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


: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. Proteases were purified by dialysis and anion exchange chromatography (Q-FF). Their properties were further investigated. For catalysis efficiency, the value of Kcat/Km of Kex2-667-K225L was 3 folds higher than that of Kex2-667. Both were quite stable at 25 °C and 37 °C after 8 h of incubation at pH5.6, while Kex2-667 remained nearly 90% of the total activity while Kex2-667-K225L remained only 80%. The stability of Kex2-667-K225L was lower than that of Kex2-667 from pH4.0 to pH9.0. Due to the mutation site K225 was located at one of the calcium ion binding sites, it resulted in a tighter calcium ion binding region, which may be the reason why the catalytic efficiency of Kex2-667-K225L was improved while the stability was a little decreased.


Protein Expr Purif


Yang F,Liu L,Liu Y,Li S




Has Abstract


2020-12-01 00:00:00












  • Expression, purification, and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1.

    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


    authors: Ménard LP,Lussier JG,Lépine F,Paiva de Sousa C,Dubreuil JD

    更新日期:2004-02-01 00:00:00

  • Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification.

    abstract::Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxid...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Naested H,Kramhøft B,Lok F,Bojsen K,Yu S,Svensson B

    更新日期:2006-03-01 00:00:00

  • The molecular design of a recombinant antimicrobial peptide CP and its in vitro activity.

    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


    authors: Niu M,Li X,Wei J,Cao R,Zhou B,Chen P

    更新日期:2008-01-01 00:00:00

  • Streamlined procedure for the production of normal and altered versions of recombinant human proinsulin.

    abstract::A method for the simplified, reproducible production of both normal and altered versions of human proinsulin has been developed. A polyhistidine/proinsulin fusion protein was expressed using a prokaryotic expression system and partially purified by affinity chromatography. Disulfide bonds within the polypeptide were f...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Mackin RB

    更新日期:1999-04-01 00:00:00

  • Reprint of: Immobilized-Metal Affinity Chromatography (IMAC): A Review.

    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


    authors: Block H,Maertens B,Spriestersbach A,Brinker N,Kubicek J,Fabis R,Labahn J,Schäfer F

    更新日期:2011-09-03 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

  • The screening of expression and purification conditions for replicative DNA polymerase associated B-subunits, assignment of the exonuclease activity to the C-terminus of archaeal pol D DP1 subunit.

    abstract::The B-subunits of replicative DNA polymerases belong to the superfamily of calcineurin-like phosphoesterases and are conserved from Archaea to humans. Recently we and others have shown that the B-subunit (DP1) of the archaeal family D DNA polymerase is responsible for proofreading 3'-5' exonuclease activity. The simil...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Jokela M,Raki M,Heikkinen K,Sepponen K,Eskelinen A,Syväoja JE

    更新日期:2005-09-01 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

  • Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus.

    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


    authors: Kumar GS,Sarma SP

    更新日期:2010-04-01 00:00:00

  • Recombinant β-Glucocerebrosidase specific immunoaffinity ligands selected from phage-displayed combinatorial scFv libraries.

    abstract::Antibodies specific to β-Glucocerebrosidase were selected from phage displayed naïve scFv libraries. Biopannings were performed against recombinant human protein β-Glucocerebrosidase immobilized on polystyrene surface, specific phages were eluted with 50% ethylene glycol in citrate buffer (pH 6.0). Several specific bi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Anisimov RL,Ershova OA,Ershov AV,Filatova MA,Katorkin SA,Simonov VM

    更新日期:2020-06-01 00:00:00

  • An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies.

    abstract::Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein-protein, and DNA-protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Aatsinki JT,Rajaniemi HJ

    更新日期:2005-04-01 00:00:00

  • Over-expression, purification, and characterization of recombinant NAD-malic enzyme from Escherichia coli K12.

    abstract::NAD(+)-dependent malic enzyme (NAD-ME) gene from Escherichia coli K12 was inserted into an expression vector pET24b(+) and transformed into E. coli BL21 (DE3). Recombinant NAD-ME was expressed upon IPTG induction, purified with affinity chromatography, and biochemically characterized. The results showed that recombina...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wang J,Tan H,Zhao ZK

    更新日期:2007-05-01 00:00:00

  • Remedial strategies in structural proteomics: expression, purification, and crystallization of the Vav1/Rac1 complex.

    abstract::The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 bindi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Brooun A,Foster SA,Chrencik JE,Chien EY,Kolatkar AR,Streiff M,Ramage P,Widmer H,Weckbecker G,Kuhn P

    更新日期:2007-05-01 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

  • Transmembrane-sequence-dependent overexpression and secretion of glycoproteins in Saccharomyces cerevisiae.

    abstract::Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time. These post-translational modified expression products can be purified up to >90% in a single step. The overexpression and secreti...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Schuster M,Wasserbauer E,Aversa G,Jungbauer A

    更新日期:2001-02-01 00:00:00

  • An improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies.

    abstract::Amyloid-beta (Aβ) peptide mediates several neurodegenerative diseases. The 42 amino acid (Aβ1-42) is the predominant form of peptide found in the neuritic plaques and has been demonstrated to be neurotoxic in vivo and in vitro. The availability of large quantities of Aβ peptide will help in several biochemical and bio...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chhetri G,Pandey T,Chinta R,Kumar A,Tripathi T

