McCabe, Matthew and Klaas, Manfred and Gonzalez-Rabade, Nuria and Poage, Miranda and Badillo-Corona, Jesus A. and Zhou, Fei and Karcher, Daniel and Bock, Ralph and Gray, John C. and Dix, Philip (2008) Plastid transformation of high-biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV-1) p24 antigen. Plant Biotechnology Journal, 6 (9). pp. 914-929. ISSN 1467-7644

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Abstract

Chloroplast transformation of the high-biomass tobacco variety Maryland Mammoth has been assessed as a production platform for the human immunodeficiency virus type 1 (HIV-1) p24 antigen. Maryland Mammoth offers the prospect of higher yields of intact functional protein per unit floor area of contained glasshouse per unit time prior to flowering. Two different transformation constructs, pZSJH1p24 (for the insertion of a native p24 cDNA between the rbcL and accD genes) and pZF5 (for the insertion of a chloroplast-codon-optimized p24 gene between trnfM and trnG) were examined for the production of p24. Plants generated with construct pZSJH1p24 exhibited a normal green phenotype, but p24 protein accumulated only in the youngest leaves (up to approximately 350 microg/g fresh weight or approximately 2.5% total soluble protein) and was undetectable in mature leaves. In contrast, some of the plants generated with pZF5 exhibited a yellow phenotype (pZF5-yellow) with detectable p24 accumulation (up to approximately 450 microg/g fresh weight or approximately 4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5-yellow leaves was reduced by approximately 40%. The pZF5-yellow phenotype was associated with recombination between native and introduced direct repeat sequences of the rbcL 3' untransformed region in the plastid genome. Chloroplast-expressed p24 was recognized by a conformation-dependent monoclonal antibody to p24, and p24 protein could be purified from pZF5-yellow leaves using a simple procedure, involving ammonium sulphate precipitation and ion-exchange chromatography, without the use of an affinity tag. The purified p24 was shown to be full length with no modifications, such as glycosylation or phosphorylation, using N-terminal sequencing and mass spectrometry.

Item Type:	Article
Keywords:	chloroplast transformation; high-biomass tobacco; human immunodeficiency virus type 1 (HIV-1) p24 antigen; HIV;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	7377
Identification Number:	https://doi.org/10.1111/j.1467-7652.2008.00365.x
Depositing User:	Prof. Philip J. Dix
Date Deposited:	25 Aug 2016 15:57
Journal or Publication Title:	Plant Biotechnology Journal
Publisher:	Blackwell Publishing Ltd
Refereed:	Yes
URI:	http://onlinelibrary.wiley.com
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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