Chemical synthesis of biologically active tat trans-activating protein of human immunodeficiency virus type 1.
Full-length (86-residue) polypeptide corresponding to the human immunodeficiency virus type 1 tat trans-activating protein was chemically synthesized on a semiautomated apparatus, using an Fmoc amino acid continuous-flow strategy. The bulk material was relatively homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, and it showed trans-activating activity when scrape loaded into cells containing a human immunodeficiency virus long terminal repeat-chloramphenicol acetyl-transferase reporter plasmid. Reverse-phase high-pressure liquid chromatography yielded a rather broad elution profile, and assays across the column for biological activity indicated a sharper peak. Thus, high-pressure liquid chromatography provided for enrichment of biological activity. Fast atom bombardment-mass spectrometry of tryptic digests of synthetic tat identified several of the predicted tryptic peptides, consistent with accurate chemical synthesis.
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
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D000595:Amino Acid Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D015696:Gene Products, tat / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D015534:Trans-Activators / Q000138:chemical synthesis*; D015533:Transcriptional Activation; D014357:Trypsin; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D007202:Indicators and Reagents; D010446:Peptide Fragments; D012333:RNA, Messenger; D015534:Trans-Activators; D054322:tat Gene Products, Human Immunodeficiency Virus; D015500:Chloramphenicol O-Acetyltransferase; D014357:Trypsin