Mori T, Shoemaker R H, Gulakowski R J, Krepps B L, McMahon J B, Gustafson K R, Pannell L K, Boyd M R
Laboratory of Drug Discovery Research and Development, National Cancer Institute-FCRDC, Frederick, Maryland 21702-1201, USA.
Biochem Biophys Res Commun. 1997 Sep 8;238(1):218-22. doi: 10.1006/bbrc.1997.7202.
Site-directed mutagenesis of DNA constructs coding for the novel, HIV-inactivating proteins cyanovirin-N (CV-N) and FLAG-cyanovirin-N (F-CV-N) was performed using mutagenic oligonucleotide primers in the polymerase chain reaction or by a restriction site elimination maneuver. The mutant constructs were expressed in Escherichia coli and the recombinant protein products were tested for binding to the HIV surface envelope glycoprotein gp 120 and for antiviral activity against infectious HIV. Results showed an overall very high correlation (r2 > 0.9) between the relative gp120 binding affinities and the anti-HIV activities of CV-N, F-CV-N, and the various mutants. An outlier, however, was a mutant which lacked one of the internal disulfide linkages normally present in CV-N and which showed modest gp120 binding but no antiviral activity against HIV. These findings are consistent with the view that gp120 binding is a necessary but not sufficient requirement for the HIV-inactivating activity of CV-N and related proteins; the sequence specificities for gp120 binding and anti-HIV activity are not identical.
利用诱变寡核苷酸引物，通过聚合酶链反应或限制性位点消除操作，对编码新型HIV灭活蛋白蓝藻素-N（CV-N）和FLAG-蓝藻素-N（F-CV-N）的DNA构建体进行定点诱变。突变构建体在大肠杆菌中表达，并对重组蛋白产物进行与HIV表面包膜糖蛋白gp120结合的测试以及针对感染性HIV的抗病毒活性测试。结果显示，CV-N、F-CV-N及各种突变体的相对gp120结合亲和力与抗HIV活性之间总体具有非常高的相关性（r2>0.9）。然而，一个异常值是一种突变体，它缺乏CV-N中通常存在的一个内部二硫键，显示出适度的gp120结合，但对HIV没有抗病毒活性。这些发现与以下观点一致，即gp120结合是CV-N及相关蛋白的HIV灭活活性的必要但非充分条件；gp120结合和抗HIV活性的序列特异性并不相同。
