Abstract：
:Chemokine receptors are a specific class of G-protein-coupled receptors (GPCRs) that control cell migration associated with routine immune surveillance, inflammation and development. In addition to their roles in normal physiology, these receptors and their ligands are involved in a large number of inflammatory diseases, cancer and AIDS, making them prime therapeutic targets in the pharmaceutical industry. Like other GPCRs, a significant obstacle in determining structures and characterizing mechanisms of activation has been the difficulty in obtaining high levels of pure, functional receptor. Here we describe a systematic effort to express the chemokine receptor CCR1 in mammalian cells, and to purify and reconstitute it in functional form. The highest expression levels were obtained using an inducible HEK293 system. The receptor was purified using a combination of N- (StrepII or Hemagglutinin) and C-terminal (His8) affinity tags. Function was assessed by ligand binding using a novel fluorescence polarization assay with fluorescein-labeled chemokine. A strict dependence of function on the detergent composition was observed, as solubilization of CCR1 in n-dodecyl-beta-D-maltopyranoside/cholesteryl hemisuccinate yielded functional receptor with a K(d) of 21 nM for the chemokine CCL14, whereas it was non-functional in phosphocholine detergents. Differences in function were observed despite the fact that both these detergent types maintained the receptor in a state characterized by monomers and small oligomers, but not large aggregates. While optimization is still warranted, yields of approximately 0.1-0.2mg of pure functional receptor per 10(9) cells will permit biophysical studies of this medically important receptor.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Allen SJ,Ribeiro S,Horuk R,Handel TMdoi
10.1016/j.pep.2009.03.001subject
Has Abstractpub_date
2009-07-01 00:00:00pages
73-81issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(09)00058-8journal_volume
66pub_type
杂志文章abstract：:Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：
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abstract：:Telomerase is a specialized reverse transcriptase that catalyzes the addition of telomeric repeats, TTAGGG in all vertebrates, to the ends of chromosomes. The lack of recombinant purified human telomerase reverse transcriptase (hTERT) has hampered biochemical and structural studies. The primary problem in generating a...
journal_title：Protein expression and purification
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abstract：:A method for the simplified, reproducible production of both normal and altered versions of human proinsulin has been developed. A polyhistidine/proinsulin fusion protein was expressed using a prokaryotic expression system and partially purified by affinity chromatography. Disulfide bonds within the polypeptide were f...
journal_title：Protein expression and purification
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doi：10.1006/prep.1998.1024
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abstract：:Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains, among which stearoyl-acyl carrier protein desaturase (S-ACP-DES) was widely distributed in the plant kingdom. We cloned the cDNA coding for fab2/ssi2, an S-ACP-DES from Arabidopsis thaliana, into the vector pET30a and heterologously...
journal_title：Protein expression and purification
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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
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doi：10.1016/j.pep.2019.02.013
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journal_title：Protein expression and purification
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doi：10.1016/j.pep.2015.09.024
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journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.06.013
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abstract：:The human iron-binding protein lactoferrin (hLf) has been implicated in a number of important physiological pathways, including those regulating immune function and tumor growth. In an effort to develop an efficient system for production of recombinant hLf (rhLf) that is structurally and functionally equivalent to the...
journal_title：Protein expression and purification
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doi：10.1016/j.pep.2009.01.003
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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
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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：:The zoonotic Rift Valley fever virus affects livestock and humans in Africa and on the Arabian Peninsula. The economic impact of this pathogen due to livestock losses, as well as its relevance to public health, underscores the importance of developing effective and easily distributed vaccines. Vaccines that can be del...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2016.07.003
更新日期：2016-11-01 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：:High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZalphaA with the Saccharomyces cerevisiae alpha-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.06.021
更新日期：2009-11-01 00:00:00
abstract：:Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297-394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2010.07.013
更新日期：2010-12-01 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：: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：:The UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli, an enzyme involved in the biosynthesis of the bacterial peptidoglycan monomer unit, was overproduced and purified to homogeneity on a large scale, yielding 4 mg of protein per liter of bacterial culture. Crystals of the complex with the subst...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1997.0850
更新日期：1998-06-01 00:00:00
abstract：:Human β-glucuronidase (GUS; EC 3.2.1.31) is a lysosomal enzyme that catalyzes the hydrolysis of β-d-glucuronic acid residues from the non-reducing termini of glycosaminoglycans. Impairment in GUS function leads to the metabolic disorder mucopolysaccharidosis type VII, also known as Sly syndrome. We produced GUS from a...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2017.07.011
更新日期：2017-12-01 00:00:00
abstract：:Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CYP1A1.4 (T461N), were expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30-90 nmol ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00672-1
更新日期：2003-04-01 00:00:00
abstract：:Human Relaxin 2 is an insulin-related peptide hormone with a mass of 19,084 Da. The mRNA contains a number of arginine codons that are rarely used by Escherichia coli to produce highly expressed proteins. As a result, expressing this recombinant protein in E. coli is problematic. When human Relaxin 2 was expressed in ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.02.016
更新日期：2008-08-01 00:00:00
abstract：:Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening b...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1565
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abstract：:Glycolate oxidase is a flavin-dependent enzyme in the photorespiratory pathway in plants. Here we report the heterologous expression of glycolate oxidase in Escherichia coli and an isolation procedure which results in 4 mg pure protein per gram cell paste in only 1.5 days. This corresponds to a more than 50-fold impro...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0103
更新日期：1996-11-01 00:00:00
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
更新日期：2016-04-01 00:00:00
abstract：:Ketol-acid reductoisomerase (EC 1.1.1.86) catalyzes the conversion of 2-aceto-2-hydroxyacids to 2-keto-3-hydroxyacids and their subsequent reduction by NADPH to 2,3-dihydroxyacids. The gene encoding the Escherichia coli enzyme was cloned and expressed as a hexahistidine-tagged fusion protein and the recombinant enzyme...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.0988
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abstract：:Soybean root nodules possess a developmentally regulated acid phosphatase (ACP) that exhibits the highest specificity for purine 5'-nucleoside monophosphates. The enzyme is a glycosylated dimer of 28- and 31-kDa subunits, which appear to be products of the same gene but differ in posttranslational modifications. In or...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.0935
更新日期：1998-10-01 00:00:00
abstract：:A new procedure for the large-scale purification of the recombinant thermostable chitinase (Chi40) cloned from Streptomyces thermoviolaceus in various expression vectors in Escherichia coli is described. Chi40 was overproduced in the cytosolic and secreted forms. The cytosolic form (Chi40c) was highly overproduced and...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1490
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abstract：:The Ca2+-ATPase accounts for the majority of Ca2+ removed from the cytoplasm during cardiac muscle relaxation. The Ca2+-ATPase is regulated by phospholamban, a 52 amino acid phosphoprotein, which inhibits Ca2+-ATPase activity by decreasing the apparent affinity of the ATPase for Ca2+. To study the physical mechanism o...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.11.005
更新日期：2004-03-01 00:00:00
abstract：:Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system. The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da). Thrombin digestion of fusion protein was expected at a clone...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.1015
更新日期：1999-03-01 00:00:00
abstract：:A fibrinolytic enzyme was purified from the cultured mycelia of Armillaria mellea by ion-exchange chromatography followed by gel filtration, and was designated A. mellea metalloprotease (AMMP). The purification protocol resulted in a 627-fold purification of the enzyme, with a final yield of 6.05%. The apparent molecu...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.05.004
更新日期：2005-09-01 00:00:00
abstract：:Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly ex...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.01.019
更新日期：2004-06-01 00:00:00
