Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins
Version of Record online: 18 OCT 2007
Plant Biotechnology Journal
Volume 6, Issue 1, pages 46–61, January 2008
How to Cite
Bally, J., Paget, E., Droux, M., Job, C., Job, D. and Dubald, M. (2008), Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins. Plant Biotechnology Journal, 6: 46–61. doi: 10.1111/j.1467-7652.2007.00298.x
- Issue online: 18 OCT 2007
- Version of Record online: 18 OCT 2007
- Received 30 May 2007; revised 7 August 2007; accepted 9 August 2007.
The authors have provided the following supplementary material, which can be accessed alongside the article at http://www.blackwell-synergy.com. It contains two figures and two tables.
Figure S1 Southern blot analyses. Total DNA extracted from transformants expressing the short (PhoA-S) (pCLT515, lanes 1–4) or long (PhoA-L) (pCLT516, lanes 5–8) form of alkaline phosphatase was analysed by Southern blotting with the rbcL (a) or phoA (b) probe. The analysis was conducted with total DNA from generation T0 in lanes 1 and 5, from one T1 progeny in lanes 2 and 6, and from two T2 progenies in lanes 3, 4, 7 and 8. WT, wild-type sample. The samples were digested by the restriction enzyme HindIII, separated by agarose gel electrophoresis, blotted on to a nylon membrane and hybridized with 32P-labelled probes covering the rbcL and phoA coding regions, as depicted in Figure 1. A unique DNA fragment at around 7 kb is detected in all the lanes using the rbcL probe, confirming the integration of the transgenes at the predicted locus between the rbcL and accD plastome genes (arrow). The phoA probe reveals a major band at around 8 kb for the transgenic samples (arrow), testifying to the presence of the phoA gene in the plastid genome of these lines. A lower molecular weight band at around 3 kb is also visible in all lanes, except for wild-type tobacco, which is always in the same ratio with the predominant band at 8 kb. This unexpected fragment might correspond to recombinations between introduced and resident tobacco plastid sequences, as shown by Eibl et al., (1999). Such events have been detected in the first tobacco plastid transformants generated by Svab and Maliga (1993) using the aadA selectable marker gene placed under the control of tobacco plastid expression elements. Finally, no difference is observed in this pattern between T0, T1 and T2 generations, showing that the introduced transgenes are stably inherited.
Figure S2 Characterization of chloroplast alkaline phosphatase (PhoA) after native polyacrylamide gel electrophoresis. (a) PhoA activity after protein transfer on to nitrocellulose membrane and incubation with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chloride (BCIP/NBT) substrate. (b) Corresponding Western blot analysis using antibodies directed against PhoA. Twenty micrograms of tobacco leaf proteins were loaded from wild-type extracts (wt) and independent extracts (1 and 2) from transgenic lines generated with vectors pCLT515 (PhoA-S) and pCLT516 (PhoA-L). Two micrograms of PhoA from Escherichia coli (standard) were loaded as control. MWM, molecular weight marker from Bio-Rad (dual colour). These data show that recombinant PhoA is expressed in transgenic tobacco as an active homodimer (the monomeric unfolded form is inactive; Sone et al., 1997).
Table S1 Peptide sequences corresponding to recombinant alkaline phosphatase expressed in tobacco chloroplasts. Spot numbers correspond to Figure 3 in the main text
Table S2 Peptide sequences corresponding to recombinant alkaline phosphatase expressed in bacterial strains BL21 and BL21 trxB/gor. Spot numbers correspond to Figure 4 in the main text
This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/10.1111/j.1467-7652.2007.00298.x
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