Title

Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections

Authors

Authors

S. B. Lee; B. C. Li; S. X. Jin;H. Daniell

Comments

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Abbreviated Journal Title

Plant Biotechnol. J.

Keywords

antimicrobial peptide; chloroplast genetic engineering; molecular; farming; plant-made biopharmaceuticals; TRANSGENIC TOBACCO CHLOROPLASTS; ANTHRAX LETHAL TOXIN; PROTECTIVE; ANTIGEN; BACILLUS-ANTHRACIS; THYLAKOID LUMEN; DISULFIDE BONDS; VACCINE; RESISTANCE; PROTEINS; DEFENSINS; Biotechnology & Applied Microbiology; Plant Sciences

Abstract

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%similar to 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.

Journal Title

Plant Biotechnology Journal

Volume

9

Issue/Number

1

Publication Date

1-1-2011

Document Type

Article

Language

English

First Page

100

Last Page

115

WOS Identifier

WOS:000285012600010

ISSN

1467-7644

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