Document 0450
 DOCN  M9460450
 TI    Large-scale production and characterization of recombinant human
       immunodeficiency virus type 1 Nef.
 DT    9404
 AU    Azad AA; Failla P; Lucantoni A; Bentley J; Mardon C; Wolfe A; Fuller K;
       Hewish D; Sengupta S; Sankovich S; et al; Biomolecular Research
       Institute, Parkville, Victoria, Australia.
 SO    J Gen Virol. 1994 Mar;75 ( Pt 3):651-5. Unique Identifier : AIDSLINE
       MED/94172341
 AB    Sequences encoding the 27K and 25K nef gene products (Nef 27 and Nef 25)
       were amplified by PCR from a human immunodeficiency virus type 1
       infectious clone and subcloned directly into Escherichia coli, 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 E. coli-derived glutathione S-transferase-Nef fusion
       proteins were very high and a major portion was soluble. Large-scale
       production of E. coli-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 non-reducing 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 E. coli-derived
       Nef 27 and yeast-derived myristylated 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.
 DE    Base Sequence  Escherichia coli/GROWTH & DEVELOPMENT/GENETICS  Gene
       Products, nef/*BIOSYNTHESIS  Genes, nef/*GENETICS  HIV-1/*GENETICS
       Molecular Sequence Data  Recombinant Proteins/*BIOSYNTHESIS  Support,
       Non-U.S. Gov't  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).