Expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans and some properties of the recombinant enzyme

2002 ◽  
Vol 362 (2) ◽  
pp. 223-229 ◽  
Author(s):  
Benjamin ADAMS ◽  
David J. LOWE ◽  
Andrew T. SMITH ◽  
Claudio SCAZZOCCHIO ◽  
Stephane DEMAIS ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Aspergillus Nidulans ◽  
Specific Activity ◽  
Recombinant Expression ◽  
Expression System ◽  
Complex Mixture ◽  
Xanthine Dehydrogenase ◽  
Cell System ◽  
Post Translational Modification ◽  
Enzyme Molecules

Recent crystal structures of xanthine dehydrogenase, xanthine oxidase and related enzymes have paved the way for a detailed structural and functional analysis of these enzymes. One problem encountered when working with these proteins, especially with recombinant protein, is that the preparations tend to be heterogeneous, with only a fraction of the enzyme molecules being active. This is due to the incompleteness of post-translational modification, which for this protein is a complex, and incompletely understood, process involving incorporation of the Mo and Fe/S centres. The enzyme has been expressed previously in both Drosophila and insect cells using baculovirus. The insect cell system has been exploited by Iwasaki et al. [Iwasaki, Okamoto, Nishino, Mizushima and Hori (2000) J. Biochem (Tokyo) 127, 771–778], but, for the rat enzyme, yields a complex mixture of enzyme forms, containing around 10% of functional enzyme. The expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans is described. The purified protein has been analysed both functionally and spectroscopically. Its specific activity is indistinguishable from that of the enzyme purified from fruit flies [Doyle, Burke, Chovnick, Dutton, Whittle and Bray (1996) Eur. J. Biochem. 239, 782–795], and it appears to be more active than recombinant xanthine dehydrogenase produced with the baculovirus system. EPR spectra of the recombinant Drosophila enzyme are reported, including parameters for the Fe/S centres. Only a very weak ‘Fe/SIII’ signal (g1,2,3, 2.057, 1.930, 1.858) was observed, in contrast to the strong analogous signal reported for the enzyme from baculovirus. Since this signal appears to be associated with incomplete post-translational modification, this is consistent with relatively more complete cofactor incorporation in the Aspergillus-produced enzyme. Thus we have developed a recombinant expression system for D. melanogaster xanthine dehydrogenase, which can be used for the production of site-specific mutations of this enzyme.

scholarly journals POST-TRANSLATIONAL MODIFICATION OF XANTHINE DEHYDROGENASE IN A NATURAL POPULATION OF DROSOPHILA MELANOGASTER

Genetics ◽  
1981 ◽  
Vol 98 (4) ◽  
pp. 817-831
Author(s):  
George Johnson ◽  
Victoria Finnerty ◽  
Daniel Hartl
Keyword(s):  
Drosophila Melanogaster ◽  
Natural Population ◽  
Aldehyde Oxidase ◽  
Xanthine Dehydrogenase ◽  
Chromosome 3 ◽  
Chromosome 2 ◽  
Structural Genes ◽  
Post Translational Modification ◽  
Polymorphic Loci

ABSTRACT Second chromosomes of D. melanogaster were isolated from a single natural population, and 40 were analyzed by gel-sieving electrophoresis for the presence of polymorphic loci on chromosome 2 that act to modify xanthine dehydrogenase and/or aldehyde oxidase, whose structural genes map to chromosome 3. Clear evidence of polymorphism for one or more xanthine dehydrogenase modifier loci was obtained.

scholarly journals POST-TRANSLATIONAL MODIFICATION AS A POTENTIAL EXPLANATION OF HIGH LEVELS OF ENZYME POLYMORPHISM: XANTHINE DEHYDROGENASE AND ALDEHYDE OXIDASE IN DROSOPHILA MELANOGASTER

