当前位置： SCI文献检索 > PROTEIN EXPRESSION AND PURIFICATION期刊下所有文献 > Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris.

Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris.

Abstract：

:The bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T cell leukemia, autoimmune diseases, and tolerance induction for transplantation. The multi-domain structure of the bivalent immunotoxin hinders efficient production in Escherichia coli and most eukaryotes are sensitive to the toxin. However, Pichia pastoris has a tolerance to levels of DT (diphtheria toxin) that were previously observed to intoxicate wild type eukaryotic cells, including Saccharomyces cerevisiae. This tolerance has permitted the optimization of the secreted expression of A-dmDT390-bisFv(G(4)S) in P. pastoris under the control of AOX1 (alcohol oxidase 1) promoter. The original DNA sequence of A-dmDT390-bisFv(G(4)S) was not expressed in P. pastoris because of several AT-rich regions, which induce an early termination of transcription. After DNA rebuilding for abolishing AT-rich regions and codon optimization, the immunotoxin could be expressed up to 10mg/L in the shake flask culture. No differences in the expression levels of immunotoxin were observed by using different secretional signal sequences, Mut(s) (methanol utilization slow phenotype) or Mut(+) (methanol utilization plus phenotype) phenotypes. Buffered complex medium (pH 7.0) having 1% casamino acids provided the highest expression in shake flask culture and PMSF (phenylmethylsulfonyl fluoride) in the range of 1 to 3mM further improved the expression level presumably by inhibiting protein degradation. The immunotoxin was purified by DEAE (diethylaminoethyl) Sepharose ion exchange chromatography and Protein L affinity chromatography. The immunotoxin purified from P. pastoris culture was as fully functional as that expressed in a toxin resistant mutant CHO (Chinese hamster ovary) cell line. Our results demonstrate that P. pastoris is an ideal system for expression of toxin-based fusion proteins.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Woo JH,Liu YY,Mathias A,Stavrou S,Wang Z,Thompson J,Neville DM Jr

doi

10.1016/s1046-5928(02)00009-8

keywords:

subject

Has Abstract

pub_date

2002-07-01 00:00:00

pages

270-82

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046592802000098

journal_volume

25

pub_type

杂志文章

相关文献

PROTEIN EXPRESSION AND PURIFICATION文献大全
  • 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

    doi：10.1016/j.pep.2005.09.003

    authors： Geisbrecht BV,Bouyain S,Pop M

    更新日期：2006-03-01 00:00:00

  • Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway.

    abstract：:Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.1998.0870

    authors： Huang KX,Huang QL,Wildung MR,Croteau R,Scott AI

    更新日期：1998-06-01 00:00:00

  • Biochemical characterization of Arabidopsis developmentally regulated G-proteins (DRGs).

    abstract：:Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP-binding proteins found in archaea, plants, fungi and animals, indicating important roles in fundamental pathways. Their function is poorly understood, but they have been implicated in cell division, proliferation, and growth, as well as s...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2009.05.009

    authors： O'Connell A,Robin G,Kobe B,Botella JR

    更新日期：2009-10-01 00:00:00

  • Identification of pigment epithelium-derived factor in the interphotoreceptor matrix of bovine eyes.

    abstract：:Pigment epithelium-derived factor (PEDF) is a neurotrophic protein and a member of the serine protease inhibitor superfamily. Here we describe the identification of PEDF in bovine eyes and optimization of its purification from this natural source. We have developed a polyclonal antibody to recombinant human PEDF, Ab-r...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.1995.1060

    authors： Wu YQ,Notario V,Chader GJ,Becerra SP

    更新日期：1995-08-01 00:00:00

  • Production and purification of the heavy chain fragment C of botulinum neurotoxin, serotype A, expressed in the methylotrophic yeast Pichia pastoris.

    abstract：:A recombinant H(C) fragment of botulinum neurotoxin, serotype A (rBoNTA(H(C))), has been successfully expressed in a Mut(+) strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to ac...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.2000.1256

    authors： Potter KJ,Zhang W,Smith LA,Meagher MM

    更新日期：2000-08-01 00:00:00

  • Expression of the Trichoderma reesei tyrosinase 2 in Pichia pastoris: isotopic labeling and physicochemical characterization.

    abstract：:Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic compounds and also peptide and protein bound tyrosine, and thus is of great interest for different biotechnological applications. In order to understand the reaction mechanism of the enzyme it would be essential to solve it...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2007.04.014

    authors： Westerholm-Parvinen A,Selinheimo E,Boer H,Kalkkinen N,Mattinen M,Saloheimo M

    更新日期：2007-09-01 00:00:00

  • Human CYP1A1 allelic variants: baculovirus expression and purification, hydrodynamic, spectral, and catalytical properties and their potency in the formation of all-trans-retinoic acid.

