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J Virol Methods. 1997 Dec;69(1-2):159-69.

The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris.

Author information

1
Dengue Virus Group, Institute of Molecular and Cell Biology, National University of Singapore, Singapore. rjsu@mail.nerc-oxford.ac.uk

Abstract

The dengue virus envelope protein was expressed as a GST fusion protein using E. coli and P. pastoris as expression hosts. In E. coli the recombinant E protein is expressed initially as a soluble 81 kDa GST fusion protein. Treatment of the fusion protein with thrombin released a 55 kDa protein, which is the expected size for correctly processed, non-glycosylated recombinant E protein. The antiserum from animals immunised with this recombinant E protein was found to specifically recognise the dengue virus E protein in virus-infected cells, thus demonstrating the immunogenic nature of the recombinant E protein. This expression system allowed production of up to 2 mg of purified recombinant E protein from a 1 1 bacterial culture. In contrast, expression of this GST fusion protein in P. pastoris is associated with extensive proteolytic degradation of the recombinant E protein. However, this proteolytic degradation was not observed in the truncated E protein sequences which were expressed. One of these recombinant fusion proteins, GST E401 was secreted into the culture medium at levels of up to 100 microg/l of growth medium.

PMID:
9504761
DOI:
10.1016/s0166-0934(97)00151-1
[Indexed for MEDLINE]

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