    更新日期:2015-10-01 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

  • Plasma fibronectin: three steps to purification and stability.

    abstract::Large amounts of soluble fibronectin were easily purified from cryoprecipitated or fresh citrated human blood plasma by a three-step combination of gelatin and heparin-cellufine affinity chromatography. The elution conditions were optimized to obtain a homogeneous fraction on SDS-PAGE and Western blot under reducing c...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Poulouin L,Gallet O,Rouahi M,Imhoff JM

    更新日期:1999-10-01 00:00:00

  • Purification and characterization of human dehydrodolychil diphosphate synthase (DHDDS) overexpressed in E. coli.

    abstract::Protein asparagine (N)-linked glycosylation is a post-translational modification that occurs in the endoplasmic reticulum; it plays an important role in protein folding, oligomerization, quality control, sorting, and transport. Accordingly, disorders of glycosylation may affect practically every organ system. Dehydrod...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Giladi M,Edri I,Goldenberg M,Newman H,Strulovich R,Khananshvili D,Haitin Y,Loewenstein A

    更新日期:2017-04-01 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

  • Purification and biochemical characterization of a recombinant mouse seminal vesicle trypsin inhibitor produced in Escherichia coli.

    abstract::Escherichia coli cells were transformed with an expression vector constructed by inserting a DNA fragment encoding a Kazal-type trypsin inhibitor from mouse seminal vesicle into pGEX-2. The cloned cells were able to produce a high yield of a chimeric polypeptide made by fusing the trypsin inhibitor to glutathione S-tr...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lai ML,Li SH,Chen YH

    更新日期:1994-02-01 00:00:00

  • Intein-mediated expression, purification, and characterization of thymosin α1-thymopentin fusion peptide in Escherichia coli.

    abstract::Thymosin α1-thymopentin (Tα1-TP5) fusion peptide has been proved to be an immune regulator based on its higher immunoregulatory activity than Tα1 and TP5. To obtain Tα1-TP5 more effectively and economically, Tα1-TP5 was genetically fused to a self-cleaving intein-chitin binding domain tag for purification via chitin b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Li J,Zheng L,Li P,Wang F

    更新日期:2012-07-01 00:00:00

  • Purification and characterization of recombinant forms of murine Tcl1 proteins.

    abstract::The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Du Bois GC,Song SP,Kulikovskaya I,Rothstein JL,Germann MW,Croce CM

    更新日期:2000-04-01 00:00:00

  • Identification and characterization of a novel amidase signature family amidase from Parvibaculum lavamentivorans ZJB14001.

    abstract::Amidase signature (AS) family amidases are known to exhibit broad substrate specificity. According to the available genome sequence data, a novel AS family amidase, Pl-Ami, was identified and cloned from the genome of Parvibaculum lavamentivorans ZJB14001. The recombinant amidase was overexpressed in Escherichia coli ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wu ZM,Zheng RC,Zheng YG

    更新日期:2017-01-01 00:00:00

  • Isolation, overexpression, and biochemical characterization of the two isoforms of glutamic acid decarboxylase from Escherichia coli.

    abstract::Escherichia coli glutamic acid decarboxylase is a pyridoxal phosphate-dependent enzyme that catalyzes the alpha-decarboxylation of glutamate to yield 4-amino-butyrate and CO2. The E. coli chromosome contains two genes encoding for this enzyme, gadA and gadB, which map at distinct loci. Their protein products differ in...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: De Biase D,Tramonti A,John RA,Bossa F

    更新日期:1996-12-01 00:00:00

  • High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli.

    abstract::Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II)-mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at approximately 30mg of crude PDO and approximately 1mg ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Jaganaman S,Pinto A,Tarasev M,Ballou DP

    更新日期:2007-04-01 00:00:00

  • Expression of a functional cold active β-galactosidase from Planococcus sp-L4 in Pichia pastoris.

    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


    authors: Mahdian SM,Karimi E,Tanipour MH,Parizadeh SM,Ghayour-Mobarhan M,Bazaz MM,Mashkani B

    更新日期:2016-09-01 00:00:00

  • Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme.

    abstract::The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeB gene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of the gpd promoter and trpC terminator and expressed in a protease defi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Levasseur A,Benoit I,Asther M,Asther M,Record E

    更新日期:2004-09-01 00:00:00

  • Isolation, purification and characterization of a novel solvent stable lipase from Pseudomonas reinekei.

    abstract::The Pseudomonas sp. have been long recognized for their exogenous lipolytic activities yet the genus still contains a lot of unexplored strains. Due to the versatile metabolic machinery and their potential for adaptation to fluctuating environmental conditions Pseudomonas sp. are of great interest for biotechnological...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Priyanka P,Kinsella G,Henehan GT,Ryan BJ

    更新日期:2019-01-01 00:00:00

  • Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells.

    abstract::Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of an...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zhao Y,Gutshall L,Jiang H,Baker A,Beil E,Obmolova G,Carton J,Taudte S,Amegadzie B

    更新日期:2009-10-01 00:00:00