Abstract:
:Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Kurz M,Cowieson NP,Robin G,Hume DA,Martin JL,Kobe B,Listwan Pdoi
10.1016/j.pep.2006.05.006subject
Has Abstractpub_date
2006-11-01 00:00:00pages
68-73issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(06)00154-9journal_volume
50pub_type
杂志文章abstract::2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia col...
journal_title:Protein expression and purification
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更新日期:2016-10-01 00:00:00
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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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
pub_type: 杂志文章
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journal_title:Protein expression and purification
pub_type: 杂志文章
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(02)00672-1
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2016.08.011
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abstract::Human Fas ligand is a medically important membrane glycoprotein that induces the apoptosis of harmful cells. A new secretory expression and purification method was devised for the production of a large amount of recombinant human Fas ligand extracellular domain (hFasLECD) by Pichia pastoris. The expression plasmid con...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.08.006
更新日期:2006-12-01 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::We report the molecular cloning, expression, and single-step homogeneous purification of a full-length asparaginyl tRNA synthetase (NRS) from Fasciola gigantica (FgNRS). Fasciola gigantica is a parasitic liver fluke of the class Trematoda. It causes fascioliasis that infects the liver of various mammals, including hum...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2017.10.009
更新日期:2018-03-01 00:00:00
abstract::Acyl carrier protein (ACP) was purified from Euglena gracilis variety bacillaris in yields of about 1 mg/100 g (wet wt) of cells. Antibodies against the purified protein were raised in hens and isolated from eggs. Antibodies raised against Euglena ACP inhibited the Euglena chloroplast nonaggregated fatty acid syntheta...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(91)90072-q
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abstract::In Pisum sativum, the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). Expression of two of these genes (SAD-A and -C) in Escherichia coli or Pichia pastoris resulted in full-length soluble proteins. Purified SAD-A was used as antigen for antibody...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.09.004
更新日期:2009-01-01 00:00:00
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journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.11.018
更新日期:2008-04-01 00:00:00
abstract::A method for purifying acylation stimulating protein (ASP) from porcine serum is described. The mRNA encoding ASP was cloned by reverse transcriptase-polymerase chain reaction which predicted a 76 residue peptide. Based on this sequence, we generated antisera to a C-terminal peptide (ASP(1-20)) which aided ASP purific...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(02)00019-0
更新日期:2002-07-01 00:00:00
abstract::Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed,...
journal_title:Protein expression and purification
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abstract::Recombinant cytochrome P450 (CYP or P450) enzymes are useful for drug metabolism research and thereby many expression and purification systems have been developed. Here, we provide a method for the purification of human P450s 3A4 and 1A2 expressed in Escherichia coli using mixed micelles containing anionic phospholipi...
journal_title:Protein expression and purification
pub_type: 杂志文章
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journal_title:Protein expression and purification
pub_type: 杂志文章
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2010.02.005
更新日期:2010-06-01 00:00:00
abstract::Human small nuclear (sn) RNA genes are transcribed by either RNA polymerase II or III depending upon the arrangement of their core promoter elements. Regardless of polymerase specificity, these genes share a requirement for a general transcription factor called the snRNA activating protein complex or SNAP(C). This mul...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.02.015
更新日期:2006-08-01 00:00:00
abstract::Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) is a 4.1k Da protein originally discovered in EAEC but known to be scattered in other diarrheagenic E. coli as well, possibly causing diarrhea in humans and animals. We report for the first time a method to express and purify EAST1 using the G...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2003.09.008
更新日期:2004-02-01 00:00:00
abstract::Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase (Protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the large subunit of Rubisco. In this paper, an affinity purification procedure for pea (Pisum sativum L. cv Laxton'...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1995.1070
更新日期:1995-08-01 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
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journal_title:Protein expression and purification
pub_type: 杂志文章
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journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2010.10.013
更新日期:2011-03-01 00:00:00
abstract::HtrA2 is an apoptosis-activating protein to enhance the apoptotic process by preventing the formation of the IAP-caspase complex, thus freeing caspase to trigger the apoptosis pathway. Here, we presented the full-length sequence of HtrA2 from the black tiger shrimp (PmHtrA2). The full-length PmHtrA2 transcript was 140...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2013.09.012
更新日期:2013-12-01 00:00:00
abstract::p53 protein is an important regulation factor that can bind to p53 mRNA to regulate its translation in human and murine. To determine if a similar interaction exists in zebrafish and if the interaction affects zebrafish development, we cloned and expressed p53 protein from zebrafish in Escherichia coli. Soluble p53 pr...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2010.03.028
更新日期:2010-08-01 00:00:00