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：:The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 bindi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.10.027
更新日期：2007-05-01 00:00:00
abstract：:A highly efficient cell-free translation system has been combined with suppressor tRNA technology to substitute nor-Tyr and 3-fluoro-Tyr in place of Tyr183 at the DNA polymerase active site of p66 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Supplementing the wild-type HIV-1 p51 RT subunit ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.07.019
更新日期：2004-11-01 00:00:00
abstract：:Cystathionine beta-synthase (CBS) catalyzes the pyridoxal-50-phosphate-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chrom...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.10.012
更新日期：2009-04-01 00:00:00
abstract：:In Pisum sativum, the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). Expression of two of these genes (SAD-A and -C) in Escherichia coli or Pichia pastoris resulted in full-length soluble proteins. Purified SAD-A was used as antigen for antibody...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.09.004
更新日期：2009-01-01 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：:The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim50 is a major receptor in TIM23 complex, which spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the interme...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.06.012
更新日期：2011-11-01 00:00:00
abstract：:Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in chemoenzymatic peptide synthes...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2013.01.004
更新日期：2013-05-01 00:00:00
abstract：:PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.02.015
更新日期：2014-06-01 00:00:00
abstract：:The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2012.11.005
更新日期：2013-07-01 00:00:00
abstract：:We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella entericaprpBCDE promoter (P(prpB)) and compared it to that from the strongest IPTG-inducible promoter, P(T7). In contrast to our previous study showing slight...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.06.008
更新日期：2008-10-01 00:00:00
abstract：:A tremendous increase has taken place over the last decades in the biochemical and clinical use of antibodies. Unfortunately, the constantly growing demand has not been matched by a corresponding easy access to pure immunoglobulin, as purification procedures tend to be either laborious, expensive, or inefficient. We p...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(03)00130-x
更新日期：2003-08-01 00:00:00
abstract：:Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market....
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2013.01.011
更新日期：2013-04-01 00:00:00
abstract：:The expression of recombinant proteins in bacterial hosts may alter the biophysical properties of the protein of interest as a result of differences in post-translational processing from that observed when produced in the native cell. For example, recombinant human interleukin-1beta (IL-1beta) is produced as three iso...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.01.010
更新日期：2005-05-01 00:00:00
abstract：:The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Protea...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2020.105725
更新日期：2020-12-01 00:00:00
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
更新日期：1992-10-01 00:00:00
abstract：:The gene that was inferred to encode the TyrR protein of Haemophilus influenzae Rd was synthesized by polymerase chain reaction and inserted into a T7-based expression vector. Methods were developed to overexpress the TyrR protein of H. influenzae in Escherichia coli and to purify the protein on a large scale. Both in...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1997.0757
更新日期：1997-07-01 00:00:00
abstract：:The guanine nucleotide exchange factor EF-1beta gene from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1beta) was amplified by PCR and cloned into the pT7-7 expression vector. One of four selected clones harbored the T160C nucleotide substitution leading to the Y54H amino acid change in a hydrophobic r...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1997.0806
更新日期：1998-02-01 00:00:00
abstract：:Polyhistidine tags enable the facile purification of proteins by immobilized metal affinity chromatography (IMAC). Both the type and position of purification tags can affect significantly properties of a protein such as its expression level, behavior in solution, and its ability to form suitable samples (esp. suitable...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.10.010
更新日期：2004-02-01 00:00:00
abstract：:In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, aspartate kinase-homoserine dehydrogenase (AK-HSDH). In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from plants (Arabidopsis ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1539
更新日期：2002-02-01 00:00:00
abstract：:The market of therapeutic glycoproteins (including coagulation factors, antibodies, cytokines and hormones) is one of the profitable, fast-growing and challenging sectors of the biopharmaceutical industry. Although mammalian cell culture is still expensive and technically complex, the ability to produce desired post-t...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.08.008
更新日期：2015-11-01 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：:Over-expression of heterologous proteins in Escherichia coli is commonly hindered by the formation of inclusion bodies. Nevertheless, refolding of proteins in vitro has become an essential requirement in the development of structural genomics (proteomics) and as a means of recovering functional proteins from inclusion...
journal_title：Protein expression and purification
pub_type： 杂志文章,评审
doi：10.1016/j.pep.2007.05.014
更新日期：2007-10-01 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：:Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.09.015
更新日期：2015-01-01 00:00:00
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：: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
更新日期：2004-11-01 00:00:00
abstract：:Vascular endothelial growth factor (VEGF) is one of the most significant mediators of angiogenesis, which interacts with a specific membrane receptor: VEGF receptor 2 (VEGFR2). Studies elsewhere have shown that, a VEGF-blocker can regulate several vital processes of tumor promotion. However, there is no literature evi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2013.04.010
更新日期：2013-08-01 00:00:00
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
更新日期：2007-03-01 00:00:00
abstract：:Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.02.005
更新日期：2018-07-01 00:00:00
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
更新日期：2019-01-01 00:00:00
