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


: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 cloned thrombin consensus sequence (. LVPRGS. ) located between glutathione S-transferase and mMRP14. Analysis of products of digestion by C4 reverse-phase HPLC and SDS-PAGE/Western blotting revealed two immunoreactive cleavage products with molecular weights around 13, 000. Masses of the two proteins determined by ESI/MS were 13,062 and 11,919 Da. The larger product corresponded to the expected mass of recombinant mMRP14 (13,061.9 Da). Analysis of the protein sequence of recombinant mMRP14 revealed a thrombin-like consensus sequence (. NNPRGH. ) located close to the C-terminus. The smaller protein corresponded to a truncated form of rec mMRP14 (rec MRP141-102) with a calculated mass of 11,918.6 Da. Optimization of the cleavage conditions resulted in >95% full-length rec mMRP14. Native mMRP14 contains one intramolecular disulfide bond between Cys79 and Cys90. The full-length recombinant protein was renatured and oxidized in ammonium acetate (pH approximately 7) for 96 h and formed >95% of the native intramolecular disulfide-bonded form. MRP141-102 bound substantially less 65Zn2+ compared to native mMRP14 or rec mMRP14 after transfer to polyvinylidene difluoride and incubation with 65ZnCl2, implicating the His residues located within the C-terminal domain in Zn2+ binding.


Protein Expr Purif


Raftery MJ,Collinson L,Geczy CL





Has Abstract


1999-03-01 00:00:00














  • Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

    abstract::An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatogra...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: He X,Zhou X,Yang Z,Xu L,Yu Y,Jia L,Li G

    更新日期:2015-10-01 00:00:00

  • High-level synthesis of recombinant murine endostatin in Chinese hamster ovary cells.

    abstract::Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fus...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chura-Chambi RM,Tornieri PH,Spencer PJ,Nascimento PA,Mathor MB,Morganti L

    更新日期:2004-05-01 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

  • Optimization of a thermostable lipase from Bacillus stearothermophilus P1: overexpression, purification, and characterization.

    abstract::An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open re...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sinchaikul S,Sookkheo B,Phutrakul S,Pan FM,Chen ST

    更新日期:2001-08-01 00:00:00

  • Cloning, heterologous expression, and enzymatic characterization of a novel glucoamylase GlucaM from Corallococcus sp. strain EGB.

    abstract::The gene encoding a novel glucoamylase (GlucaM) from the Corallococcus sp. strain EGB was cloned and heterologous expressed in Escherichia coli BL21(DE3), and the enzymatic characterization of recombinant GlucaM (rGlucaM) was determined in the study. The glucaM had an open reading frame of 1938 bp encoding GlucaM of 6...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Li Z,Ji K,Dong W,Ye X,Wu J,Zhou J,Wang F,Chen Q,Fu L,Li S,Huang Y,Cui Z

    更新日期:2017-01-01 00:00:00

  • Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli.

    abstract::Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively hig...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Seddi R,Chaix JC,Puigserver A,Guo XJ

    更新日期:2003-02-01 00:00:00

  • Molecular cloning, characterization, and heterologous expression of an acetyl-CoA acetyl transferase gene from Sanghuangporus baumii.

    abstract::Acetyl-CoA C-acetyltransferase synthase gene (AACT) cDNA, DNA and promoter were cloned from Sanghuangporus baumii. The gene ORF (1260 bp) encoded 419 amino acids. The AACT DNA includes five exons (1-84 bp, 140-513 bp, 570-1027 bp, 1090-1282 bp, 1344-1494 bp) and four introns (85-139 bp, 514-569 bp, 1028-1089 bp, 1283-...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wang X,Wang S,Xu X,Sun J,Ma Y,Liu Z,Sun T,Zou L

    更新日期:2020-06-01 00:00:00

  • High-level soluble expression of recombinant human manganese superoxide dismutase in Escherichia coli, and its effects on proliferation of the leukemia cell.

    abstract::Manganese superoxide dismutase (Mn-SOD) is one of the major enzymes responsible for the defense against oxidative damage due to reactive oxygen species (ROS) in the mitochondria. The present study aimed to produce and evaluate the genetically engineered manganese superoxide dismutase protein. A recombinant plasmid con...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Feng W,Mei S,Wenjie Y,Luyuan H

    更新日期:2011-05-01 00:00:00

  • Purification and characterization of a long-acting ciliary neurotrophic factor via genetically fused with an albumin-binding domain.

    abstract::Ciliary neurotrophic factor (CNTF) is a promising candidate for the treatment of neurodegenerative or metabolic diseases, but suffers rapid clearance in body. Herein we constructed a new long-acting recombinant human CNTF (rhCNTF) by genetic fusion with an albumin-binding domain (ABD) through a flexible peptide linker...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Xu L,Zhang C,Liu L,Zhang Y,Wang Q,Wang J,Liu Y,Su Z

