The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris.
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.
Sugrue
R J
RJ
Dengue Virus Group, Institute of Molecular and Cell Biology, National University of Singapore, Singapore. rjsu@mail.nerc-oxford.ac.uk
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Chan
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D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000276:immunology; D015153:Blotting, Western; D002847:Chromatography, Agarose; D003716:Dengue Virus / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011233:Precipitin Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D014170:Transformation, Genetic; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C072281:E protein TH Sman, Dengue virus; D005982:Glutathione Transferase