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


: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. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase.


Protein Expr Purif


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




Has Abstract


2012-01-01 00:00:00














  • Enhancing the soluble expression of an amylase in Escherichia coli by the mutations related to its domain interactions.

    abstract::The sequence and structure of the target protein exert a marked effect on its soluble expression in Escherichia coli. The effects of the mutation of an amylase isolated from Bacillus licheniformis (BLA) on its soluble expression in E. coli were investigated. A random mutation library of BLA was constructed to screen f...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wang P,Qin W,Xu J,Yan Y,Tian J,Wu N,Yao B

    更新日期:2016-04-01 00:00:00

  • Cloning, purification, and refolding of human paraoxonase-3 expressed in Escherichia coli and its characterization.

    abstract::Human paraoxonase (hPON3) is a high density lipoprotein-related glycoprotein with multi-enzymatic properties and antioxidant activity which is proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. In this study, hPON3 gene was amplified from Human Fetal Liver Marathon-Ready cDNA and exp...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lu H,Zhu J,Zang Y,Ze Y,Qin J

    更新日期:2006-03-01 00:00:00

  • Expression and purification of stable 33-kDa soluble human CD23 using the Drosophila S2 expression system.

    abstract::CD23, a 45-kDa type II membrane glycoprotein present on B cells, monocytes, and other human immune cells, is a low-affinity receptor for IgE. The extracellular region of the membrane-bound human CD23 is processed into at least four soluble (s) CD23 forms, with apparent molecular masses of 37, 33, 29, and 25 kDa. High ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Khandekar SS,Mayer RJ,Cusimano DM,Katchur SR,Appelbaum ER

    更新日期:2001-07-01 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

  • Expression and purification of Arg196 and Lys272 mutants of mevalonate kinase from Methanococcus jannaschii.

    abstract::In microorganisms and plants, mevalonate kinase is involved in the biosynthesis of isoprenoid derivatives, one of the largest groups of natural products. We subcloned the gene of mevalonate kinase from Methanococcus jannaschii into a bacterial expression vector pLM1 with six continuous histidine codons attached to the...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chu X,Liu X,Yau M,Leung YC,Li D

    更新日期:2003-08-01 00:00:00

  • Heterologous expression of Class IIb bacteriocin Plantaricin JK in Lactococcus Lactis.

    abstract::Plantaricin JK (PlnJK) is a Class IIb LAB bacteriocin that includes two peptides; i.e., PlnJ and PlnK, which can synergistically halt many types of gram-positive bacteria, including food spoilage organisms. Purification of these peptides from natural lactic acid bacteria is difficult therefore, their application remai...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Xu Y,Yang L,Li P,Gu Q

    更新日期:2019-07-01 00:00:00

  • Purification, characterization, and crystallization of human pyrroline-5-carboxylate reductase.

    abstract::Pyrroline-5-carboxylate reductase (P5CR) catalyzes the reduction of Delta1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is very important in many physiological and pathological processes. Human P5CR was over-expressed in Escher...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Meng Z,Lou Z,Liu Z,Hui D,Bartlam M,Rao Z

    更新日期:2006-09-01 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

  • TRAIL-CM4 fusion protein shows in vitro antibacterial activity and a stronger antitumor activity than solo TRAIL protein.

    abstract::A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sang M,Zhang J,Li B,Chen Y

    更新日期:2016-06-01 00:00:00

  • Codon engineering for improved antibody expression in mammalian cells.

    abstract::While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Carton JM,Sauerwald T,Hawley-Nelson P,Morse B,Peffer N,Beck H,Lu J,Cotty A,Amegadzie B,Sweet R

    更新日期:2007-10-01 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

  • Peptidyl-prolyl isomerase and the biological activities of recombinant protein cyclophilin from Pyropia yezoensis (PyCyp).

    abstract::Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ulagesan S,Choi JW,Nam TJ,Choi YH

    更新日期:2020-08-01 00:00:00

  • Production of human pancreatic ribonuclease in Saccharomyces cerevisiae and Escherichia coli.

    abstract::Human pancreatic ribonuclease (HP-RNase) has considerable promise as a therapeutic agent. Structure-function analyses of HP-RNase have been impeded by the difficulty of obtaining the enzyme from its host. Here, a gene encoding HP-RNase was designed, synthesized, and inserted into two expression vectors that then direc...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ribó M,delCardayré SB,Raines RT,de Llorens R,Cuchillo CM

    更新日期:1996-05-01 00:00:00

  • Expression and purification of amyloid-beta peptides from Escherichia coli.

    abstract::Soluble oligomers and fibrillar deposits of amyloid beta (Abeta) are key agents of Alzheimer's disease pathogenesis. However, the mechanism of amyloid aggregation and its interaction with live cells still remain unclear requiring the preparation of large amounts of pure and different Abeta peptides. Here we describe a...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Garai K,Crick SL,Mustafi SM,Frieden C

    更新日期:2009-07-01 00:00:00

  • Affinity chromatographic purification of a protein which binds specifically to the yeast leucine tRNA gene.

    abstract::A crude cell extract from yeast Saccharomyces cerevisae was fractionated by affinity chromatography using the leucine tRNA gene as the recognition site. This approach enables the rapid purification of a protein, which retained its full DNA binding capacity during the enrichment procedure. The active fraction contains ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Kaufmann E

    更新日期:1990-11-01 00:00:00

  • Kinetic characterization of recombinant human cystathionine beta-synthase purified from E. coli.

