Böttcher M, Grosse F
Institut für Molekulare Biotechnologie, Abteilung Biochemie, Postfach 100 813, D-07708 Jena, Germany.
Nucleic Acids Res. 1997 May 1;25(9):1709-14. doi: 10.1093/nar/25.9.1709.
The reading frame of the HIV-1 pol gene, encoding protease (PR) and reverse transcriptase (RT), including RNase H as well as integrase, was fused to the bacterialbeta-galactosidase gene and overexpressed in Escherichia coli cells. The resulting fusion protein was cleaved autocatalytically leading to PR, RT and integrase. Immunoprecipitations of bacterial crude extracts with anti-RT antibodies precipitated both RT and PR. Co-precipitation of PR and RT was also observed with anti-PR antibodies, strongly suggesting a physical interaction between fully processed RT and PR within the bacterial cell. Physical interactions were confirmed with purified components by means of an ELISA assay. Furthermore, purified PR inhibited the DNA synthesis activity of purified RT, while its RNase H activity remained unaffected. The type of inhibition was uncompetitive with respect to poly(rA).oligo(dT); the inhibition constant was 50-100 nM. A possible physiological significance of this type of interaction is discussed.
编码蛋白酶（PR）、逆转录酶（RT）（包括核糖核酸酶H）以及整合酶的HIV-1 pol基因的阅读框与细菌β-半乳糖苷酶基因融合，并在大肠杆菌细胞中过表达。产生的融合蛋白会自动催化裂解，产生PR、RT和整合酶。用抗RT抗体对细菌粗提物进行免疫沉淀，沉淀出了RT和PR。用抗PR抗体也观察到PR和RT的共沉淀，这强烈表明在细菌细胞内完全加工的RT和PR之间存在物理相互作用。通过酶联免疫吸附测定法用纯化的组分证实了物理相互作用。此外，纯化的PR抑制了纯化的RT的DNA合成活性，而其核糖核酸酶H活性未受影响。这种抑制类型对聚（rA）·寡聚（dT）而言是非竞争性的；抑制常数为50 - 100 nM。讨论了这种相互作用类型可能的生理意义。
