Functional Expression and Purification of CYP93C20,a Plant Membrane-Associated Cytochrome P450 from Medicago truncatula.


: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.


Protein Expr Purif


Chang Z,Wang X,Wei R,Liu Z,Shan H,Fan G,Hu H




Has Abstract


2010-12-04 00:00:00








  • Expression in E. coli and purification of the nucleoside diphosphate kinase b from Leishmania major.

    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


    authors: de Oliveira AH,Ruiz JC,Cruz AK,Greene LJ,Rosa JC,Ward RJ

    更新日期:2006-10-01 00:00:00

  • The heterologous expression strategies of antimicrobial peptides in microbial systems.

    abstract::Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章,评审


    authors: Deng T,Ge H,He H,Liu Y,Zhai C,Feng L,Yi L

    更新日期:2017-12-01 00:00:00

  • Identification, expression, and purification of a unique stable domain from human HSPC144 protein.

    abstract::HSPC144 is a newly identified gene in human CD34(+) hematopoietic stem/progenitor cells. In this work, we have expressed and purified the 225-residue protein from Escherichia coli BL21 (DE3) and identified a stable fragment HSPC144-P (residues 44-225) by limited proteolysis method. The HSPC144-P fragment exhibits high...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Song AX,Chang YG,Gao YG,Lin XJ,Shi YH,Lin DH,Hang QH,Hu HY

    更新日期:2005-07-01 00:00:00

  • Confronting high-throughput protein refolding using high pressure and solution screens.

    abstract::Over-expression of heterologous proteins in Escherichia coli is commonly hindered by the formation of inclusion bodies. Nevertheless, refolding of proteins in vitro has become an essential requirement in the development of structural genomics (proteomics) and as a means of recovering functional proteins from inclusion...

    journal_title:Protein expression and purification

    pub_type: 杂志文章,评审


    authors: Qoronfleh MW,Hesterberg LK,Seefeldt MB

    更新日期:2007-10-01 00:00:00

  • Expression of active human GRO beta and GRO gamma neutrophil chemotactic proteins in E. coli.

    abstract::Human GRO alpha, GRO beta, and GRO gamma are neutrophil chemoattractants structurally related to IL-8 and compete with IL-8 for binding to IL-8 receptors on neutrophils. These proteins are part of a large superfamily of chemotactic cytokines, the "chemokines," members of which share striking structural similarities. W...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zagorski J,DeLarco JE

    更新日期:1994-08-01 00:00:00

  • Production and single-step purification of EGFP and a biotinylated version of the Human Rhinovirus 14 3C protease.

    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


    authors: Youell J,Fordham D,Firman K

    更新日期:2011-10-01 00:00:00

  • Purification and characterization of the L-Ara4N transferase protein ArnT from Salmonella typhimurium.

    abstract::The covalent addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) groups to lipid A, which resides in the outer membranes of bacteria such as Salmonella typhimurium and Escherichia coli, is the final step in the polymyxin-resistance pathway in these organisms. This modification is catalyzed by the inner membrane protein ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Bretscher LE,Morrell MT,Funk AL,Klug CS

    更新日期:2006-03-01 00:00:00

  • Comparison of the expression of native and mutant bovine annexin IV in Escherichia coli using four different expression systems.

    abstract::Bovine annexin IV, a Ca(2+)-dependent, membrane-binding protein, was expressed in E. coli using four different prokaryotic expression vector systems. An annexin IV cDNA was mutated in the 5' noncoding region to introduce an NcoI restriction site at the translation initiation site. The coding sequence was then excised ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Nelson MR,Creutz CE

    更新日期:1995-04-01 00:00:00

  • Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli.

    abstract::We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of criti...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Joshi BH,Puri RK

    更新日期:2005-02-01 00:00:00

  • Facile production of Aspergillus niger α-N-acetylgalactosaminidase in yeast.

