Abstract：
:Acyl-acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein. The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine acyltransferase. The E. coli aas open reading frame was inserted into the expression plasmid pET28a so that, upon expression, a 21-amino-acid extension containing 6 consecutive histidine residues was added to the carboxyl terminus. The plasmid was designated pAasH. The activity of Aas in membranes was assessed from several cell lines. Membranes from the commonly used host line BL21(DE3) containing pAasH accumulated 30-fold and 38-fold more Aas activity than membranes from BL21(DE3) cells lacking the plasmid, when induced with isopropyl beta-d-thiogalactopyranoside (IPTG) or lactose, respectively. When pAasH was expressed under IPTG induction in cell line C41(DE3), a previously described cell line selected to enhance the expression of membrane proteins, Aas levels accumulated to 135-fold higher levels than in the cell line lacking the plasmid. Functional Aas can be isolated from either BL21(DE3) or C41(DE3) cell lines by differential centrifugation, followed by detergent extraction with Triton X-100 and nickel nitrilotriacetic acid affinity chromatography. The overexpression of Aas in cell line C41(DE3) is noteworthy compared to cell line BL21(DE3) because it results in a 3- to 4-fold higher accumulation of active enzyme in the membrane fraction and a lower proportion of inactive protein in the inclusion body.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Shanklin Jdoi
10.1006/prep.2000.1206keywords:
subject
Has Abstractpub_date
2000-04-01 00:00:00pages
355-60issue
3eissn
1046-5928issn
1096-0279pii
S1046-5928(00)91206-3journal_volume
18pub_type
杂志文章abstract：:A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpresse...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.05.016
更新日期：2006-01-01 00:00:00
abstract：:Production of VCSM13 phage displaying a high density of CD147 ectodomain (CD147Ex) was achieved when culturing conditions were modulated. A phagemid expressing CD147Ex was constructed and used to produce phage display CD147Ex gpVIII fusion protein in TG1 Escherichia coli. Displaying of CD147Ex via gpVIII was successfu...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.08.019
更新日期：2003-12-01 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：:Human lysosomal beta-hexosaminidase exists in two major forms: the A isoform is composed of both alpha and beta chains, while the B form is a homopolymer of beta chains. Deficiency of beta-hexosaminidase underlies the GM2 gangliosidoses. We have produced active beta-hexosaminidase B in cultured insect (Sf9) cells by i...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/1046-5928(90)90003-h
更新日期：1990-11-01 00:00:00
abstract：:The aim of this study was to establish a method of purifying intact complement factor H (CFH) from human plasma. CFH was isolated from human plasma by polyethylene glycol (PEG) precipitation, following three sequential chromatographic columns, which consisted of l-lysine Sepharose column, Resource Q column and Sephacr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2013.07.014
更新日期：2013-10-01 00:00:00
abstract：:The main principles of higher-order protein oligomerization are elucidated by many structural and biophysical studies. An astonishing number of proteins self-associate to form dimers or higher-order quaternary structures which further interact with other biomolecules to elicit complex cellular responses. In this study...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.08.010
更新日期：2019-01-01 00:00:00
abstract：:Escherichia coli cells were transformed with an expression vector constructed by inserting a DNA fragment encoding a Kazal-type trypsin inhibitor from mouse seminal vesicle into pGEX-2. The cloned cells were able to produce a high yield of a chimeric polypeptide made by fusing the trypsin inhibitor to glutathione S-tr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1994.1003
更新日期：1994-02-01 00:00:00
abstract：:While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.05.017
更新日期：2007-10-01 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：: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：: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：:Ndt80 is a Saccharomyces cerevisiae meiosis-specific transcription factor responsible for promoting the stage-specific expression of a family of genes referred to as middle sporulation genes. Many members of this gene family are essential for the completion of meiotic chromosome segregation. Thus, Ndt80 is essential f...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.08.025
更新日期：2004-01-01 00:00:00
abstract：:Integrated studies that address proteins structure and function in the new era of systems biology and genomics often require the application of high-throughput approaches for parallel production of many different purified proteins from the same organism. Cytochromes c-electron transfer proteins carrying one or more he...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.06.014
更新日期：2008-11-01 00:00:00
abstract：:In order to produce sufficient quantities of fibroblast growth factor-saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large-scale expression, purification, and characterization of the recombinant molecule. The fusion gene encoding rF...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0079
更新日期：1996-08-01 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：: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
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：: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
更新日期：2002-03-01 00:00:00
abstract：:Pokeweed antiviral protein (PAP)-I from the spring leaves of Phytolacca americana is a naturally occurring RNA-depurinating enzyme with broad-spectrum antiviral activity. Interest in PAP is growing due to its use as a potential anti-HIV agent. However, the clinical use of native PAP is limited due to inherent difficul...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1999.1181
更新日期：2000-03-01 00:00:00
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
pub_type： 杂志文章
doi：10.1016/j.pep.2007.05.016
更新日期：2007-11-01 00:00:00
abstract：:Periplasmic expression of recombinant proteins ensures the production of biologically active proteins in a correctly folded state with several key advantages. This research focused on the in-frame cloning of rhIL-15 in pET-20 (+) vector with pelB-leader sequence to direct the protein to the bacterial periplasm. The ta...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2020.105746
更新日期：2021-01-01 00:00:00
abstract：:Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proli...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.01.001
更新日期：2011-05-01 00:00:00
abstract：:We have developed a family of cloning vectors that direct expression of fusion proteins that mimic aggregated immunoglobulin (IgG) (AIG) and immune complex function with respect to their interactions with FcgammaR and that allow for the inclusion and targeting of a second protein domain to cells expressing FcgammaR. T...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1406
更新日期：2001-04-01 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：:A full-length hexokinase cDNA was cloned from Solanum chacoense, a wild relative of the cultivated potato. Analysis of the predicted primary sequence suggested that the protein product, ScHK2, may be targeted to the secretory pathway and inserted in the plant plasma membrane, facing the cytosol. ScHK2 was expressed as...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.11.003
更新日期：2006-05-01 00:00:00
abstract：:Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. The GM-CSF cDNA was carried by a binary vector under the control of the CaMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated region from the tobacco...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1232
更新日期：2000-06-01 00:00:00
abstract：:To investigate the expression and purification of an unstable heterologous protein in Pichia pastoris, the cDNA of H5-lysozyme, a hen egg lysozyme mutant with a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) fused to the carboxyl terminus, was integrated into the genome of P. pastoris. It was found that medium composi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00642-3
更新日期：2003-02-01 00:00:00
abstract：:Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, fu...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.09.014
更新日期：2006-04-01 00:00:00
abstract：:Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypept...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/1046-5928(91)90095-z
更新日期：1991-10-01 00:00:00
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