    abstract：:Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CYP1A1.4 (T461N), were expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30-90 nmol ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(02)00672-1

    authors： Chernogolov A,Behlke J,Schunck WH,Roots I,Schwarz D

    更新日期：2003-04-01 00:00:00

  • A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2.

    abstract：:Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signal...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2020.105628

    authors： Ådén J,Mushtaq AU,Dingeldein A,Wallgren M,Gröbner G

    更新日期：2020-08-01 00:00:00

  • Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis L-asparaginase.

    abstract：:We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichiacoli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We e...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2011.12.005

    authors： Pokrovskaya MV,Aleksandrova SS,Pokrovsky VS,Omeljanjuk NM,Borisova AA,Anisimova NY,Sokolov NN

    更新日期：2012-03-01 00:00:00

  • Bacterially expressed recombinant p68 RNA helicase is phosphorylated on serine, threonine, and tyrosine residues.

    abstract：:We previously reported the expression and purification of recombinant p68 RNA helicase in a bacterial expression system. The recombinant p68 is an RNA-dependent ATPase and ATP-dependent RNA helicase. In the process of characterizing the ATPase and RNA unwinding activities of the recombinant p68, we observed that the b...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2004.02.004

    authors： Yang L,Liu ZR

    更新日期：2004-06-01 00:00:00

  • Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase.

    abstract：:A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked b...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.1998.0890

    authors： di Salvo ML,Delle Fratte S,De Biase D,Bossa F,Schirch V

    更新日期：1998-07-01 00:00:00

  • A hydrophobic interaction chromatography strategy for purification of inactivated foot-and-mouth disease virus.

    abstract：:A purification scheme based on hydrophobic interaction chromatography was developed to separate inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and 8.8-fold purification were achieved on Butyl Sepharose 4 FF. Further purification on Superdex 200 resulted in another 29-fold pu...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2015.04.011

    authors： Li H,Yang Y,Zhang Y,Zhang S,Zhao Q,Zhu Y,Zou X,Yu M,Ma G,Su Z

    更新日期：2015-09-01 00:00:00

  • Molecular cloning and functional expression of hemolysin from the sea anemone Actineria villosa.

    abstract：:The full-length cDNA that encodes the hemolytic toxin Avt-I, with 226 amino acids, from the venomous sea anemone Actineria villosa has been cloned using the oligo-capping method. The cDNA contains 681bp open reading frame and its predicted amino acid sequences revealed that Avt-I was basic polypeptides without cystein...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2004.12.003

    authors： Uechi G,Toma H,Arakawa T,Sato Y

    更新日期：2005-04-01 00:00:00

  • Heme conjugated magnetic beads to isolate heme-binding proteins from complex mixtures.

    abstract：:The cofactor heme (Fe-protoporphyrin IX) plays many important roles in biology. Identification of novel proteins for the transport, chaperoning and delivery of heme in cells is of widespread interest. Here, we describe the use of heme conjugated magnetic beads for the isolation of heme-binding proteins from complex pr...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2010.10.001

    authors： Jones R,Allen JW

    更新日期：2011-03-01 00:00:00

  • Development of a bacterial system for high yield expression of fully functional adrenal cytochrome b561.

    abstract：:Adrenal cytochrome b561 (cyt b561) is the prototypical member of an emerging family of proteins that are distributed widely in vertebrate, invertebrate and plant tissues. The adrenal cytochrome is an integral membrane protein with two b-type hemes and six predicted transmembrane helices. Adrenal cyt b561 is involved i...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2007.04.010

    authors： Liu W,Rogge CE,Kamensky Y,Tsai AL,Kulmacz RJ

    更新日期：2007-12-01 00:00:00

  • Improved solubility of replication factor C (RFC) Walker A mutants.

    abstract：:Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously do...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2012.03.010

    authors： Marzahn MR,Bloom LB

    更新日期：2012-06-01 00:00:00

  • Zebrafish Sp1-like protein is structurally and functionally comparable to human Sp1.

    abstract：:The transcription factor Sp1 is a regulator of TATA-less genes. It belongs to the Cys₂-His₂ zinc finger domain-containing family. A zebrafish cDNA encoding a peptide homologous to mammalian Sp1 was cloned and inserted into a pET43.1a vector and expressed in Escherichia coli Rosetta (DE3) cells as a Nus-His-tag fusion ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2010.10.010

    authors： Lin CJ,Hsiao TH,Chung YS,Chang WN,Yeh TM,Chen BH,Fu TF

    更新日期：2011-03-01 00:00:00

  • Expression in Escherichia coli: purification and characterization of cyclin H, a subunit of the human general transcription/DNA repair factor TFIIH.

    abstract：:The human cyclin H, a protein normally associated with the cyclin-dependent kinase cdk7, was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity. The purified recombinant cyclin H is similar to the endogenous one according to the following criteria...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.1996.0693

    authors： Poterszman A,Andersen G,Busso D,Rossignol M,Egly JM,Thierry JC

    更新日期：1997-03-01 00:00:00

  • Identification of UDP-linked murein precursors as contaminants in recombinant proteins of low molecular weight.

