Direct refolding of inclusion bodies using reversed micelles

Masafumi Sakono, Yu Mi Kawashima, Hirofumi Ichinose, Tatsuo Maruyama, Noriho Kamiya, Masahiro Goto

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.

Original languageEnglish
Pages (from-to)1783-1787
Number of pages5
JournalBiotechnology Progress
Volume20
Issue number6
DOIs
Publication statusPublished - Nov 1 2004

Fingerprint

Inclusion Bodies
inclusion bodies
Micelles
micelles
Pancreatic Ribonuclease
ribonucleases
Protein Refolding
molecular chaperones
Molecular Chaperones
Dioctyl Sulfosuccinic Acid
Acetone
aerosols
solubilization
acetone
Adenosine Triphosphate
Escherichia coli
protein refolding

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Direct refolding of inclusion bodies using reversed micelles. / Sakono, Masafumi; Kawashima, Yu Mi; Ichinose, Hirofumi; Maruyama, Tatsuo; Kamiya, Noriho; Goto, Masahiro.

In: Biotechnology Progress, Vol. 20, No. 6, 01.11.2004, p. 1783-1787.

Research output: Contribution to journalArticle

Sakono, Masafumi ; Kawashima, Yu Mi ; Ichinose, Hirofumi ; Maruyama, Tatsuo ; Kamiya, Noriho ; Goto, Masahiro. / Direct refolding of inclusion bodies using reversed micelles. In: Biotechnology Progress. 2004 ; Vol. 20, No. 6. pp. 1783-1787.
@article{6fd3b6a900df4e4b916b1180c4e37d1f,
title = "Direct refolding of inclusion bodies using reversed micelles",
abstract = "The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90{\%} recovery of the renatured RNase A.",
author = "Masafumi Sakono and Kawashima, {Yu Mi} and Hirofumi Ichinose and Tatsuo Maruyama and Noriho Kamiya and Masahiro Goto",
year = "2004",
month = "11",
day = "1",
doi = "10.1021/bp049887j",
language = "English",
volume = "20",
pages = "1783--1787",
journal = "Biotechnology Progress",
issn = "8756-7938",
publisher = "John Wiley and Sons Ltd",
number = "6",

}

TY - JOUR

T1 - Direct refolding of inclusion bodies using reversed micelles

AU - Sakono, Masafumi

AU - Kawashima, Yu Mi

AU - Ichinose, Hirofumi

AU - Maruyama, Tatsuo

AU - Kamiya, Noriho

AU - Goto, Masahiro

PY - 2004/11/1

Y1 - 2004/11/1

N2 - The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.

AB - The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.

UR - http://www.scopus.com/inward/record.url?scp=10044289445&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=10044289445&partnerID=8YFLogxK

U2 - 10.1021/bp049887j

DO - 10.1021/bp049887j

M3 - Article

C2 - 15575712

AN - SCOPUS:10044289445

VL - 20

SP - 1783

EP - 1787

JO - Biotechnology Progress

JF - Biotechnology Progress

SN - 8756-7938

IS - 6

ER -