1887

Abstract

Sequences encoding the 27K and 25K gene products (Nef 27 and Nef 25) were amplified by PCR from a human immunodeficiency virus type 1 infectious clone and subcloned directly into , yeast and baculovirus expression vectors. The yeast- and baculovirus-derived Nef had native N termini but the expression levels were low. The expression levels of the derived glutathione V-transferase-Nef fusion proteins were very high and a major portion was soluble. Large-scale production of -derived Nef 27 and Nef 25 was carried out by growing recombinant cells in a fermenter under fed-batch conditions followed by affinity purification on glutathione-Sepharose before and after thrombin cleavage. Large quantities of highly purified recombinant Nef proteins have been produced for functional and structural studies. Under nonreducing conditions both Nef 27 and Nef 25 existed as a mixture of monomers, dimers and small amounts of higher oligomers, but when reduced were monomeric. The highly purified Nef proteins had no G protein activities, however Nef 27 was biologically active. When electroporated into uninfected CD4 T lymphocytes both -derived Nef 27 and yeast-derived myristy-lated Nef 27 down-regulated the surface expression of CD4, demonstrating that this method can be used to assess the biological activity of purified recombinant Nef.

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1994-03-01
2021-10-18
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