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Protease I from Pyrococcus furiosus

Published on Jan 1, 2001in Methods in Enzymology1.86
· DOI :10.1016/S0076-6879(01)30392-0
Lara S Chang3
Estimated H-index: 3
,
Paula M Hicks4
Estimated H-index: 4
,
Robert M. Kelly51
Estimated H-index: 51
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Abstract
Publisher Summary Pyrococcus furiosus is a hyperthermophilic archaeon from the order Thermococcales capable of growth on a variety of proteinaceous and carbohydrate-containing substrates. Analysis of gelatin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) gels indicates that at least 11 endoproteinases are active in the cell extracts of this organism 2'3 and the following proteases have been characterized: protease I (PfpI), pyrolysin, proteasome, prolyl oligopeptidase, and proline dipeptidase. The gene encoding the 19-kDa subunit of PfpI has homologs in nearly every organism and cell examined to date, ranging from Escherichia coli to Homo sapiens; this ubiquity and evolutionary conservation indicates that it may play a fundamental physiological role. Efforts to study this issue have been exacerbated by difficulties encountered in obtaining significant amounts of a particular assembly of PfpI in either a native or a recombinant form. Native PfpI undergoes autoproteolysis and/or disassembly during direct purification from P. furiosus biomass and exists in multiple (singleto multisubunit) forms in vitro. The production of a recombinant form of PfpI is also problematic due to its toxicity toward mesophilic hosts. This chapter describes several methods that have been used to purify PfpI directly from P. Furiosus cell extracts are described here, together with an assay to detect proteolytic activity, a procedure to determine its molecular mass, and approaches to minimize PfpI-catalyzed proteolysis of other P. furious proteins.
  • References (11)
  • Citations (9)
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References11
Newest
Published on Sep 1, 1998in Journal of Bacteriology3.23
Mousumi Ghosh2
Estimated H-index: 2
(UGA: University of Georgia),
Amy M. Grunden5
Estimated H-index: 5
(UGA: University of Georgia)
+ 2 AuthorsMichael W. W. Adams65
Estimated H-index: 65
(UGA: University of Georgia)
Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+,...
Published on Jun 1, 1997in Journal of Bacteriology3.23
Valerie J. Harwood2
Estimated H-index: 2
(UMB: University of Maryland, Baltimore),
Jackie D. Denson1
Estimated H-index: 1
(UMB: University of Maryland, Baltimore)
+ 1 AuthorsHarold J. Schreier4
Estimated H-index: 4
(UMB: University of Maryland, Baltimore)
The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90 degrees C. Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70...
Published on Jan 1, 1997in Applied and Environmental Microbiology4.08
Sheryl B. Halio3
Estimated H-index: 3
,
Michael W. Bauer10
Estimated H-index: 10
+ 2 AuthorsRobert M. Kelly51
Estimated H-index: 51
The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100(deg)C by the fermentation of peptides and carbohydrates. From this organism, we have purified to homogeneity an intracellular protease, previously designated PfpI (P. furiosus protease I) (S. B. Halio, I. I. Blumentals, S. A. Short, B. M. Merrill, and R. M. Kelly, J. Bacteriol. 178:2605-2612, 1996). The protease contains a single subunit with a molecular mass of approximately 19 kDa and exists in at least two functional co...
Published on Aug 23, 1996in Journal of Biological Chemistry
W.G.B. Voorhorst6
Estimated H-index: 6
,
Rik I. L. Eggen40
Estimated H-index: 40
+ 3 AuthorsW.M. de Vos128
Estimated H-index: 128
Abstract The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin a...
Published on May 1, 1996in Journal of Bacteriology3.23
S B Halio1
Estimated H-index: 1
(NCSU: North Carolina State University),
I I Blumentals1
Estimated H-index: 1
(NCSU: North Carolina State University)
+ 2 AuthorsRobert M. Kelly51
Estimated H-index: 51
(NCSU: North Carolina State University)
A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95...
Published on Jan 1, 1995in Gene2.64
Kelly A. Robinson3
Estimated H-index: 3
(UMBC: University of Maryland, Baltimore County),
Duane A. Bartley1
Estimated H-index: 1
(UMBC: University of Maryland, Baltimore County)
+ 1 AuthorsHarold J. Schreier16
Estimated H-index: 16
(UMBC: University of Maryland, Baltimore County)
Abstract The mlr-2 gene from the hyperthermophilic archaeum Pyrococcus furiosus was identified from a family of clones whose expression was influenced by the presence of maltose in the medium. The sequence of 2100 bp of DNA containing mlr-2 and its flanking regions revealed a 616-amino-acid (71 kDa) open reading frame (ORF). The ORF's initiation codon appeared 10 nt into the mlr-2 message and was not preceded by any apparent ribosome-binding site. The deduced product shared homology with prolyl ...
Published on Jan 1, 1992
Bořivoj Keil1
Estimated H-index: 1
1 Introduction.- 2 Nomenclature and Conventions.- 2.1 EC Numbers.- 2.2 Enzyme Names.- 2.3 Enzyme and Substrate Codes.- 2.4 Subsite Nomenclature.- 2.5 Bibliography.- 3 Data Treatment.- 3.1 Data Bank LYSIS.- 3.2 Statistical Approach to Specificity.- 3.2.1 Calculations.- 3.2.2 Sources of Errors and Limits.- 3.2.3 Influence of Subsites on Chymotryptic Cleavages.- 3.2.4 Roles of Subsites P4-P3' in Cleavages by Pepsin.- 3.2.5 Fixation Preference of Chymotrypsin and Pepsin.- 3.2.6 Predictions of Cleava...