    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


    authors: Belew MS,Quazi FI,Willmore WG,Aitken SM

    更新日期:2009-04-01 00:00:00

  • Expression and purification of the D4 region of PLD1 and characterization of its interaction with PED-PEA15.

    abstract::PLD's (Phospholipases D) are ubiquitously expressed proteins involved in many transphosphatidylation reactions. They have a bi-lobed structure composed by two similar domains which at their interface reconstitute the catalytic site through the association of the two conserved HxKx(4)Dx(6)GSxN motifs. PLD1 interacts wi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Viparelli F,Doti N,Sandomenico A,Marasco D,Dathan NA,Miele C,Beguinot F,Monti SM,Ruvo M

    更新日期:2008-06-01 00:00:00

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

    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 catalyzi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


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

    更新日期:2010-12-04 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

  • Overexpression and reconstitution of a Rieske iron-sulfur protein from the higher plant.

    abstract::The iron-sulfur protein subunit, known as the Rieske protein, is one of the central components of the cytochrome b(6)f complex residing in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overexpression in Escherichia coli of full-length and truncated Rieske (PetC) proteins from the...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Gubernator B,Seidler A,Rögner M,Szczepaniak A

    更新日期:2003-05-01 00:00:00

  • On-resin cleavage of bacterially expressed fusion proteins for purification of active recombinant peptides SK-29, KR-20, LL-29, and LL-23 from human sweat or skin.

    abstract::Post-translational processing of host defense cathelicidin peptide LL-37 in human sweat and skin generates new antimicrobial peptides. To understand structure and mechanism of action of these LL-37 derivatives, this article presents the cloning and expression of SK-29, KR-20, LL-29, and LL-23. We also provide a two-st...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Li Y,Li X,Wang G

    更新日期:2007-10-01 00:00:00

  • Prokaryotic expression, polyclonal antibody preparation, and sub-cellular localization analysis of Na+, K+-ATPase beta2 subunit.

    abstract::Na+, K+-ATPase beta2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule. To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chen TF,Zhang YL,Xu WL,Li ZQ,Hou B,Wang CL,Fan M,Qian LJ,Zhou RP,Zhang CG

    更新日期:2004-09-01 00:00:00

  • Co-expression as a convenient method for the production and purification of core histones in bacteria.

    abstract::Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Anderson M,Huh JH,Ngo T,Lee A,Hernandez G,Pang J,Perkins J,Dutnall RN

    更新日期:2010-08-01 00:00:00

  • Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis.

    abstract::The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containin...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Mishra S,Bhagavat R,Chandra N,Vijayarangan N,Rajeswari H,Ajitkumar P

    更新日期:2012-11-01 00:00:00

  • Overexpression and purification of the Escherichia coli inner membrane enzyme acyl-acyl carrier protein synthase in an active form.

    abstract::Acyl-acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein. The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Shanklin J

    更新日期:2000-04-01 00:00:00

  • Expression and reconstitution of NF-kappaB from insect cells using a baculovirus vector.

    abstract::NF-kappaB is a pleiotropic transcriptional activator originally identified by its ability to regulate immunoglogulin kappa light chain expression. Purification of this DNA-binding complex demonstrated that NF-kappaB is a heterodimer composed of two subunits, NFKB1 and RelA. Previous studies have shown that truncated v...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Coleman TA,Huddleston KA,Ruben SM,Rosen CA,Gentz R

    更新日期:1997-02-01 00:00:00

  • High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.

    abstract::The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secr...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Truan D,Vasil A,Stonehouse M,Vasil ML,Pohl E

    更新日期:2013-07-01 00:00:00

  • Heterologous expression and purification of plantaricin NC8, a two-peptide bacteriocin against Salmonella spp. from Lactobacillus plantarum ZJ316.

    abstract::Bacteriocin, which is produced by lactic acid bacteria (LAB), has the potential to act as natural preservatives in the food industry. To develop strategies to overproduce such peptides, plantaricin NC8, a class IIb LAB bacteriocin that consists of two peptides, PLNC8α and PLNC8β, was successfully heterologously expres...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Jiang H,Li P,Gu Q

    更新日期:2016-11-01 00:00:00

  • A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli.

    abstract::To facilitate studies of multicomponent protein complexes, I have developed an Escherichia coli expression system which coexpresses up to four polypeptides from a single plasmid. The modular nature of the system enables efficient subcloning of a gene into each of the 4 cassettes in the polycistronic expression vector....

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Tan S

    更新日期:2001-02-01 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