    abstract::α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC. 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


    authors: Mrázek H,Benada O,Man P,Vaněk O,Křen V,Bezouška K,Weignerová L

    更新日期:2012-01-01 00:00:00

  • Effect of K225 residue to the catalytic efficiency of Kex2 protease.

    abstract::The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Protea...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Yang F,Liu L,Liu Y,Li S

    更新日期:2020-12-01 00:00:00

  • Purification and characterization of the oxygen-sensitive 4-hydroxybutanoate dehydrogenase from Clostridium kluyveri.

    abstract::Cell extracts of Clostridium kluyveri grown on ethanol plus succinate contained a NAD(H) dependent 4-hydroxybutanoate dehydrogenase (EC at 66 U/mg. This enzyme was purified 42-fold under anaerobic conditions to homogeneity. Heat treatment, ion exchange chromatography on DEAE-cellulose, nondenaturing polyacry...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wolff RA,Kenealy WR

    更新日期:1995-04-01 00:00:00

  • Secretory production and purification of functional full-length streptavidin from Bacillus subtilis.

    abstract::Streptavidin is a versatile molecule for many in vitro and in vivo applications. To optimize the production of the full-length streptavidin in a soluble and functional form via secretion using Bacillus subtilis as the expression host, three different strategies were used. These strategies include the construction of a...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wu SC,Hassan Qureshi M,Wong SL

    更新日期:2002-04-01 00:00:00

  • Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylobacter jejuni and Campylobacter coli.

    abstract::Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviou...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wenbap P,Seetang-Nun Y,Luangtongkum T,Khunrae P,Tuitemwong P,Rattanarojpong T

    更新日期:2021-04-01 00:00:00

  • Expression, purification, and characterization of His-tagged human mitochondrial 2,4-dienoyl-CoA reductase.

    abstract::Mitochondrial 2,4-dienoyl-CoA reductase is a key enzyme for the beta-oxidation of unsaturated fatty acids. The cDNA of the full-length human mitochondrial 2,4-dienoyl-CoA reductase was previously cloned as pUC18::DECR. PCR methodologies were used to subclone the genes encoding various truncated human mitochondrial 2,4...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chu X,Yu W,Chen G,Li D

    更新日期:2003-10-01 00:00:00

  • Increased peptide deformylase activity for N-formylmethionine processing of proteins overexpressed in Escherichia coli: application to homogeneous rubredoxin production.

    abstract::Deformylation of the initiator N-formylmethionine does not always proceed to completion for proteins overexpressed in Escherichia coli. To overcome this limitation, the def gene encoding the Escherichia coli peptide deformylase was cloned into the plysS plasmid under the tetracycline (Tc) promoter control. The efficie...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Tang J,Hernández G,LeMaster DM

    更新日期:2004-07-01 00:00:00

  • Co-expression of multiple subunits enables recombinant SNAPC assembly and function for transcription by human RNA polymerases II and III.

    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


    authors: Hanzlowsky A,Jelencic B,Jawdekar G,Hinkley CS,Geiger JH,Henry RW

    更新日期:2006-08-01 00:00:00

  • Overexpression, oxidative refolding, and zinc binding of recombinant forms of the murine S100 protein MRP14 (S100A9).

    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


    authors: Raftery MJ,Collinson L,Geczy CL

    更新日期:1999-03-01 00:00:00

  • Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli.

    abstract::Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Pearce LA,Yu M,Waddington LJ,Barr JA,Scoble JA,Crameri GS,McKinstry WJ

    更新日期:2015-12-01 00:00:00

  • Thermostable tyrosine phenol-lyase of Symbiobacterium sp. SC-1: gene cloning, sequence determination, and overproduction in Escherichia coli.

    abstract::During the screening for tyrosine phenol-lyase-producing thermophiles, we isolated an obligatory symbiotic thermophile, Symbiobacterium sp. SC-1, which grew only in coculture with Bacillus sp. SK-1. A gene encoding thermostable tyrosine phenol-lyase (TPL) was cloned from the genomic DNA of the Symbiobacterium sp. SC-1...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lee SG,Hong SP,Choi YH,Chung YJ,Sung MH

