Guenet C, Leppik R A, Pelton J T, Moelling K, Lovenberg W, Harris B A
Merrell Dow Research Institute, Strasbourg, France.
Eur J Pharmacol. 1989 Dec 5;172(6):443-51. doi: 10.1016/0922-4106(89)90027-8.
Considerable interest exists in the HIV-1 protease for biochemical studies as a potential therapeutic target of acquired immunodeficiency syndrome. We have produced the retroviral enzyme in E. coli from a synthetic gene encoding the protease that was constructed by assembling six overlapping and complementary oligonucleotides into the vector pKK223-3. When expressed in E. coli, the recombinant protease was able to correctly process the HIV-1 core protein p24 from a beta-galactosidase-gag fusion protein and to use a heptapeptide as a substrate for proteolytic cleavage. A single base pair mutation was identified in a recombinant that resulted in the substitution of lysine for asparagine at position 88 and a significant loss of enzyme activity. Through site-directed mutagenesis, the Asn88 was changed to five other residues representative of all classes of amino acids. The correlation between enzyme activity and amino acid substitution suggests that the protease domain surrounding position 88 affects the protein's potential for forming an active homodimeric protein and hence, indicates a biochemical interaction that could be inhibited by novel antiviral compounds.
作为获得性免疫缺陷综合征的潜在治疗靶点，HIV-1蛋白酶在生化研究中备受关注。我们通过将六个重叠且互补的寡核苷酸组装到载体pKK223-3中构建了编码该蛋白酶的合成基因，并在大肠杆菌中生产了这种逆转录病毒酶。当在大肠杆菌中表达时，重组蛋白酶能够正确地从β-半乳糖苷酶-gag融合蛋白中加工HIV-1核心蛋白p24，并将一种七肽用作蛋白水解切割的底物。在一个重组体中鉴定出一个单碱基对突变，该突变导致88位的天冬酰胺被赖氨酸取代，酶活性显著丧失。通过定点诱变，将88位的天冬酰胺改变为代表所有氨基酸类别的其他五个残基。酶活性与氨基酸取代之间的相关性表明，88位周围的蛋白酶结构域影响蛋白质形成活性同二聚体蛋白的潜力，因此，表明了一种可能被新型抗病毒化合物抑制的生化相互作用。
