TY - JOUR
T1 - Plastid transformation of high-biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV-1) p24 antigen
AU - McCabe, Matthew S.
AU - Klaas, Manfred
AU - Gonzalez-Rabade, Nuria
AU - Poage, Miranda
AU - Badillo-Corona, Jesus A.
AU - Zhou, Fei
AU - Karcher, Daniel
AU - Bock, Ralph
AU - Gray, John C.
AU - Dix, Philip J.
PY - 2008/12
Y1 - 2008/12
N2 - 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 μg/g fresh weight or ∼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 μg/g fresh weight or ∼4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5-yellow leaves was reduced by ∼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.
AB - 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 μg/g fresh weight or ∼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 μg/g fresh weight or ∼4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5-yellow leaves was reduced by ∼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.
KW - Chloroplast transformation
KW - High-biomass tobacco
KW - Human immunodeficiency virus type 1 (HIV-1) p24 antigen
UR - http://www.scopus.com/inward/record.url?scp=55949087097&partnerID=8YFLogxK
U2 - 10.1111/j.1467-7652.2008.00365.x
DO - 10.1111/j.1467-7652.2008.00365.x
M3 - Artículo
SN - 1467-7644
VL - 6
SP - 914
EP - 929
JO - Plant Biotechnology Journal
JF - Plant Biotechnology Journal
IS - 9
ER -