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Title 

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

Authors 

Kandula SunithaBong Hyun ChungKi Hyo JangKi Bang SongChul Ho KimSang Ki Rhee

Publisher 

Elsevier

Issue Date 

2000

Citation 

Protein Expression & Purification, vol. 18, no. 3, pp. 388-393

Keywords 

cell inclusionenzyme purificationfed batch culturelevansucraseculture mediaEscherichia coliinclusion bodiesrecombinant proteinszymomonas

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

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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