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 CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination. Finally, using silver staining, Western blotting and LC-MS/MS we compare the components of INT of purifications from CRISPR derived lines to 293T cells overexpressing FLAG-INTS11 to define a highly resolved constituency of mammalian INT.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Baillat D,Russell WK,Wagner EJdoi
10.1016/j.pep.2016.08.011subject
Has Abstractpub_date
2016-12-01 00:00:00pages
101-8eissn
1046-5928issn
1096-0279pii
S1046-5928(16)30167-Xjournal_volume
128pub_type
杂志文章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：:The sequence and structure of the target protein exert a marked effect on its soluble expression in Escherichia coli. The effects of the mutation of an amylase isolated from Bacillus licheniformis (BLA) on its soluble expression in E. coli were investigated. A random mutation library of BLA was constructed to screen f...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.12.010
更新日期：2016-04-01 00:00:00
abstract：:Chinese hamster dihydrofolate reductase (ch-DHFR) was overexpressed in Escherichia coli DH5 alpha under the transcriptional control of PRPL promoters regulated by temperature-sensitive repressors. The desired recombinant product is soluble and constitutes about 30% of the total soluble proteins of the bacterial cell. ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0676
更新日期：1997-03-01 00:00:00
abstract：:The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeB gene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of the gpd promoter and trpC terminator and expressed in a protease defi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.05.019
更新日期：2004-09-01 00:00:00
abstract：:BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome b(5) fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage b...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.10.017
更新日期：2010-04-01 00:00:00
abstract：:This study describes comparison between IPTG and lactose induction on expression of caprine growth hormone (cGH), enhancing cell densities of Escherichia coli cultures and refolding the recombinant cGH, produced as inclusion bodies, to biologically active state. 2-3 times higher cell densities were obtained in shake f...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.05.011
更新日期：2009-11-01 00:00:00
abstract：:The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stres...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.01.016
更新日期：2008-05-01 00:00:00
abstract：:Mannan outer chain N-glycan structures are yeast/fungal-specific typically found on secreted and cell wall glycoproteins. Mannan outer chains consist of an alpha-1,6 polymannose backbone attached to a Man(8-10)(GlcNAc)(2) core. The backbone contains branches of alpha-1,2 mannose residues, terminated with alpha-1,3 man...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.02.013
更新日期：2009-07-01 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：:Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer po...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.03.011
更新日期：2014-07-01 00:00:00
abstract：:Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3'-5' exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E. coli, but not in higher organisms, and close homologues are present in other genomes from ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.10.005
更新日期：2008-02-01 00:00:00
abstract：:Quinolinate synthetase catalyzes the second step of the de novo biosynthetic pathway of pyridine nucleotide formation. In particular, quinolinate synthetase is involved in the condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinic acid. To study the mechanism of action, the specificity of the...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1999.1153
更新日期：2000-02-01 00:00:00
abstract：:We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.08.014
更新日期：2004-01-01 00:00:00
abstract：:The mRNA encoding the 51-kDa subunit of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver was reverse-transcribed and amplified. The resulting cDNA was reamplified in N-terminal and C-terminal segments and spliced to generate a full-length clone, and an internal cDNA fragment was also amplified. The full-lengt...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.0896
更新日期：1998-07-01 00:00:00
abstract：:Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP-binding proteins found in archaea, plants, fungi and animals, indicating important roles in fundamental pathways. Their function is poorly understood, but they have been implicated in cell division, proliferation, and growth, as well as s...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.05.009
更新日期：2009-10-01 00:00:00
abstract：:We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichiacoli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We e...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.12.005
更新日期：2012-03-01 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：:Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(03)00159-1
更新日期：2003-09-01 00:00:00
abstract：:Ciliary neurotrophic factor (CNTF) is a promising candidate for the treatment of neurodegenerative or metabolic diseases, but suffers rapid clearance in body. Herein we constructed a new long-acting recombinant human CNTF (rhCNTF) by genetic fusion with an albumin-binding domain (ABD) through a flexible peptide linker...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2017.07.006
更新日期：2017-11-01 00:00:00
abstract：:Protein convertase 1/3 is a serine endoproteinase present in the regulated secretory pathway of endocrine and neuroendocrine cells. It is responsible for the processing of numerous prohormones and proneuropeptides into their biologically active moieties, often following cleavage at pairs of basic residues. The determi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.06.014
更新日期：2004-10-01 00:00:00
abstract：:Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in the methylotrophic yeast Pichia pastoris and established the purification pr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.03.025
更新日期：2008-08-01 00:00:00
abstract：:The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2alpha at Ser51. In the...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.0863
更新日期：1998-04-01 00:00:00
abstract：:Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2010.03.013
更新日期：2010-08-01 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：: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：: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：:Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic an...
journal_title：Protein expression and purification
pub_type： 杂志文章,评审
doi：10.1006/prep.2001.1465
更新日期：2001-07-01 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：:The human cyclin H, a protein normally associated with the cyclin-dependent kinase cdk7, was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity. The purified recombinant cyclin H is similar to the endogenous one according to the following criteria...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0693
更新日期：1997-03-01 00:00:00
abstract：:Heterologous protein expression in Escherichia coli is commonly used to obtain recombinant proteins for a variety of downstream applications. However, many proteins are not, or are only poorly, expressed in soluble form. High level expression often leads to the formation of inclusion bodies and an inactive product tha...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.08.030
更新日期：2012-01-01 00:00:00
