Abstract：
:The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Lawrie AM,Tito P,Hernandez H,Brown NR,Robinson CV,Endicott JA,Noble ME,Johnson LNdoi
10.1006/prep.2001.1504keywords:
subject
Has Abstractpub_date
2001-11-01 00:00:00pages
252-60issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(01)91504-9journal_volume
23pub_type
杂志文章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：:Post-translational processing of host defense cathelicidin peptide LL-37 in human sweat and skin generates new antimicrobial peptides. To understand structure and mechanism of action of these LL-37 derivatives, this article presents the cloning and expression of SK-29, KR-20, LL-29, and LL-23. We also provide a two-st...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.04.023
更新日期：2007-10-01 00:00:00
abstract：:This paper reports the overproduction and the details of a rapid method to purify active sigmaS monomers from a T7 RNA polymerase-based protein expression system. This 2-day procedure involves solubilizing inclusion bodies in sarkosyl detergent, removal of sarkosyl by dialysis, and a single gel filtration column chrom...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0069
更新日期：1996-08-01 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：: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：:Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activitie...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.10.002
更新日期：2019-02-01 00:00:00
abstract：:The ubiquitin system represents a selective mechanism for intracellular proteolysis in eukaryotic cells that involves the sequential activity of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The identification of these proteins and their cellular...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.08.018
更新日期：2006-02-01 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：:HSPC144 is a newly identified gene in human CD34(+) hematopoietic stem/progenitor cells. In this work, we have expressed and purified the 225-residue protein from Escherichia coli BL21 (DE3) and identified a stable fragment HSPC144-P (residues 44-225) by limited proteolysis method. The HSPC144-P fragment exhibits high...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.03.008
更新日期：2005-07-01 00:00:00
abstract：:High-level constitutive expression of xylanase GH11 from Penicillium occitanis Pol6 termed PoXyn2 was achieved using the methylotrophic yeast Pichia pastoris. The PoXyn2 cDNA encoding for a mature xylanase of 320 amino acids was subcloned into the pGAPZαA vector, to construct recombinant xylanse with six histidine res...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2012.02.012
更新日期：2012-05-01 00:00:00
abstract：:Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic compounds and also peptide and protein bound tyrosine, and thus is of great interest for different biotechnological applications. In order to understand the reaction mechanism of the enzyme it would be essential to solve it...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.04.014
更新日期：2007-09-01 00:00:00
abstract：:The SaeRS two-component system in Staphylococcus aureus controls the expression of a series of virulence factors, such as hemolysins, proteases, and coagulase. The response regulator, SaeR, belongs to the OmpR family with an N-terminal regulatory domain and a C-terminal DNA binding domain. To improve the production an...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2020.105765
更新日期：2021-01-01 00:00:00
abstract：:FcgammaRs are involved in regulating a multitude of innate and adaptive immune responses, which makes them attractive targets for the development of novel immunotherapeutic approaches. In this report, we describe a simple method for the production of a large quantity of recombinant porcine FcgammaRII. The extracellula...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.06.020
更新日期：2009-11-01 00:00:00
abstract：:Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, a...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1610
更新日期：2002-06-01 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：: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：:Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overprodu...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.09.001
更新日期：2012-01-01 00:00:00
abstract：:Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.03.013
更新日期：2004-07-01 00:00:00
abstract：:We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhe...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1290
更新日期：2000-11-01 00:00:00
abstract：:The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CR...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2016.08.011
更新日期：2016-12-01 00:00:00
abstract：:The fluorescent reporter enhanced Green Fluorescent Protein (EGFP) has been used for assaying a wide range of biological activities ranging from gene expression, or localization of target proteins through to intermolecular interactions. However, over-production of this protein in Escherichia coli has resulted in the p...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.05.003
更新日期：2011-10-01 00:00:00
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：:To study the subunit structure and the active site of human immunodeficiency virus reverse transcriptase (RT), the enzyme was expressed in E. coli and purified to homogeneity in large quantities. The recombinant enzyme consists of two major polypeptides of 66,000 and 53,000 Da in equimolar amounts and a minor species ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/1046-5928(92)90005-h
更新日期：1992-08-01 00:00:00
abstract：:A soluble N-terminal domain of the Escherichia coli adenylyl transferase (ATase) is responsible for deadenylylation activity of the intact enzyme. Previous studies of the deadenylylation activity have involved a fragment, AT-N423 (residues 1 to 423), which was extended by 17 amino acids to give AT-N440. This new domai...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.11.001
更新日期：2004-03-01 00:00:00
abstract：:Magnolia officinalis Rehder et Wilson is a traditional Chinese herbal medicine that is used to treat various diseases such as neurosis, anxiety, and stroke. The main secondary metabolites in magnolia bark are phenolic compounds and terpenoids. Squalene synthase plays a significant role in catalyzing two farnesyl dipho...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.12.008
更新日期：2016-04-01 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：:Antibodies specific to β-Glucocerebrosidase were selected from phage displayed naïve scFv libraries. Biopannings were performed against recombinant human protein β-Glucocerebrosidase immobilized on polystyrene surface, specific phages were eluted with 50% ethylene glycol in citrate buffer (pH 6.0). Several specific bi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2020.105573
更新日期：2020-06-01 00:00:00
abstract：:This is the first report of an insect esterase efficiently expressed in the methylotrophic yeast Pichia pastoris (so far insect esterases have been produced only in the baculovirus system). Having isolated a Tribolium castaneum carboxylesterase cDNA (TCE), we were initially unable to express it in Escherichia coli or ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.04.011
更新日期：2005-08-01 00:00:00
abstract：:The 42 kDa cleavage product from the carboxyl end of the Plasmodium falciparum merozoite surface protein 1 (MSP1(42)) is an important blood-stage malaria vaccine target. Several recombinant protein expression systems have been used for production of MSP1(42) including yeast (Saccharomyces cerevisiae and Pichia pastori...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.06.018
更新日期：2006-11-01 00:00:00
abstract：:A gene encoding a glutamate-specific endopeptidase (GSE) from Bacillus licheniformis (BL) has been cloned in Escherichia coli cells. The recombinant protein was expressed as cytoplasmic insoluble inclusion bodies. Immobilized metal affinity chromatography was employed to purify the protein, and then a 27-kDa GSE inter...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.12.001
更新日期：2012-03-01 00:00:00