Genetics ◽  
1979 ◽  
Vol 91 (4) ◽  
pp. 695-722
Author(s):  
Victoria Finnerty ◽  
George Johnson
Keyword(s):  
Drosophila Melanogaster ◽  
Natural Populations ◽  
Aldehyde Oxidase ◽  
Xanthine Dehydrogenase ◽  
Enzyme Polymorphism ◽  
The Body ◽  
Post Translational Modification ◽  
Heritable Variation ◽  
Thermal Stabilities ◽  
Partial Restoration

ABSTRACT Xanthine dehydrogenase (XDH) and aldehyde oxidase (AO) in Drosophila melanogaster require for their activity the action of another unlinked locus, maroon-like (mal), While the XDH and A 0 loci are on chromosome 3, mal maps to the X chromosome. Although functional mal gene product is required for XDH and A 0 activity, it is possible to examine the effects of mutant mal alleles in those cases when pairs of mutants complement to produce a partial restoration of activity. To test whether mal mediates a post-translational modification of the XDH and A0 proteins, we constructed several mal heteroallelic complementing stocks of Drosophila in which the third chromosomes were co-isogenic. Since all lines were co-isogenic for the XDH and A0 structural genes, any variation in these enzymes seen when comparing these stocks must have been produced by post-translational modification by mal. We examined the XDH and A 0 proteins in these stocks by gel-sieving electrophoresis, a procedure that permits independent characterization of a protein's charge and shape, and is capable of discriminating many variants not detected in routine electrophoresis. In every mal heteroallelic combination, there is a significant alteration in protein shape, when compared to wild type. The magnitude of differences in shape of XDH and AO is correlated both with differences in their enzyme activities and with differences in their thermal stabilities. As the body of this variation appears heritable, any functional differences resulting from these variants are of real genetic and evolutionary interest. A similar post-translational modification of XDH and A0 by yet another locus, lxd, was subsequently documented in an analogous manner. The pattern of electrophoretic differences produced by mal and lxd modification is similar to that reported for electrophoretic "alleles" of XDH in natural populations. The implication is that heritable variation in electrophoretic mobility at these two enzyme loci, and potentially at other loci, is not necessarily allelic to the structural gene loci.

Purification of Recombinant Mouse C-Reactive Protein from Pichia Pastoris GS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography and P-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem

2021 ◽  
Author(s):  
Bin Cheng ◽  
Di Wu ◽  
Ke Wu ◽  
Xiao-Ping Huang ◽  
Jian-Min Lv ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Affinity Chromatography ◽  
Protein Function ◽  
Recombinant Expression ◽  
Expression System ◽  
Post Translational Modification ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Phosphoryl Choline ◽  
Fast Flow

Abstract C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p-Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands.

CHANGES IN LEVELS OF ALCOHOL DEHYDROGENASE DURING THE DEVELOPMENT OF DROSOPHILA MELANOGASTER

1981 ◽  
Vol 23 (2) ◽  
pp. 305-313 ◽  
Author(s):  
Steven M. Anderson ◽  
John F. McDonald
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Specific Activity ◽  
Biochemical Properties ◽  
Developmental Changes ◽  
Post Translational Modification ◽  
Biochemical Basis ◽  
Quantitative Changes ◽  
Adh Activity ◽  
Qualitative Changes

The results of an analysis of the biochemical basis of changes in alcohol dehydrogenase (E.C.1.1.1.1) activity over Drosophila development are presented. The data indicate that (1) the characteristic changes that occur in ADH activity over development are predominantly, if not exclusively, the result of quantitative changes in the amount of enzyme present rather than qualitative changes affecting the enzyme's specific activity and (2) the fluctuations in amount of ADH which occur during development are not the result of the only known form of post-translational modification capable of affecting the biochemical properties of the enzyme. We conclude that developmental changes in amount of ADH are most likely the result of fluctuations in the turnover of the ADH protein.

