Expression of eukaryotic peptides in plant plastids
Constructs and methods are provided for expressing peptides derived from eukaryotic organisms in plant plastids. Constructs have a promoter functional in a plant plastid, a DNA sequence encoding a peptide derived from an eukaryotic organism and a transcription termination region. Other elements include a selectable marker for selection of plant cells comprising a plastid expressing the marker and DNA regions of homology to the genome of the plastid and optionally a ribosome binding site joined to the promoter. By methods using such constructs high levels of eukaryotic peptides, such as mammalian proteins, are produced in a plant cell by growing plant cells under conditions whereby the DNA encoding sequences are expressed to produce eukaryotic peptide in said plastid.
1. A method for producing a non-methionine N-terminus protein in a plastid, wherein said method comprises:
transforming a plastid with a construct comprising, as operably joined components in the 5′ to 3′ direction of transcription,
(a) a promoter functional in a plastid,
(b) a DNA sequence encoding a cleavable ubiquitin peptide,
(c) a DNA sequence encoding a protein of interest,
(d) a transcription termination region, and
(e) at least two DNA regions of homology to the genome of said plastid flanking said operably joined components of (a), (b), (c) and (d); and
growing a plant cell comprising said transformed plastid under suitable conditions for expression of said protein of interest and said cleavable ubiquitin sequence in said plastid.
2. The method according to claim 1 , wherein said construct further comprises (f) a gene encoding a selectable marker for selection of a plant cell comprising a plastid expressing said marker.
3. The method according to claim 1 , wherein said construct further comprises (g) a ribosome binding site joined to said promoter (a).
4. The method according to claim 3 , wherein said ribosome binding site is derived from a leader sequence selected from the group consisting of a plastid leader sequence, a bacterial leader sequence and a bacteriophage leader sequence.
5. The method according to claim 3 , wherein said ribosome binding site is selected from the group consisting of the binding site of the T7 bacteriophage gene 10 leader and the rbcL RBS.
6. A plastid having a protein of interest produced according to the method of claim 1 .
7. The plastid according to claim 6 wherein said construct further comprises (g) a T7 bacteriophage gene 10 leader ribosome binding site that is operably joined to a plastid promoter, and wherein said protein of interest comprises at least about 1.0% of total soluble protein in said plastid.
8. The plastid according to claim 7 wherein said protein of interest comprises at least about 7.0% of total soluble protein in said plastid.
9. A plastid comprising stably incorporated and operably joined components (a), (b), (c), and (d) contained within said DNA regions of homology of the construct of claim 1 within its genome.
10. A plant cell comprising a plastid comprising stably incorporated and operably joined components (a), (b), (c), and (d) contained within said DNA regions of homology of the construct of claim 1 within its genome.
11. A plant cell comprising a transformed plastid produced according to the method of claim 1 .
12. A plant, plant seed or plant part each comprising a plastid according to claim 6 .
13. A plant, plant seed or plant part each comprising a plastid according to claim 9 .
14. A plant, plant seed or plant part each comprising a plant cell according to claim 10 .
15. A plant, plant seed or plant part each comprising a plant cell according to claim 11 .
16. The method according to claim 1 wherein said protein of interest is folded with the correct number of disulfide bonds.
17. The method according to claim 1 wherein said protein of interest is hGH.
18. The method according to claim 1 wherein said protein of interest is bioactive when isolated from said plant cell.
19. The method according to claim 18 wherein said protein of interest is hGH.
20. A plastid comprising:
operably joined components (a), (b), (c), and (d) contained within said DNA regions of homology of a construct according to claim 1 stably integrated into its genome; and
a non-methionine N-terminus protein.
21. A plant cell comprising a plastid having:
operably joined components (a), (b), (c), and (d) contained within said DNA regions of homology of a construct according to claim 1 stably integrated into its genome; and
a non-methionine N-terminus protein.