    abstract：:The A(280)/A(260) ratio of a purified protein is frequently used as an indication of the purity of the preparation with respect to nucleic acids. We show here that for low-molecular-weight recombinant proteins purified from Escherichia coli, a low A(280)/A(260) ratio can also result from contamination with UDP-linked ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.1999.1145

    authors： Ram MK,Andrade LJ,Phillips TB,van Schravendijk MR

    更新日期：1999-11-01 00:00:00

  • Expression and purification of receptor for activated C-kinase 1 (RACK1).

    abstract：:Receptor for activated C-kinase (RACK1) binds to protein kinase C and functions as an anchor for several other cellular components. Most in vitro studies of RACK1 have been carried out with RACK1 fused to a soluble fusion protein partner, such as GST or MBP. Here, we show that fusion complexes may exist as large solub...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(03)00135-9

    authors： Bjørndal B,Trave G,Hageberg I,Lillehaug JR,Raae AJ

    更新日期：2003-09-01 00:00:00

  • Expression, purification, and characterization of the recombinant NAD-malic enzyme from Ascaris suum.

    abstract：:The cDNA encoding the 65-kDa subunit of malic enzyme from Ascaris suum was cloned into the bacterial expression vector pKK223-3 and overproduced in Escherichia coli. A protein with a subunit molecular mass of 65,000 was expressed at a level of up to 3% of the total soluble protein in JM109, as judged by SDS-PAGE. The ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.1996.0705

    authors： Chooback L,Karsten WE,Kulkarni G,Nalabolu SR,Harris BG,Cook PF

    更新日期：1997-06-01 00:00:00

  • Expression and biochemical characterization of a type I methionine aminopeptidase of Plasmodium vivax.

    abstract：:Methionine aminopeptidases (MetAPs), ubiquitous enzymes that play an important role in nascent protein maturation, have been recognized as attractive targets for the development of drugs against pathogenic protozoa including Plasmodium spp. Here, we characterized partial biochemical properties of a type I MetAP of Pla...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2015.01.003

    authors： Kang JM,Ju JW,Kim JY,Ju HL,Lee J,Lee KH,Lee WJ,Sohn WM,Kim TS,Na BK

    更新日期：2015-04-01 00:00:00

  • Expression and purification of a cold-adapted group III trypsin in Escherichia coli.

    abstract：:The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from t...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2006.06.008

    authors： Pálsdóttir HM,Gudmundsdóttir A

    更新日期：2007-02-01 00:00:00

  • Expression, purification, and characterization of human malonyl-CoA decarboxylase.

    abstract：:The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2003.11.023

    authors： Zhou D,Yuen P,Chu D,Thon V,McConnell S,Brown S,Tsang A,Pena M,Russell A,Cheng JF,Nadzan AM,Barbosa MS,Dyck JR,Lopaschuk GD,Yang G

    更新日期：2004-04-01 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

    doi：10.1006/prep.1995.1034

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

    更新日期：1995-06-01 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

    doi：10.1016/j.pep.2021.105818

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

    更新日期：2021-04-01 00:00:00

  • Increasing the expression levels of papillomavirus major capsid protein in Escherichia coli by N-terminal deletion.

    abstract：:The major capsid protein L1 of human papillomavirus (HPV) contains the immunodominant neutralization epitopes of the virus and can auto-assembles to form virus-like particles (VLPs). Therefore, HPV L1 capsid proteins have been well investigated as potential vaccine candidates. To express large quantities of human papi...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2007.05.010

    authors： Ma Z,Chen B,Zhang F,Yu M,Liu T,Liu L

    更新日期：2007-11-01 00:00:00

  • Purification of rat pro-atrial natriuretic factor: a simplified scheme using reversed-phase high-performance liquid chromatography.

    abstract：:A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-s...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/1046-5928(90)90041-v

    authors： Belcourt D,Varma DR,Toney K,Bennett HP

    更新日期：1990-09-01 00:00:00

  • Heterologous expression of enzymopenic methemoglobinemia variants using a novel NADH:cytochrome c reductase fusion protein.

    abstract：:Hereditary enzymopenic methemoglobinemia is a rare disease that predominantly results from defects in either the erythrocytic (type I) or microsomal (type II) forms of the enzyme NADH:cytochrome b5 reductase (EC 1.6.2.2). All 25 currently identified type I and type II methemoglobinemia mutants have been expressed in E...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(03)00046-9

    authors： Davis CA,Barber MJ

    更新日期：2003-07-01 00:00:00

  • Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse.

    abstract：:Acyl coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-t...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2007.11.010

    authors： Petrescu AD,Huang H,Hostetler HA,Schroeder F,Kier AB

    更新日期：2008-04-01 00:00:00