Abstract:
:Na+, K+-ATPase beta2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule. To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl portion segment of NKA1b2. The coding region for amino acids 190-290 at the carboxyl portion of NKA1b2 (NKA1b2-CP) was sub-cloned into the vector pGEX-4T-2 and introduced into the Escherichia coli BL21(DE3) cell for efficient soluble expression. The amino acid sequence of expressed protein was determined using mass spectrometry following Mascot analysis. After purification, GST-NKA-beta2-CP was used to immunize the adult rabbits following standard protocols. The produced antiserum could detect the NKA1b2 protein expressed not only in the prokaryotic cells (E. coli) but also in the eukaryotic cells (COS7) transfected with NKA1b2 expression vector (pEGFP-NKA1b2). Furthermore, the antiserum was used for determining the localization of NKA1b2 in primary culture of neonatal rat neurons using immunohistochemical technique. Results demonstrated that NKA1b2 was localized both in the cytoplasm and cellular membrane. The preparation of anti-NKA-beta2-CP polyclonal antibody will facilitate further functional study on NKA1b2.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Chen TF,Zhang YL,Xu WL,Li ZQ,Hou B,Wang CL,Fan M,Qian LJ,Zhou RP,Zhang CGdoi
10.1016/j.pep.2004.05.003keywords:
subject
Has Abstractpub_date
2004-09-01 00:00:00pages
47-52issue
1eissn
1046-5928issn
1096-0279pii
S1046592804001664journal_volume
37pub_type
杂志文章abstract::To facilitate studies of multicomponent protein complexes, I have developed an Escherichia coli expression system which coexpresses up to four polypeptides from a single plasmid. The modular nature of the system enables efficient subcloning of a gene into each of the 4 cassettes in the polycistronic expression vector....
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2000.1363
更新日期:2001-02-01 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::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
更新日期:2020-08-01 00:00:00
abstract::Membrane proteins, including solute transporters play crucial roles in cellular function and have been implicated in a variety of important diseases, and as such are considered important targets for drug development. Currently the drug discovery process is heavily reliant on the structural and functional information d...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.07.003
更新日期:2014-09-01 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::Human interleukin-7 (IL-7) is a member of the interleukin family. Numerous studies have demonstrated IL-7's effect on B- and T-cell development as well as its potential in various clinical applications. Previously, a study reported that IL-7 could be purified from inclusion bodies using a prokaryotic system, however, ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.08.009
更新日期:2009-01-01 00:00:00
abstract::Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that acts specifically on epithelial cells in a paracrine mode. We employed a mammalian expression system to synthesize recombinant human KGF and isolated two preparations, KGF-a and KGF-b, from medium conditioned by Chinese hamster ov...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1997.0840
更新日期:1998-03-01 00:00:00
abstract::Mitochondrial 2,4-dienoyl-CoA reductase is a key enzyme for the beta-oxidation of unsaturated fatty acids. The cDNA of the full-length human mitochondrial 2,4-dienoyl-CoA reductase was previously cloned as pUC18::DECR. PCR methodologies were used to subclone the genes encoding various truncated human mitochondrial 2,4...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(03)00191-8
更新日期:2003-10-01 00:00:00
abstract::Expression and purification of human beta-secretase (BACE1) in bacteria have been plagued with issues concerning solubility, inhomogeneous N-terminus, and lack of enzymic activity. Several forms of the mature human BACE1 have been expressed in Escherichia coli with different N-terminal extensions and without the C-ter...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2003.12.014
更新日期:2004-04-01 00:00:00
abstract::Mammalian target of rapamycin (TOR) controls cell growth and metabolism in response to the availability of nutrients, growth factors, and the cellular energy status. Misregulation of TOR can result in a pathogenic increase or decrease in organ size and in cancer. TOR can be inhibited by binding of a complex of rapamyc...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.01.014
更新日期:2008-05-01 00:00:00
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
更新日期:2017-07-01 00:00:00
abstract::Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, mo...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.10.007
更新日期:2013-02-01 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::L-Threonine dehydrogenase was purified 10,000-fold to a specific activity approximately 300 mumol.min-1.mg-1 protein from porcine liver mitochondria. Purification to apparent homogeneity was achieved by sequential chromatography on DEAE Sepharose FF, Affi-Gel Blue, Sephacryl S-200, Matrex Gel Red A, and Matrex Gel Gre...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1994.1061
更新日期:1994-10-01 00:00:00
abstract::Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high leve...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1999.1138
更新日期:1999-12-01 00:00:00
abstract::Pullulanase is crucial to the specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in the starch-processing industry. Recombinant Bacillus subtilis that employs an inducible promoter would be a suitable candidate for pullulanase expression because of its safety and contr...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2018.03.012
更新日期:2018-08-01 00:00:00
abstract::A new method for the purification of manganese superoxide dismutase from human liver is described. The procedure involves essentially three steps: DEAE-cellulose, hydroxylapatite, and butyl-Toyopearl chromatographies. The method has several advantages: (i) its simplicity and rapidity (it takes less than 3 days), (ii) ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(91)90067-s
更新日期:1991-04-01 00:00:00
abstract::The YUH1 gene coding for ubiquitin C-terminal hydrolase 1, a deubiquitinating enzyme, was cloned from the Saccharomyces cerevisiae genomic DNA and expressed in Escherichia coli. YUH1 was fused with the 6 histidine tag at the N-terminus (H6YUH1) or C-terminus (YUH1H6) and purified by an immobilized metal affinity chrom...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.07.005
更新日期:2007-11-01 00:00:00
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
更新日期:2011-12-01 00:00:00
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
更新日期:2020-09-01 00:00:00
abstract::The gene encoding a novel glucoamylase (GlucaM) from the Corallococcus sp. strain EGB was cloned and heterologous expressed in Escherichia coli BL21(DE3), and the enzymatic characterization of recombinant GlucaM (rGlucaM) was determined in the study. The glucaM had an open reading frame of 1938 bp encoding GlucaM of 6...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.06.009
更新日期:2017-01-01 00:00:00
abstract::The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.03.010
更新日期:2009-08-01 00:00:00
abstract::High affinity interaction between S-protein and S-peptide fragments of bovine pancreatic RNase A has been recently used for construction of molecular vehicles for targeted drug delivery. The vehicle is assembled as a complex of drug carrier conjugated S-protein with S-peptide-tagged targeting protein. To avoid random ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(02)00546-6
更新日期:2002-12-01 00:00:00
abstract::A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2016.02.015
更新日期:2016-06-01 00:00:00
abstract::NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein. This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1. Different heterologous expression sy...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1999.1098
更新日期:1999-10-01 00:00:00
abstract::Antimicrobial peptide LL-37 plays an important role in human body's first line of defense against infection. To better understand the mechanism of action, it is critical to elucidate the three-dimensional structure of LL-37 in complex with bacterial membranes. We present a bacterial expression system that allows the i...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.10.022
更新日期:2006-06-01 00:00:00
abstract::We present a straightforward, versatile method for expressing and purifying β-amyloid (Aβ40) and transmembrane peptides derived from β-amyloid precursor protein (Aβ55). In principle, these methods should be applicable to other types of strongly aggregating peptides. We start with a DNA plasmid encoding a HexaHis tag w...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2019.04.006
更新日期:2019-10-01 00:00:00
abstract::Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7 kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.05.002
更新日期:2014-08-01 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::A purification scheme based on hydrophobic interaction chromatography was developed to separate inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and 8.8-fold purification were achieved on Butyl Sepharose 4 FF. Further purification on Superdex 200 resulted in another 29-fold pu...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.04.011
更新日期:2015-09-01 00:00:00