Expression in Escherichia coli of an immunoreactive visna virus transactivating protein.
We have cloned the gene encoding the visna 1514 transactivating protein, Tat, into the Escherichia coli lambda pR expression plasmid, pRIT2T. Efficient synthesis of the protein A::Tat fusion protein was obtained in host strains which carried either wild-type or temperature-sensitive (ts) lambda repressors. However, constitutive synthesis of the fusion protein in these host strains resulted in selection against plasmids which synthesized the fusion protein. Efficient repression of the lambda pR promoter was obtained using a ts repressor gene carried on a multicopy plasmid. Synthesis of the fusion protein in this strain was efficient on induction, and reproducible after subculture. Antisera generated against the termini of visna Tat were used to demonstrate that the fusion protein retained the antigenicity of both the N and C termini of the transactivating protein.
Watson
G
G
Department of Veterinary Pathology, University of Edinburgh, Summerhall, U.K.
Harkiss
G D
GD
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D015696:Gene Products, tat / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014760:Viral Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D014790:Visna-maedi virus / Q000235:genetics*
D015696:Gene Products, tat; D014760:Viral Fusion Proteins