U.S. patent number RE44,266 [Application Number 11/197,730] was granted by the patent office on 2013-06-04 for expression of eukaryotic polypeptides in chloroplasts.
This patent grant is currently assigned to The Scripps Research Institute. The grantee listed for this patent is Stephen P. Mayfield. Invention is credited to Stephen P. Mayfield.
United States Patent |
RE44,266 |
Mayfield |
June 4, 2013 |
Expression of eukaryotic polypeptides in chloroplasts
Abstract
The present invention relates to a gene expression system in
eukaryotic and prokaryotic cells, preferably plant cells and intact
plants. In particular, the invention relates to an expression
system having a RB47 binding site upstream of a translation
initiation site for regulation of translation mediated by binding
of RB47 protein, a member of the poly(A) binding protein family.
Regulation is further effected by RB60, a protein disulfide
isomerase. The expression system is capable of functioning in the
nuclear/cytoplasm of cells and in the chloroplast of plants.
Translation regulation of a desired molecule is enhanced
approximately 100 fold over that obtained without RB47 binding site
activation.
Inventors: |
Mayfield; Stephen P. (Cardiff,
CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Mayfield; Stephen P. |
Cardiff |
CA |
US |
|
|
Assignee: |
The Scripps Research Institute
(La Jolla, CA)
|
Family
ID: |
26712647 |
Appl.
No.: |
11/197,730 |
Filed: |
January 16, 1998 |
PCT
Filed: |
January 16, 1998 |
PCT No.: |
PCT/US98/00840 |
371(c)(1),(2),(4) Date: |
July 13, 1999 |
PCT
Pub. No.: |
WO98/31823 |
PCT
Pub. Date: |
July 23, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10310587 |
Dec 4, 2002 |
Re. 39350 |
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60035955 |
Jan 17, 1997 |
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60069400 |
Dec 12, 1997 |
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Reissue of: |
09341550 |
Jul 13, 1999 |
6156517 |
Dec 5, 2000 |
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Current U.S.
Class: |
435/69.1;
435/419; 435/375; 435/320.1; 435/468; 536/23.1 |
Current CPC
Class: |
C12N
15/63 (20130101); C12P 21/02 (20130101); C12N
9/90 (20130101); C07K 14/405 (20130101); C12N
15/8214 (20130101); C07K 16/1282 (20130101); C12N
15/8216 (20130101); C12N 15/8258 (20130101); C07K
14/415 (20130101); C12N 15/70 (20130101); C12N
15/79 (20130101) |
Current International
Class: |
C12P
21/02 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 97/41228 |
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Nov 1997 |
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WO |
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WO 01/64929 |
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Feb 2001 |
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WO |
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Other References
Blowers et al., The Plant Cell, vol. 1,(1989) pp. 123-132. cited by
examiner .
Tavladoraki et al, Nature, vol. 366 (1993) pp. 469-472. cited by
examiner .
Sidorov et al., "Stable chloroplast transformation in potato: use
of green fluorescent protein as a plastid marker." The Plant
Journal, vol. 19 (2) pp. 209-216 (1999). cited by applicant .
Daniell et al., "Transient Foreign Gene Expression in Chloroplasts
of Cultured Tobacco Cells After Biolistic Delivery of Chloroplast
Vectors", Proc. Natl. Acad. Sci., 87:88-92 (1990). cited by
applicant .
Danon and Mayfield, "Light Regulated Translational Activators:
Identification of Chloroplast Gene Specific mRNA Binding Proteins"
The EMBO Journal, 10(13):3993-4001 (1991). cited by applicant .
Danon and Mayfield, "ADP-Dependent Phosphorylation Regulates
RNA-Binding in vitro: Implications in Light-Modulated Translation",
The EMBO Journal, 13(9):2227-2235 (1994). cited by applicant .
Hauser et al., "Translational Regulation of Chloroplast Genes", The
Journal of Biological Chemistry, 271(3):1486-1497 (1996). cited by
applicant .
Kim and Mayfield, "Protein Disulfide Isomerase as a Regulator of
Chloroplast Translational Activation", Science, 278:1954-1957
(1997). cited by applicant .
Kin-Ying et al., "Introduction and Expression of Foreign DNA in
Isolated Spinach Chloroplasts by Electroporation" The Plant
Journal, 10(4):737-743 (1996). cited by applicant .
Le et al., "Triticum Aestivum Poly(A)-Binding Protein (wheatpab)
mRNA, Complete Cds", Database EBI (online), p. 1-2 (1996). cited by
applicant .
Pihlajaniemi et al., "Molecular Cloning of the .beta.-Subunit of
Human Prolyl 4-Hygroxylase. This Subunit and Protein Disulphide
Isomerase are Products of the Same Gene" The EMBO Journal,
6(3):643-649 (1987). cited by applicant .
Yohn et al., "A Poly(A) Binding Protein Functions in the
Chloroplast as a Message-Specific Translation Factor", Proc. Natl.
Acad. Sci., 95:2238-2243 (1998). cited by applicant .
Yohn et al., "Translation of the Chloroplast psbA mRNA Requires the
Nuclear-Encoded Poly(A)-Binding Protein, RB47", The Journal of Cell
Biology, 142:435-442 (1998). cited by applicant .
