Recombinant Human Pancreatic Ribonuclease Produced in E. coli

Importance of the Amino-Terminal Sequence

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31 Citations (Scopus)

Abstract

Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3′,5′-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein.

Original languageEnglish
Pages (from-to)406-413
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume216
Issue number1
DOIs
Publication statusPublished - Nov 2 1995

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Pancreatic Ribonuclease
Escherichia coli
Secondary Protein Structure
Proteins
Disintegration
Inclusion Bodies
Circular Dichroism
Yeast
Adenosine
Oxidation-Reduction
Glutathione
Cations
Yeasts
RNA
Amino Acids
placental ribonuclease inhibitor

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology
  • Biophysics
  • Biochemistry

Cite this

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title = "Recombinant Human Pancreatic Ribonuclease Produced in E. coli: Importance of the Amino-Terminal Sequence",
abstract = "Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3′,5′-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein.",
author = "Junichiro Futami and Masaharu Seno and Megumi Kosaka and Hiroko Tada and S. Seno and H. Yamada",
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T1 - Recombinant Human Pancreatic Ribonuclease Produced in E. coli

T2 - Importance of the Amino-Terminal Sequence

AU - Futami, Junichiro

AU - Seno, Masaharu

AU - Kosaka, Megumi

AU - Tada, Hiroko

AU - Seno, S.

AU - Yamada, H.

PY - 1995/11/2

Y1 - 1995/11/2

N2 - Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3′,5′-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein.

AB - Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3′,5′-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein.

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