Abstract：
:Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coli XL1-blue cells and was purified by Ni(2+)-NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Chang Z,Wang X,Wei R,Liu Z,Shan H,Fan G,Hu Hdoi
10.1016/j.pep.2010.11.012subject
Has Abstractpub_date
2010-12-04 00:00:00eissn
1046-5928issn
1096-0279pii
S1046-5928(10)00322-0pub_type
杂志文章abstract：:A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the millig...
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：:Transmembrane and coiled-coil domains 1 (TMCO1) has a highly conserved amino acid sequence among species, indicating a critical role of TMCO1 in cell physiology. The deficiency of TMCO1 in humans is associated with cerebrofaciothoracic dysplasia (CFTD), glaucoma, osteogenesis and the occurrence of cancer. TMCO1 was re...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2020.105803
更新日期：2021-03-01 00:00:00
abstract：:The gene for the extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed structural and functional studies of the enzyme. The recombinant enzyme was purified to homogenei...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1999.1076
更新日期：1999-06-01 00:00:00
abstract：:Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant protei...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(03)00075-5
更新日期：2003-07-01 00:00:00
abstract：:Lactase deficiency problem discourages many adults from consuming milk as a major source of micro- and macronutrients. Enzymatic hydrolysis of lactose is an ideal solution for this problem but such processing adds significant costs. In this study, a cold active β-galactosidase from Planococcus sp-L4 (bgal) was optimiz...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.09.008
更新日期：2016-09-01 00:00:00
abstract：:We describe an efficient strategy to produce high-quality proteins by using a single large IMAC chromatography column and enzymatic His-tag removal via the TAGZyme system in pilot scale. Numerous quality assays demonstrated a high purity of the final product, the human cytokine Interleukin-1beta (IL-1beta). The protei...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.09.019
更新日期：2008-02-01 00:00:00
abstract：:The supply of many valuable proteins that have potential clinical or industrial use is often limited by their low natural availability. With the modern advances in genomics, proteomics and bioinformatics, the number of proteins being produced using recombinant techniques is exponentially increasing and seems to guaran...
journal_title：Protein expression and purification
pub_type： 杂志文章,评审
doi：10.1016/j.pep.2013.10.016
更新日期：2014-02-01 00:00:00
abstract：:In this work we communicate the heterologous expression of a laccase from Coriolopsis gallica in Pichia pastoris. This enzyme has been reported to efficiently degrade a variety of pollutants such as industrial dyes. The expression strategy included using a previously reported modified α-factor preproleader for enhance...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2017.06.001
更新日期：2017-08-01 00:00:00
abstract：:Notch signaling plays a key role in cell differentiation and is very well conserved from Drosophila to humans. Ligands of Notch receptors are type I, membrane spanning proteins composed of a large extracellular region and a 100-150 residue cytoplasmic tail. We report here, for the first time, the expression, purificat...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.11.027
更新日期：2006-06-01 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：:A relatively inexpensive yet highly efficient and extremely rapid procedure has been developed for the isolation and purification of estrogen receptor from the goat uterine cytosol. Greater than 1 mg of purified receptor protein could be obtained from 75 g of uterine tissue within a period of < 24 h, following this pr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1993.1070
更新日期：1993-12-01 00:00:00
abstract：:The transcription factor IIIC (TFIIIC) is a multisubunit DNA-binding factor required for promoter recognition and TFIIIB assembly on tRNA genes transcribed by RNA polymerase III. Yeast TFIIIC consists of six subunits, organized in the two globular subcomplexes tauA and tauB, which recognize two internal tDNA promoter ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.06.013
更新日期：2006-02-01 00:00:00
abstract：:Replacing the chymotrypsin inhibitory loop of soybean Bowman-Birk inhibitor (sBBI) with a VEGF binding peptide (BBI-AV) significantly reduces the overall purification yield when BBI-AV is produced as a fusion protein in a Bacillussubtilis expression system. The low purification yield is primarily due to a higher fract...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.08.006
更新日期：2009-12-01 00:00:00
abstract：:Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7 kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.05.002
更新日期：2014-08-01 00:00:00
abstract：:The hexokinase (ATP;D-hexose 6-phosphotransferase, EC 2.7.1.1) of Schistosoma mansoni has been expressed in Escherichia coli as a fusion protein including an N-terminal polyhistidine tag and enterokinase cleavage site. The enzyme was purified by metal chelate chromatography to > 95% homogeneity, based on analysis by S...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0113
更新日期：1996-11-01 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：:Cystathionine beta-synthase (CBS) catalyzes the pyridoxal-50-phosphate-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chrom...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.10.012
更新日期：2009-04-01 00:00:00
abstract：:The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B(max) values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.06.018
更新日期：2004-10-01 00:00:00
abstract：:The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His(6) secreted by S. cerevisiae migrated as a broad diffuse ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00035-9
更新日期：2002-08-01 00:00:00
abstract：:The kinetic locking-on strategy improves the selectivity of protein purification procedures based on immobilized cofactor derivatives through use of enzyme-specific substrate analogues in irrigants to promote biospecific adsorption. This paper describes the development and application of this strategy to the one-chrom...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.0995
更新日期：1999-02-01 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：: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
abstract：:Listeriolysin O (LLO) is a cholesterol-binding sulfhydryl-activated hemolysin encoded by Listeria monocytogenes hlyA gene. After analyzing the nucleotide coding sequence of this gene from the ATCC 9525 L. monocytogenes strain, we cloned it in a pET vector for expression in Escherichia coli. Thanks to the optimization ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00682-4
更新日期：2003-03-01 00:00:00
abstract：:Serine hydroxymethyltransferase (SHMT) plays a key role in cell physiology as it participates in the different interconversion pathway of folate coenzymes, provides almost exclusively folate one carbon fragments for the biosynthesis of a variety of end products. For the first time, Mycobacterium leprae glyA gene, enco...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.04.017
更新日期：2007-09-01 00:00:00
abstract：:Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications....
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.05.011
更新日期：2011-09-01 00:00:00
abstract：:Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.08.004
更新日期：2014-10-01 00:00:00
abstract：:Human D-amino acid oxidase (hDAAO) is a flavoprotein that plays a key role in the pathophysiology of schizophrenia. So far, the biochemical characterization of this enzyme has been hampered by the difficulty of expressing it in a common heterologous host such as Escherichia coli. Increasing amounts of recombinant hDAA...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.05.013
更新日期：2009-11-01 00:00:00
abstract：:Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with speci...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.12.003
更新日期：2015-06-01 00:00:00
abstract：:Refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. We will review key parameters associated with (1) conformation of the protein solubilized from inc...
journal_title：Protein expression and purification
pub_type： 杂志文章,评审
doi：10.1016/s1046-5928(02)00641-1
更新日期：2003-03-01 00:00:00
abstract：:General procedures for the rapid, large-scale purification of recombinant Lactobacillus casei thymidylate synthase and its mutants have been established. An effective method employs sequential phosphocellulose and hydroxylapatite chromatography. Crude cell extracts are directly applied to phosphocellulose, and the enz...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(05)80039-7
更新日期：1992-10-01 00:00:00