Published on May 1, 1991in Microbiology1.03
Helen Connaris1
Estimated H-index: 1
,
Don A. Cowan47
Estimated H-index: 47
,
Richard J. Sharp23
Estimated H-index: 23
Intracellular and extracellular samples from the extremely thermophilic archaeobacterium Pyrococcus furiosus showed the presence of multiple active proteinases. Using gelatin-containing SDS-PAGE, up to 13 activity bands were visualized with apparent molecular masses of between 66 and 135 kDa. Characterization studies revealed these bands to be due to discrete polypeptides, and not artefacts. Results from gel permeation chromatography, sucrose density gradient centrifugation and non-denaturing PA...
Published on Sep 1, 1990in Fems Microbiology Letters1.99
Rik I. L. Eggen40
Estimated H-index: 40
,
A.C.M. Geerling13
Estimated H-index: 13
+ 1 AuthorsW.M. de Vos128
Estimated H-index: 128
From the hyperthermophilic archaebacterium Pyrococcus furiosus an oxygen-stable, extremely thermostable protease activity, which we designate pyrolysin, has been identified and characterized. Pyrolysin is a cell-envelope associated protease activity high thermo-activity and stability. The temperature optimum is 115°C and half-life values in the absence of substrate are: at least 96 h at 80°C, 9 h at 95°C, 4h at 100°C, 20 min at 105°C and 3 min at 110°C. Pyrolysin is active at a broad pH range be...
Published on Jul 1, 1990in Applied and Environmental Microbiology4.08
Ilse I. Blumentals6
Estimated H-index: 6
,
Anne S. Robinson25
Estimated H-index: 25
,
Robert M. Kelly51
Estimated H-index: 51
(Johns Hopkins University)
Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98{degree}C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by t...
Cited By9
Newest
Published on Sep 1, 2017
Steven B. Larson13
Estimated H-index: 13
(UCI: University of California, Irvine),
Alexander McPherson53
Estimated H-index: 53
(UCI: University of California, Irvine)
The Pfp1 protease, a cysteine protease of unknown specificity from the hyperthermophilic archaeon Thermococcus thioreducens, was crystallized in two distinctive crystal forms: from concentrated citrate in one case and PEG in the other. X-ray data were collected from both crystal forms at room temperature to about 1.9 A resolution using a laboratory source and detector, and the structures were solved by molecular replacement using the Pfp1 protease from Pyrococcus horikoshii as the search model. ...
Published on Jun 18, 2013in African Journal of Microbiology Research
Nouman Rasool2
Estimated H-index: 2
,
Naeem Rashid13
Estimated H-index: 13
+ 1 AuthorsMasood Ahmed Siddiqui2
Estimated H-index: 2
Published on Jan 1, 2013
P.M. Hicks (Cargill), Lara S Chang3
Estimated H-index: 3
,
R.M. Kelly
Published on Apr 1, 2005in Current Opinion in Chemical Biology8.54
Haruyuki Atomi43
Estimated H-index: 43
(Kyoto University)
The discovery of extremophiles has drastically changed our understanding towards the diversity of life itself and the conditions under which it can be sustained. Extremophiles have evolved to withstand and multiply under the extremes of temperature, pressure, pH and salinity. Hyperthermophiles are the group that have adapted to high temperature; many have been found to grow at temperatures above the boiling point of water. This review focuses on recent advances in application-based research on h...
Garabed Antranikian47
Estimated H-index: 47
,
Constantinos E. Vorgias20
Estimated H-index: 20
,
Costanzo Bertoldo10
Estimated H-index: 10
The steady increase in the number of newly isolated extremophilic microorganisms and the discovery of their enzymes by academic and industrial institutions underlines the enormous potential of extremophiles for application in future biotechnological processes. Enzymes from extremophilic microorganisms offer versatile tools for sustainable developments in a variety of industrial application as they show important environmental benefits due to their biodegradability, specific stability under extre...
Published on Mar 1, 2004in Protein Expression and Purification1.29
Linley R. Schofield5
Estimated H-index: 5
(Massey University),
Mark L. Patchett16
Estimated H-index: 16
(Massey University),
Emily J. Parker22
Estimated H-index: 22
(Massey University)
Abstract The enzyme 3-deoxy- d - arabino -heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus has been expressed in Escherichia coli . The expressed protein was insoluble but was partially solubilized as a dimer by the inclusion of 200 mM KCl in the cell lysis buffer. An effective two step purification procedure has been developed. The first step res...
Published on Jan 1, 2002in Archaea3.09
B. Franzetti1
Estimated H-index: 1
,
Guy Schoehn41
Estimated H-index: 41
+ 2 AuthorsG. Zaccai1
Estimated H-index: 1
A 20S proteasome, comprising two subunits α and β, was purified from the extreme halophilic archaeon Haloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of the H. marismortui proteasome (Hm proteasome), obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of ...
Published on Jan 1, 2002in Archaea3.09
Donald E. Ward5
Estimated H-index: 5
,
Keith R. Shockley14
Estimated H-index: 14
+ 4 AuthorsRobert M. Kelly51
Estimated H-index: 51
Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus ...
View next paperSequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus.