Methionine Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus: Molecular Cloning and Overexperssion in Escherichia coli of the Gene, and Characteristics of the Enzyme

1997 ◽  
Vol 122 (4) ◽  
pp. 843-850 ◽  
Author(s):  
S. Tsunasawa ◽  
Y. Izu ◽  
M. Miyagi ◽  
I. Kato
Keyword(s):  
Escherichia Coli ◽  
Molecular Cloning ◽  
Pyrococcus Furiosus ◽  
Methionine Aminopeptidase ◽  
Hyperthermophilic Archaeon

scholarly journals A Novel DNA Polymerase Family Found inArchaea

1998 ◽  
Vol 180 (8) ◽  
pp. 2232-2236 ◽  
Author(s):  
Yoshizumi Ishino ◽  
Kayoko Komori ◽  
Isaac K. O. Cann ◽  
Yosuke Koga
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Dna Polymerases ◽  
Open Reading Frames ◽  
Gene Products ◽  
Polymerase Gene ◽  
Hyperthermophilic Archaeon ◽  
Dna Polymerase Ii ◽  
Reading Frames

ABSTRACT One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical α-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeonPyrococcus furiosus, which has also at least one α-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499–512, 1997). The genes in M. jannaschiiencoding the proteins that are homologous to the DNA polymerase II ofP. furiosus have been located and cloned. The gene products of M. jannaschii expressed in Escherichia colihad both DNA polymerizing and 3′→5′ exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.

scholarly journals Cellobiose Uptake in the Hyperthermophilic ArchaeonPyrococcus furiosus Is Mediated by an Inducible, High-Affinity ABC Transporter

2001 ◽  
Vol 183 (17) ◽  
pp. 4979-4984 ◽  
Author(s):  
Sonja M. Koning ◽  
Marieke G. L. Elferink ◽  
Wil N. Konings ◽  
Arnold J. M. Driessen
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Abc Transporter ◽  
Transport System ◽  
Abc Transporters ◽  
Pyrococcus Furiosus ◽  
Binding Protein ◽  
Hyperthermophilic Archaeon ◽  
High Affinity ◽  
Dependent Transport

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosuscan utilize different β-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (K m = 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from P. furiosusmembranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The cbtAgene was cloned and functionally expressed in Escherichia coli. cbtA belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.

scholarly journals Biochemical adaptations of two sugar kinases from the hyperthermophilic archaeon Pyrococcus furiosus

2002 ◽  
Vol 366 (1) ◽  
pp. 121-127 ◽  
Author(s):  
Corné H. VERHEES ◽  
Denise G.M. KOOT ◽  
Thijs J.G. ETTEMA ◽  
Cor DIJKEMA ◽  
Willem M. de VOS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Pyrococcus Furiosus ◽  
Phosphoryl Group ◽  
Hyperthermophilic Archaeon ◽  
High Affinity ◽  
Optimal Activity ◽  
Genes Encoding ◽  
Modified Embden Meyerhof Pathway

The hyperthermophilic archaeon Pyrococcus furiosus possesses a modified Embden—Meyerhof pathway, including an unusual ADP-dependent glucokinase (ADP-GLK) and an ADP-dependent phosphofructokinase. In the present study, we report the characterization of a P. furiosus galactokinase (GALK) and its comparison with the P. furiosus ADP-GLK. The pyrococcal genes encoding the ADP-GLK and GALK were functionally expressed in Escherichia coli, and the proteins were subsequently purified to homogeneity. Both enzymes are specific kinases with an optimal activity at approx. 90°C. Biochemical characterization of these enzymes confirmed that the ADP-GLK is unable to use ATP as the phosphoryl group donor, but revealed that GALK is ATP-dependent and has an extremely high affinity for ATP. There is a discussion about whether the unusual features of these two classes of kinases might reflect adaptations to a relatively low intracellular ATP concentration in the hyperthermophilic archaeon P. furiosus.

Production of human interferon alpha-2b in Escherichia coli and removal of N-terminal methionine utilizing archaeal methionine aminopeptidase

Biologia ◽  
2015 ◽  
Vol 70 (7) ◽  
Author(s):  
Amina Arif ◽  
Qura-Tul-Ann A. Gardner ◽  
Naeem Rashid ◽  
Muhammad Akhtar
Keyword(s):  
Escherichia Coli ◽  
Interferon Alpha ◽  
Inclusion Bodies ◽  
High Performance ◽  
Pyrococcus Furiosus ◽  
Human Interferon ◽  
Methionine Aminopeptidase ◽  
Interferon Alpha 2B ◽  
Foreign Proteins ◽  
Insoluble Form

AbstractMethionine is the first amino acid residue in most of the recombinant proteins and sometimes it is necessary to remove the starting methionine in order to get foreign proteins with wild type sequences. Recombinant human interferon alpha-2b was produced in Escherichia coli in insoluble form as inclusion bodies, which were solubilized and refolded. Analysis of the purified recombinant protein indicated that it consisted of two types of molecules, one type, in majority, having N-terminal methionine intact, whereas the other type, in minority, having the N-terminal methionine cleaved by methionine aminopeptidase of the host. Both species were separated by high performance liquid chromatography and Nterminal methionine of the remaining molecules was removed by utilizing methionine aminopeptidase from Pyrococcus furiosus in order to get similar form of the interferon alpha-2b as produced by human body. The methodology developed in this study can be extended to other proteins of pharmaceutical importance.

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