Departmental Papers (Dental)
Expression and Characterization of Antimicrobial Peptides Retrocyclin-101 and Protegrin-1 in Chloroplasts to Control Viral and Bacterial Infections
Date of this Version
Plant Biotechnology Journal
Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC101 and PG1 accumulated up to 32%–38% and 17%~26% of the total soluble protein. Both RC101 and PG1 were cleaved from GFP by corresponding proteases in vitro, and Factor Xa–like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6-fold higher yield of RC101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to tobacco mosaic virus infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further preclinical studies.
This is the peer reviewed version of the following article: [Lee, S.-B., Li, B., Jin, S., & Daniell, H. (2011). Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections. Plant Biotechnology Journal, 9(1), 100–115. http://doi.org/10.1111/j.1467-7652.2010.00538.x], which has been published in final form at [Link to final article using the 10.1111/j.1467-7652.2010.00538.x]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving [link to http://olabout.wiley.com/WileyCDA/Section/id-820227.html#terms].
antimicrobial peptide, chloroplast genetic engineering, molecular farming, plant-made biopharmaceuticals
Lee, S., Li, B., Jin, S., & Daniell, H. (2011). Expression and Characterization of Antimicrobial Peptides Retrocyclin-101 and Protegrin-1 in Chloroplasts to Control Viral and Bacterial Infections. Plant Biotechnology Journal, 9 (1), 100-115. http://dx.doi.org/10.1111/j.1467-7652.2010.00538.x
Date Posted: 01 March 2022
This document has been peer reviewed.
At the time of publication, author Henry Daniell was affiliated with the University of Central Florida. Currently, he is a faculty member at the School of Dental Medicine at the University of Pennsylvania.