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.
Chun
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Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Glabe
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Fan
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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