    更新日期:2017-11-01 00:00:00

  • Expression, purification and characterization of a recombinant Lipomyces starkey dextranase in Pichia pastoris.

    abstract::The DEX gene encoding an extracellular dextranase from Lipomyces starkeyi was cloned into vector pPIC9k-His6 and was expressed in Pichia pastoris GS115 strain under the control of AOX1 promoter. After 107 h of the 5L-scaled fermentation, wet cells weight of the recombinant P. pastoris Mut(+) strain reached to 588.6g/L...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chen L,Zhou X,Fan W,Zhang Y

    更新日期:2008-03-01 00:00:00

  • Expression and purification recombinant antihypertensive peptide ameliorates hypertension in rats with spontaneous hypertension.

    abstract::A highly efficient Escherichia coli expression system was established to obtain an appreciable quantity of antihypertensive peptide. The DNA-coding sequence for the Gly-Val-Tyr-Pro-His-Lys peptide was chemically synthesized and linked to form a ten-copy in tandem. It was cloned into the vector pET-15b and expressed in...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wang XL,Ma SN,Yuan YH,Ding Y,Li DS

    更新日期:2015-09-01 00:00:00

  • High-level extracellular production of Rhizopus oryzae lipase in Pichia pastoris via a strategy combining optimization of gene-copy number with co-expression of ERAD-related proteins.

    abstract::Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Jiao L,Zhou Q,Su Z,Xu L,Yan Y

    更新日期:2018-07-01 00:00:00

  • An optimized system for expression and purification of secreted bacterial proteins.

    abstract::In this report, we describe an optimized system for the efficient overexpression, purification, and refolding of secreted bacterial proteins. Candidate secreted proteins were produced recombinantly in Escherichia coli as Tobacco Etch Virus protease-cleavable hexahistidine-c-myc eptiope fusion proteins. Without regard ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Geisbrecht BV,Bouyain S,Pop M

    更新日期:2006-03-01 00:00:00

  • Production and secretion of Lactobacillus crispatus β-galactosidase in Pichia pastoris.

    abstract::Lactobacillus β-galactosidases are mostly heterodimeric proteins, which are encoded by the two overlapping genes, lacL and lacM, and produced in recombinant prokaryotic systems for higher yield. This is the first report on the expression of a heterodimeric β-galactosidase from Lactobacillus crispatus B470 in Pichia pa...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Nie C,Liu B,Zhang Y,Zhao G,Fan X,Ning X,Zhang W

    更新日期:2013-11-01 00:00:00

  • A simple and efficient expression and purification system using two newly constructed vectors.

    abstract::Structural biology places a high demand on proteins both in terms of quality and quantity. Although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. Here, we report a new system which combines improved expression, solubility ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Liu H,Naismith JH

    更新日期:2009-02-01 00:00:00

  • Extracellular production of active vibriolysin engineered by random mutagenesis in Escherichia coli.

    abstract::Vibriolysin, an extracellular protease of Vibrio proteolyticus, is synthesized as a preproenzyme. The N-terminal propeptide functions as an intramolecular chaperone and an inhibitor of the mature enzyme. Extracellular production of recombinant vibriolysin has been achieved in Bacillus subtilis, but not in Escherichia ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sonoda H,Sugimura A

    更新日期:2008-12-01 00:00:00

  • Overexpression, purification, and characterization of a thermostable chitinase (Chi40) from Streptomyces thermoviolaceus OPC-520.

    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


    authors: Christodoulou E,Duffner F,Vorgias CE

    更新日期:2001-10-01 00:00:00

  • A study of the small-molecule system used to investigate the effect of arginine on antibody elution in hydrophobic charge-induction chromatography.

    abstract::Hydrophobic charge-induction chromatography (HCIC) using 4-mercaptoethylpyridine (4-MEP) as the ligand is used to purify antibodies. The 4-MEP resin ligand has high affinity for antibodies, which makes it difficult to optimize the elution conditions. Recent studies showed that arginine is effective at eluting and puri...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hirano A,Maruyama T,Shiraki K,Arakawa T,Kameda T

    更新日期:2017-01-01 00:00:00

  • Improving storage stability of recombinant organophosphorus hydrolase.

    abstract::Organophosphorus hydrolase (OPH) is a ∼38kDa enzyme encoded by opd gene of Flavobacterium sp. The enzyme can hydrolyze and inactivate variety of organophosphate (OP)-compounds, including chemical warfare nerve agents. Thus, OPH is a strong candidate for the development of therapeutic intervention against OP-poisoning ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Satvik Iyengar AR,Tripathy RK,Bajaj P,Pande AH

    更新日期:2015-07-01 00:00:00

  • Purification of the medium-chain/long-chain (COT/CPT) carnitine acyltransferase of rat liver microsomes.

    abstract::A procedure for the purification of the rat liver microsomal carnitine octanoyltransferase (COT) that catalyzes the reversible formation of medium-chain and long-chain acylcarnitines from acyl-coenzyme A is described. The K0.5 for L-carnitine is 0.6 mM and the K0.5 for both decanoyl-CoA and palmitoyl-CoA is 0.6 microM...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Murthy MS,Bieber LL

    更新日期:1992-02-01 00:00:00

  • Purification and characterization of acylation stimulating protein from porcine serum.

