Functional expression of Escherichia coli-derived recombinant plastocyanin from Canna indica L. and its anti-HIV-1 activities

Abstract

Human immunodeficiency virus (HIV) infection has been a risk to public health due to its long-life treatment. The current medications were limited due to their single therapeutic targets, drug resistance, and adverse side effects. The discovery and study of novel anti-HIV agents are necessary. From our previous study, Canna indica L. leaf extract showed anti-HIV activities with plastocyanin as a potential active compound. In this study, the plastocyanin gene of Canna indica L. was cloned using the cDNA library established, the leaf extract mRNA and expressed in pET28(+); E. coli.DE3(BL21). The 408-bp plastocyanin gene produced 14-kDa recombinant protein plastocyanin (Pc) including a histidine tag (his-tag). Pc was characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography coupled with mass spectrometry and further purified using N-terminal 6xHis-tagged fusion binding through column affinity chromatography. As expected, Pc was determined to be approximately 14 kDa on SDS-PAGE. To study the anti-HIV activity of Pc and its primary arrangement, the his-tag was removed by thrombin cleavage. Further, Pc and its product cleaved using thrombin (Pc/T) were evaluated for their cytotoxic and anti-HIV-1 activities using the syncytium reduction and anti-HIV reverse transcriptase assays. The syncytium reduction assay revealed that Pc and Pc/T exhibited anti-HIV activity at EC50 of 38.54 and 128.84 µg/ml; with calculated therapeutic indices of >6.49, active and >1.94, active; respectively. Both Pc and Pc/T also exhibited anti-HIV reverse transcriptase inhibitory activity at IC50 of 6.55 and 4.89 µg/ml, respectively. This study demonstrated that recombinant Pc could be used as an anti-HIV agent.