Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli

Cited 24 time in scopus
Metadata Downloads
Title
Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli
Author(s)
Kandula Sunitha; Bong Hyun Chung; Ki Hyo Jang; Ki Bang Song; Chul Ho Kim; Sang Ki Rhee
Bibliographic Citation
Protein Expression and Purification, vol. 18, no. 3, pp. 388-393
Publication Year
2000
Abstract
Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.
ISSN
1046-5928
Publisher
Elsevier
DOI
http://dx.doi.org/10.1006/prep.2000.1204
Type
Article
Appears in Collections:
Jeonbuk Branch Institute > Microbial Biotechnology Research Center > 1. Journal Articles
Files in This Item:
  • There are no files associated with this item.


Items in OpenAccess@KRIBB are protected by copyright, with all rights reserved, unless otherwise indicated.