control. Growth of E. coli N4830/pAHOB1 cells at 30 degrees C followed by heat induction at 40 degrees C for 5 h resulted in expression of glutaredoxin as 20% of the...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(91)90085-w
更新日期:1991-08-01 00:00:00
abstract::Human paraoxonase (hPON3) is a high density lipoprotein-related glycoprotein with multi-enzymatic properties and antioxidant activity which is proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. In this study, hPON3 gene was amplified from Human Fetal Liver Marathon-Ready cDNA and exp...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.07.021
更新日期:2006-03-01 00:00:00
abstract::We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2013.05.007
更新日期:2013-08-01 00:00:00