Abstract:
:We describe a single method to purify milligram amounts of authentic wild-type and mutant HIV-1 and HIV-2 Tat proteins overexpressed in Escherichia coli. This method takes advantage of the highly basic, positively charged RNA binding domain present in both HIV-1 and HIV-2 Tat, which also facilitated purification of HIV-1 and HIV-2 mutant Tat proteins. In contrast to previously described methods, our method does not require use of denaturing or reducing agents, since Tat is present in the soluble fraction after bacterial lysis. The activity of purified wild-type and mutant HIV-1 and HIV-2 Tat proteins was determined using cell-based uptake, in vitro transcription, and TAR binding assays. As expected, both HIV-1 and HIV-2 Tat efficiently transactivated the HIV-2 LTR, whereas HIV-2 Tat transactivated the HIV-1 LTR less efficiently than HIV-1 Tat. Purified HIV-2 Tat proteins in which the glutamic acid residue at position 77 was changed to either glycine or glutamine transactivated the HIV-1 LTR more efficiently than wild-type Tat-2, providing additional evidence that the net charge of this region may be responsible for nonreciprocal transactivation between Tat-1 and Tat-2. Our results demonstrate that this method can be used to rapidly purify authentic wild-type and mutant Tat proteins which are suitable for cell-based and in vitro functional studies.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Orsini MJ,García-Martínez LF,Mavankal G,Gaynor RB,Debouck CMdoi
10.1006/prep.1996.0096subject
Has Abstractpub_date
1996-09-01 00:00:00pages
238-46issue
2eissn
1046-5928issn
1096-0279pii
S1046592896900960journal_volume
8pub_type
杂志文章abstract::The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of...
journal_title:Protein expression and purification
pub_type: 杂志文章
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更新日期:2011-10-01 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
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abstract::BetaB2-crystallin, the major subunit of beta-crystallins, is difficult to purify either from lens homogenate or from betaH-or betaL-crystallins. It has been prepared by heterologous expression in Escherichia coli. Most often, the methods used for purifying a recombinant globular protein employ the combination of ion-e...
journal_title:Protein expression and purification
pub_type: 杂志文章
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abstract::For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus. In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of t...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.0969
更新日期:1998-12-01 00:00:00
abstract::Separation of copurifying protease activity from recombinant human Müllerian inhibiting substance (rhMIS) bound to a monoclonal antibody immunoaffinity column by a high-salt wash results in cleaner preparations of rhMIS resistant to cleavage upon storage. In addition, an inhibitor of rhMIS antiproliferative activity i...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(92)90020-w
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abstract::Human dermcidin, an anionic antimicrobial peptide expressed in the pons of the brain and the sweat glands, displays antimicrobial activity against pathogenic microorganisms such as Staphylococcus aureus and Candida albicans. Here, we describe the recombinant production of a 48 amino acid dermcidin variant with C-termi...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.07.002
更新日期:2006-02-01 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::Staphylokinase (SAK) is reported to have a serine protease domain with no proteolytic activity unlike other plasminogen activators like tissue plasminogen activator (t-PA) and urokinase. A unique protease property of Staphylokinase was observed when SAK was expressed as a fusion protein in inducible Escherichia coli e...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.07.011
更新日期:2010-02-01 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::Antibacterial peptides from various sources express different antibacterial activity. In order to obtain a high activity antibacterial peptide, the sequences of four antimicrobial peptides--Protegrin-1, 4 kDa Scorpion Defensin, Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide SMAP-29--were exploited to generate...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.08.006
更新日期:2008-01-01 00:00:00
abstract::Baculovirus expression vectors are used routinely for foreign gene expression and are under intense development as improved biological pesticides. Conventional baculovirus expression vectors are recombinant viruses that can express a foreign gene in insect cells under the control of the polyhedrin promoter, which prov...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0092
更新日期:1996-09-01 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::Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.05.010
更新日期:2006-10-01 00:00:00
abstract::Previously, we have shown that the entire extracellular domain of the granulocyte-colony stimulating factor receptor (sG-CSFr) produced in Chinese hamster ovary (CHO) cells forms a stable complex with its ligand G-CSF, at a stoichiometry of 2:2. A truncated receptor molecule consisting of the cytokine receptor homolog...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.0942
更新日期:1998-10-01 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 gram-positive bacterium Corynebacterium pseudotuberculosis is the causative agent of different diseases that cause dramatically reduced yields of wool and milk, and results in weight loss, carcass condemnation and also death mainly in sheep, equids, cattle and goats and therefore globally results in considerable e...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.04.006
更新日期:2015-08-01 00:00:00
abstract::To produce a monoclonal antibody specific to a mouse major histocompatibility complex (MHC) class II protein, we synthesized the complementary DNAs for the heavy and light chains of a monoclonal antibody by PCR amplification. These cDNAs were then introduced separately into tobacco plant cells. After performing Northe...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.02.015
更新日期:2007-07-01 00:00:00
abstract::The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragme...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/S1046-5928(03)00219-5
更新日期:2003-11-01 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::We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue bio...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.09.018
更新日期:2008-02-01 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
更新日期:2001-10-01 00:00:00
abstract::α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae....
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2011.09.009
更新日期:2012-01-01 00:00:00
abstract::Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC), troponin I (hcTnI), and troponin T (hcTnT), are well-documented. Recently, it has been recognised that human cardiac troponin (hcTn) is a sophisticated allosteric system. Therefore, the effect of drugs on this protein co...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.10.005
更新日期:2013-02-01 00:00:00
abstract::The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the wid...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2019.105546
更新日期:2020-03-01 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::This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of im...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2011.08.021
更新日期:2011-09-03 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::The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-prote...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.01.012
更新日期:2014-04-01 00:00:00
abstract::Recombinant OSU VP4 protein, and outer capsid antigen of porcine rotavirus, was purified to a high level from the spent broth of baculovirus-infected Spodoptera frugiperda insect cells. Initial clarification of the broth with a 0-60% ammonium sulfate cut retained 93% of the total VP4. Q-sepharose ion exchange chromato...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1993.1030
更新日期:1993-06-01 00:00:00
abstract::Malassezia globosa is pathogenic fungus that causes skin disorders including dandruff in humans. Many yeast cytochrome CYP enzymes are involved in the biosynthesis of sterols and are considered major targets of azole antifungal agents. Here, we report on the expression and characterization of the MGL_0310 gene product...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.07.002
更新日期:2015-10-01 00:00:00