Abstract：
:The covalent addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) groups to lipid A, which resides in the outer membranes of bacteria such as Salmonella typhimurium and Escherichia coli, is the final step in the polymyxin-resistance pathway in these organisms. This modification is catalyzed by the inner membrane protein 4-amino-4-deoxy-L-arabinose transferase (ArnT). Little is known about the ArnT protein structure because it has not previously been purified. We report here the first expression and purification of 6 x His-tagged S. typhimurium ArnT in NovaBlue cells. The enzyme was purified using sequential Q-Sepharose anion exchange and HisLink nickel affinity column chromatography. The purified protein has an apparent molecular weight of 62 kDa on SDS-PAGE and the identity of the purified ArnT was confirmed by Western blot using a monoclonal antibody against the His-tag and by MALDI-TOF mass spectrometry. Purified ArnT protein was shown to be highly alpha-helical as determined by circular dichroism analysis. A chromosomal ArnT knockout strain of E. coli BL21(DE3) was developed to allow in vivo functional analysis of plasmid-encoded ArnT constructs, and a polymyxin assay was used to confirm that the cloned ArnT proteins retained full activity. These studies provide an essential foundation for further analysis of ArnT structure and function using mutagenesis and biophysical techniques.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Bretscher LE,Morrell MT,Funk AL,Klug CSdoi
10.1016/j.pep.2005.08.028keywords:
subject
Has Abstractpub_date
2006-03-01 00:00:00pages
33-9issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(05)00286-Xjournal_volume
46pub_type
杂志文章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
更新日期：1990-09-01 00:00:00
abstract：:Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltra...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.05.022
更新日期：2011-10-01 00:00:00
abstract：:Refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. We will review key parameters associated with (1) conformation of the protein solubilized from inc...
journal_title：Protein expression and purification
pub_type： 杂志文章,评审
doi：10.1016/s1046-5928(02)00641-1
更新日期：2003-03-01 00:00:00
abstract：:The bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T cell leukemia, autoimmune diseases, and tolerance induction for transplantation. The multi-domain structure of the bivalent immunotoxin hinders efficient production in Escherichia coli and most eukaryotes are sensit...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00009-8
更新日期：2002-07-01 00:00:00
abstract：:Cell extracts of Clostridium kluyveri grown on ethanol plus succinate contained a NAD(H) dependent 4-hydroxybutanoate dehydrogenase (EC 1.1.1.61) at 66 U/mg. This enzyme was purified 42-fold under anaerobic conditions to homogeneity. Heat treatment, ion exchange chromatography on DEAE-cellulose, nondenaturing polyacry...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1995.1026
更新日期：1995-04-01 00:00:00
abstract：:The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peri...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00564-8
更新日期：2002-12-01 00:00:00
abstract：:We describe a single method to purify milligram amounts of authentic wild-type and mutant HIV-1 and HIV-2 Tat proteins overexpressed in Escherichia coli. This method takes advantage of the highly basic, positively charged RNA binding domain present in both HIV-1 and HIV-2 Tat, which also facilitated purification of HI...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0096
更新日期：1996-09-01 00:00:00
abstract：:Alginate lyase digestion is an efficient way to degrade alginate into oligosaccharides, which are useful in various areas including chemistry, pharmacy and food industry. Here we determined the sequence of Vibrio sp. QY102 sourced alginate lyase, and set up its heterologous expression in E. coli. We improved its secre...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.09.002
更新日期：2019-01-01 00:00:00
abstract：:The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the wid...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2019.105546
更新日期：2020-03-01 00:00:00
abstract：:To investigate the expression and purification of an unstable heterologous protein in Pichia pastoris, the cDNA of H5-lysozyme, a hen egg lysozyme mutant with a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) fused to the carboxyl terminus, was integrated into the genome of P. pastoris. It was found that medium composi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00642-3
更新日期：2003-02-01 00:00:00
abstract：:We have developed a new expression vector, pcI(ts) ind(+), based upon the powerful rightward promoter of bacteriophage lambda, which is controlled by a temperature-sensitive and chemically-inducible version of the lambda repressor on the same plasmid. Locating the repressor gene on the plasmid makes this vector "porta...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.09.018
