Chun R, Glabe C G, Fan H
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
J Virol. 1990 Jun;64(6):3074-7. doi: 10.1128/JVI.64.6.3074-3077.1990.
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.
采用Fmoc氨基酸连续流动策略，在半自动仪器上化学合成了与人类免疫缺陷病毒1型tat反式激活蛋白相对应的全长（86个残基）多肽。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和等电聚焦判断，大量材料相对均一，当刮取加载到含有人类免疫缺陷病毒长末端重复序列-氯霉素乙酰转移酶报告质粒的细胞中时，它表现出反式激活活性。反相高压液相色谱产生了相当宽的洗脱图谱，对柱上生物活性的测定表明有一个更尖锐的峰。因此，高压液相色谱可用于富集生物活性。合成tat的胰蛋白酶消化产物的快原子轰击质谱鉴定出了几种预测的胰蛋白酶肽段，这与精确的化学合成结果一致。