scholarly journals Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line

1992 ◽  
Vol 284 (1) ◽  
pp. 53-59 ◽  
Author(s):  
F Fossiez ◽  
G Lemay ◽  
N Labonté ◽  
F Parmentier-Lesage ◽  
G Boileau ◽  
...  
Keyword(s):  
Insect Cell ◽  
Mammalian Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Insect Cell Line ◽  
Cell Types ◽  
Neutral Endopeptidase ◽  
Cell System ◽  
Recombinant Enzyme ◽  
Soluble Form

Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.

scholarly journals Expression and Characterization ofGeobacillus stearothermophilusSR74 Recombinantα-Amylase inPichia pastoris

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Sivasangkary Gandhi ◽  
Abu Bakar Salleh ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Thean Chor Leow ◽  
Siti Nurbaya Oslan
Keyword(s):  
Specific Activity ◽  
Thermophilic Bacteria ◽  
Recombinant Expression ◽  
Expression System ◽  
Alcohol Oxidase ◽  
Product Yield ◽  
Inducible Promoter ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Gene Encoding

Geobacillus stearothermophilusSR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactiveα-amylase. Increased production and commercialization of thermostableα-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostableα-amylase inG. stearothermophilusSR74 was amplified, sequenced, and subcloned intoP. pastorisGS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinantα-amylase SR74 achieved in shake flask was 28.6 U mL−1at 120 h after induction. The recombinant 59 kDaα-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH ofα-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t1/2) of 88 min at 60°C. In conclusion, thermostableα-amylase SR74 fromG. stearothermophilusSR74 would be beneficial for industrial applications, especially in liquefying saccrification.

scholarly journals Expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans and some properties of the recombinant enzyme

2002 ◽  
Vol 362 (1) ◽  
pp. 223 ◽  
Author(s):  
Benjamin ADAMS ◽  
David J. LOWE ◽  
Andrew T. SMITH ◽  
Claudio SCAZZOCCHIO ◽  
Stephane DEMAIS ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Aspergillus Nidulans ◽  
Xanthine Dehydrogenase ◽  
Recombinant Enzyme

scholarly journals Recombinant production of active microbial transglutaminase in E. coli by using self-cleavable zymogen with mutated propeptide

2020 ◽  
Author(s):  
Ryo Sato ◽  
Kosuke Minamihata ◽  
Ryutaro Ariyoshi ◽  
Hiromasa Taniguchi ◽  
Noriho Kamiya
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Recombinant Expression ◽  
Expression System ◽  
Chimeric Protein ◽  
Microbial Transglutaminase ◽  
Cross Linking ◽  
E Coli ◽  
Tev Protease ◽  
Recombinant Expression System

AbstractMicrobial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7±2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.

High Level Expression of Active Human Prothrombin in a Vaccina Virus Expression System

1992 ◽  
Vol 68 (02) ◽  
pp. 119-124 ◽  
Author(s):  
F G Falkner ◽  
P L Turecek ◽  
R T A MacGillivray ◽  
W Bodemer ◽  
F Scheiflinger ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cell Lines ◽  
Expression System ◽  
Human Cell Line ◽  
Cell System ◽  
Time Saving ◽  
Expression Levels ◽  
Mammalian Cell Lines ◽  
Cell Line Hela ◽  
Virus Expression

SummaryWe have worked out an efficient and time saving procedure for the expression of recombinant human prothrombin. The glycoprotein was expressed in the vaccinia virus expression system in several mammalian cell lines. The kidney cell lines Vero and BHK and the human cell line Hela were found to efficiently secrete prothrombin. Expression levels of 3–4 µg of factor II per 106 cells per day corresponding to 18–23 mU per 106 cells per day were achieved. Since the expression levels obtained with the vaccinia virus/Vero cell system were comparable to those obtained in amplified transformed CHO cells it provides an alternative system for the efficient expression of human prothrombin and may allow to further elucidate structure-function relationships of (pro)thrombin and its various effectors.

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