更新日期：2009-02-01 00:00:00
abstract：:Endo-1,4-β-mannanase is an enzyme that can catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and offers many applications in different biotechnology industries. Purification and kinetic properties of the endo-1,4-β-mannanase from recombinant Esche...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.01.009
更新日期：2018-06-01 00:00:00
abstract：:The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragme...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/S1046-5928(03)00219-5
更新日期：2003-11-01 00:00:00
abstract：:The production of a single-chain variable fragment (scFv) antibody against bovine ribonuclease A in the cytoplasm of Escherichia coli trxB/gor double mutant was investigated. Previous reports have shown that the thioredoxin (Trx) protein fusion strategy is useful for the correct folding of scFvs and that the expressio...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.11.003
更新日期：2010-04-01 00:00:00
abstract：:Preventing protein aggregation is crucial for various protein studies, and has a large potential for remedy of protein misfolding or aggregates-linked diseases. In this study, we demonstrated the hyper-acidic protein fusion partners, which were previously reported to enhance the soluble expression of aggregation-prone...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.06.004
更新日期：2011-11-01 00:00:00
abstract：:One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purifie...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1586
更新日期：2002-04-01 00:00:00
abstract：:The aim of this study was to evaluate the parameters that affect the production of the recombinant 10 kDa culture filtrate protein (CFP10), a promising reagent of high specificity for intradermoreaction and other antigen-based methods used in the diagnosis of tuberculosis. Conditions of Escherichia coli growth tempera...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.09.016
更新日期：2019-02-01 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：:Splicing factor proline- and glutamine-rich (SFPQ) is an RNA-binding protein, playing significant roles in gene regulation and subnuclear body formation. Our recent serendipitous discovery showed that SFPQ binds zinc directly and forms an infinite polymer that is induced by zinc binding to the protein. The zinc-induce...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2020.105626
更新日期：2020-07-01 00:00:00
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
更新日期：1991-02-01 00:00:00
abstract：:The high specific lysyl endopeptidase (Lys-C; EC 3.4.21.50) is often used for the initial fragmentation of polypeptide chains during protein sequence analysis. However, due to its specificity it could be a useful tool for the production of tailor-made protein hydrolysates with for example bioactive or techno functiona...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.09.024
更新日期：2016-02-01 00:00:00
abstract：:The Plasmodium falciparum cysteine protease falcipain-2 is a potential new target for antimalarial chemotherapy. In order to obtain large quantities of active falcipain-2 for biochemical and structural analysis, a systematic assessment of optimal parameters for the expression and refolding of the protease was carried ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1416
更新日期：2001-06-01 00:00:00
abstract：:Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is cr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2019.02.004
更新日期：2019-06-01 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：:Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we repo...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2016.11.001
更新日期：2017-03-01 00:00:00
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
更新日期：2020-11-01 00:00:00
abstract：:As an insect-selective neurotoxin, scorpion long-chain BjαIT is a promising prospect for insecticidal application; however, the difficulty of obtaining natural BjαIT represents the major obstacle preventing analysis of its insecticidal activity against agricultural insect pests. Here, we screened recombinant Pichia pa...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.07.013
更新日期：2018-12-01 00:00:00
abstract：:Stromal-cell-derived factor-1 (SDF-1alpha) is an 8-kDa chemokine that is constitutively expressed in bone-marrow-derived stromal cells and has been identified as a ligand for the CXCR4 receptor. We produced the chemokine recombinantly as methionine-SDF-1alpha in Escherichia coli without the leader peptide sequence. Th...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1402
更新日期：2001-04-01 00:00:00
abstract：:The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0087
更新日期：1996-09-01 00:00:00
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
更新日期：1999-08-01 00:00:00