    更新日期:1997-12-01 00:00:00

  • High-level bacterial expression and purification of human SirT2 protein for NMR studies.

    abstract::Silent information regulator 2 (Sir2) proteins are a class of protein deacetylase enzymes that play key roles in transcriptional gene silencing, DNA repair, and aging. Here, we describe the high-level bacterial expression and purification of a human SirT2 construct that yields high resolution NMR spectra. By removing ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sun C,Song D,Marcotte PA,Richardson PL,Hajduk PJ

    更新日期:2006-07-01 00:00:00

  • Formation of well-defined soluble aggregates upon fusion to MBP is a generic property of E6 proteins from various human papillomavirus species.

    abstract::Protein aggregation is a main barrier hindering structural and functional studies of a number of interesting biological targets. The E6 oncoprotein of Human Papillomavirus strain 16 (E6(16)) is difficult to express under a native soluble form in bacteria. Produced as an unfused sequence, it forms inclusion bodies. Fus...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zanier K,Nominé Y,Charbonnier S,Ruhlmann C,Schultz P,Schweizer J,Travé G

    更新日期:2007-01-01 00:00:00

  • Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies.

    abstract::A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding p...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Kischnick S,Weber B,Verdino P,Keller W,Sanders EA,Anspach FB,Fiebig H,Cromwell O,Suck R

    更新日期:2006-06-01 00:00:00

  • Heterologous protein production in Escherichia coli using the propionate-inducible pPro system by conventional and auto-induction methods.

    abstract::We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella entericaprpBCDE promoter (P(prpB)) and compared it to that from the strongest IPTG-inducible promoter, P(T7). In contrast to our previous study showing slight...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lee SK,Keasling JD

    更新日期:2008-10-01 00:00:00

  • New high fidelity polymerases from Thermococcus species.

    abstract::Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. P...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Griffiths K,Nayak S,Park K,Mandelman D,Modrell B,Lee J,Ng B,Gibbs MD,Bergquist PL

    更新日期:2007-03-01 00:00:00

  • Large-scale preparation, purification, and crystallization of UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase from Escherichia coli.

    abstract::The UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli, an enzyme involved in the biosynthesis of the bacterial peptidoglycan monomer unit, was overproduced and purified to homogeneity on a large scale, yielding 4 mg of protein per liter of bacterial culture. Crystals of the complex with the subst...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Auger G,Martin L,Bertrand J,Ferrari P,Fanchon E,Vaganay S,Pétillot Y,van Heijenoort J,Blanot D,Dideberg O

    更新日期:1998-06-01 00:00:00

  • Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1.

    abstract::Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble re...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Khine AA,Lu PC,Ko TP,Huang KF,Chen HP

    更新日期:2020-11-01 00:00:00

  • Gene synthesis, expression, purification, and characterization of human Jagged-1 intracellular region.

    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


    authors: Popovic M,Coglievina M,Guarnaccia C,Verdone G,Esposito G,Pintar A,Pongor S

    更新日期:2006-06-01 00:00:00

  • Kinetic characterization and Mg2+ enhancement of Streptomyces griseocarneus sphingomyelinase C produced by recombinant Streptomyces lividans.

    abstract::Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550-950 U/mg) and was obtained in h...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sugimori D,Matsumoto Y,Tomita Y,Murayama K,Ogino C

    更新日期:2012-02-01 00:00:00

  • High-level bacterial expression of human glutathione transferase P1-1 encoded by semisynthetic DNA.

    abstract::A cDNA clone, lambda GTHP1del, encoding glutathione transferase (GST) P1-1, was isolated from a human K562 erythroleukemia cell line cDNA library. The coding sequence was lacking the codons for the N-terminal 34 amino acids. A DNA segment was designed in order to obtain the missing portion and a structure representing...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Kolm RH,Stenberg G,Widersten M,Mannervik B

    更新日期:1995-06-01 00:00:00