Biological activity and intracellular location of the Tat protein of equine infectious anemia virus.
The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10(11) bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by 'scrape loading'. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.
Rosin-Arbesfeld
R
R
Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel.
Mashiah
P
P
Willbold
D
D
Rosch
P
P
Tronick
S R
SR
Yaniv
A
A
Gazit
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004285:Dogs; D004926:Escherichia coli; D015696:Gene Products, tat / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism; D004860:Infectious Anemia Virus, Equine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013698:Templates, Genetic; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus