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Protease I from Pyrococcus furiosus

Published on Jan 1, 2001in Methods in Enzymology1.862
· DOI :10.1016/S0076-6879(01)30392-0
Lara S Chang3
Estimated H-index: 3
,
Paula M Hicks4
Estimated H-index: 4
,
Robert M. Kelly53
Estimated H-index: 53
Sources
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
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#1Mousumi Ghosh (UGA: University of Georgia)H-Index: 2
#2Amy M. Grunden (UGA: University of Georgia)H-Index: 5
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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+,...
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#1Valerie J. Harwood (UMB: University of Maryland, Baltimore)H-Index: 2
#2Jackie D. Denson (UMB: University of Maryland, Baltimore)H-Index: 1
Last. Harold J. Schreier (UMB: University of Maryland, Baltimore)H-Index: 4
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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...
28 CitationsSource
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...
34 Citations
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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...
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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...
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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 ...
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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...
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