Research articles
ScienceAsia 49 (2023):ID 910-917 |doi:
10.2306/scienceasia1513-1874.2023.101
Functional expression of Escherichia coli-derived recombinant
plastocyanin from Canna indica L. and its anti-HIV-1 activities
Apanchanid Thepouyporna,*, Warunya Woradulayapinijb, Chanita Napaswadc, Onrapak Reamtongd, Pimpikar Kanchanadumkernge, Chanpen Wiwatf
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 ?g/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 ?g/ml, respectively. This study demonstrated
that recombinant Pc could be used as an anti-HIV agent.
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| a |
Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok
10400 Thailand |
| b |
Thammasat University Research Unit in Mechanisms of Drug Action and Molecular Imaging, Drug Discovery and
Development Center, Office of Advanced Science and Technology, Thammasat University, Pathumthani 12120
Thailand |
| c |
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400 Thailand |
| d |
Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University,
Bangkok 10400 Thailand |
| e |
Department of Food Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok 10400 Thailand |
| f |
Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok 10400 Thailand |
* Corresponding author, E-mail: apanchanid.the@mahidol.ac.th
Received 27 Nov 2022, Accepted 5 Dec 2023
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