    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


    authors: Zhang H,Jacobi SK,Toombs CF,Cianflone KH,Nersesian N,Sarath G,Miner JL

    更新日期:2002-07-01 00:00:00

  • Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris.

    abstract::This is the first report of an insect esterase efficiently expressed in the methylotrophic yeast Pichia pastoris (so far insect esterases have been produced only in the baculovirus system). Having isolated a Tribolium castaneum carboxylesterase cDNA (TCE), we were initially unable to express it in Escherichia coli or ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Delroisse JM,Dannau M,Gilsoul JJ,El Mejdoub T,Destain J,Portetelle D,Thonart P,Haubruge E,Vandenbol M

    更新日期:2005-08-01 00:00:00

  • Expression and characterization of Pen b 26 allergen of Penicillium brevicompactum in Escherichia coli.

    abstract::Pen b 26 is one of the allergens produced by Penicillium brevicompactum which is one of the most prevalent in door airborne fungi and a major source of respiratory problems, including asthma. Pen b 26 wa scloned and expressed as an N-terminal as well as a C-terminal His6 tagged fusion protein in Escherichia coli. This...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sevinc MS,Kumar V,Abebe M,Mohottalage S,Kumarathasan P,Vincent R,Vijay HM

    更新日期:2009-05-01 00:00:00

  • Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene.

    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


    authors: Ko JH,Hahm MS,Kang HA,Nam SW,Chung BH

    更新日期:2002-08-01 00:00:00

  • A single-step purification of biologically active recombinant human interleukin-5 from a baculovirus expression system.

    abstract::Recombinant human interleukin-5 (rhIL-5) was expressed in baculovirus-infected insect cells and purified to homogeneity from the culture medium in a single chromatographic step. Beginning with a cDNA encoding the full-length precursor form of human IL-5, including the authentic secretory leader sequence, recombinant b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Brown PM,Scheid MP,O'Neill GP,Tagari PC,Nicholson DW

    更新日期:1995-02-01 00:00:00

  • Solubility of the catalytic domains of Botulinum neurotoxin serotype E subtypes.

    abstract::The Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins known to humans. There are seven serotypes of the BoNTs (A-G), among which serotypes A, B, E and F are known to cause natural human intoxication. To date, eleven subtypes of LC/E, termed E1∼E11, have been identified. The LCs of BoNT/E wer...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chen S,Barbieri JT

    更新日期:2016-02-01 00:00:00

  • Production and characterization of long-acting recombinant human albumin-EPO fusion protein expressed in CHO cell.

    abstract::A long-lasting recombinant human albumin-linker-erythropoietin (EPO) is a human albumin gene fused to the N-terminal of EPO with a (GGSGG)(n)-repeated linker inserted between albumin and EPO. Albumin-EPO fusion genes were co-transfected with the dhfr gene. Albumin-EPO fusion protein has three kinds of sub-types (IALE,...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Joung CH,Shin JY,Koo JK,Lim JJ,Wang JS,Lee SJ,Tan HK,Kim SL,Lim SM

    更新日期:2009-12-01 00:00:00

  • Production of constitutively acetylated recombinant p53 from yeast and Escherichia coli by tethered catalysis.

    abstract::Post-translational modification of proteins is a dynamic way of generating new protein-protein interaction interfaces that are critical for signaling networks in diverse cellular functions. Purified recombinant proteins frequently lack these signature modifications. Using the tumor suppressor p53 as the model protein,...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Acharya A,Xu XJ,Husain-Ponnampalam RD,Hoffmann-Benning S,Kuo MH

    更新日期:2005-06-01 00:00:00

  • Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr-Abl oncoprotein fused to the cytoplasmic transduction peptide.

    abstract::Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Huang SF,Liu DB,Zeng JM,Xiao Q,Luo M,Zhang WP,Tao K,Wen JP,Huang ZG,Feng WL

    更新日期:2009-04-01 00:00:00

  • Production and comprehensive quality control of recombinant human Interleukin-1beta: a case study for a process development strategy.

    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


    authors: Block H,Kubicek J,Labahn J,Roth U,Schäfer F

    更新日期:2008-02-01 00:00:00