Ohtani et al., "Location and nucleotide sequence of a tobacco
chloroplast DNA segment capable of replication in yeast", Mol. Gen.
Genet. 195:1-4, 1984. cited by applicant .
Rochaix et al., "Construction and Characterization of Autonomously
Replicating Plasmids in the Green Unicellular Alga Chlamydomonas
reinhardii", Cell, 36:925-931, 1984. cited by applicant .
Uchimiya et al., "Molecular Cloning of Tobacco Chromosomal and
Chloroplast DNA Segments Capable of Replication in Yeast", Mol.
Gen. Genet. 192:1-4, 1983. cited by applicant .
Danon et al., EMBO J., vol. 10, 1991, pp. 3993-4001. cited by
applicant .
Danon et al., EMBO J., vol. 13, 1994, pp. 2227-2235. cited by
applicant.
|
Primary Examiner: Ketter; Jim
Attorney, Agent or Firm: DLA Piper LLP (US)
Government Interests
This invention was made with government support under Contract No.
GM 54659 by the National Institutes of Health and Contract No.
DO-FG03-93ER20116 by the U.S. Department of Energy. The government
has certain rights in the invention.
Parent Case Text
.[.This is a stage application filed under 35 USC 371, of
PCT/US98/00840, filed Jan. 16, 1998. This application claims
benefit of provisional No. 60/035,955 filed Jan. 17, 1997 and
provisional appln No. 60/069,400 filed Dec. 12, 1997..].
.Iadd.Notice: More than one reissue application has been filed for
the reissue of U.S. Pat. No. 6,156,517. The reissue applications
are U.S. application Ser. No. 11/197,730 (the present application)
and Ser. No. 10/310,587, issued on Oct. 17, 2006 as U.S. Pat. No.
Re. 39,350, all of which are continuation reissues of U.S. Pat. No.
6,156,517. This application is a continuation reissue application
of U.S. application Ser. No. 10/310,587, filed Dec. 4, 2002 now
U.S. Pat. No. Re. 39,350, which is a reissue application of U.S.
Pat. No. 6,156,517, issued Dec. 5, 2000, which is a national stage
application filed under 35 U.S.C. .sctn.371, of PCT/US98/00840,
filed Jan. 16, 1998. This application claims benefit of priority to
U.S. Provisional Application Ser. No. 60/035,955, filed Jan. 17,
1997 and U.S. Provisional Patent Application Ser. No. 60/069,400,
filed Dec. 12, 1997..Iaddend.
Claims
What is claimed is:
.[.1. An expression cassette for expression of a desired molecule,
which cassette comprises: a) an RB47 binding site nucleotide
sequence upstream of a restriction endonuclease site for insertion
of a desired coding sequence to be expressed; and b) a nucleotide
sequence encoding a polypeptide which binds RB47 binding
site..].
.[.2. The expression cassette of claim 1 further comprising a
promoter sequence operably linked to and positioned upstream of the
RB47 binding site nucleotide sequence..].
.[.3. The expression cassette of claim 2 wherein the promoter
sequence is derived from a psbA gene..].
.[.4. The expression cassette of claim 3 wherein the coding
sequence is heterologous to the psbA gene..].
.[.5. The expression cassette of claim 1 wherein the cassette
comprises a plasmid or virus..].
.[.6. The expression cassette of claim 1 further comprising and
operably linked thereto a nucleotide sequence encoding RB60..].
.[.7. The expression cassette of claim 1 wherein the RB47 binding
polypeptide is selected from the group consisting of RB47, RB47
precursor and a histidine-modified RB47..].
.[.8. An expression cassette for expression of a desired molecule,
which cassette comprises: a) an RB47 binding site nucleotide
sequence upstream of a restriction endonuclease site for insertion
of a desired coding sequence to be expressed; and b) a nucleotide
sequence encoding a polypeptide which regulates the binding of RB47
to the RB47 binding site..].
.[.9. The expression cassette of claim 8 wherein the regulatory
polypeptide is RB60..].
.[.10. A method of screening for agonists or antagonists of RB47
binding to RB47 binding site, the method comprising the steps: a)
providing a cell expression system containing: 1) a promoter
sequence; 2) a RB47 binding site sequence; 3) a coding sequence for
an indicator polypeptide; and 4) a polypeptide which binds to the
RB47 binding site sequence; b) introducing an antagonist or agonist
into the cell; and c) detecting the amount of indicator polypeptide
expressed in the cell..].
.[.11. A method of screening for agonists or antagonists of RB60 in
regulating RB47 binding to RB47 binding site, the method comprising
the steps: a) providing an expression system in a cell containing:
1) a promoter sequence; 2) a RB47 binding site sequence; 3) a
coding sequence for an indicator polypeptide; 4) a polypeptide
which binds to the RB47 binding site sequence; and 5) a RB60
polypeptide; b) introducing an agonist or antagonist into the cell;
and c) detecting the amount of indicator polypeptide expressed in
the cell..].
.[.12. An isolated nucleotide sequence encoding RB47..].
.[.13. An isolated nucleotide sequence encoding a
histidine-modified RB47..].
.[.14. An isolated nucleotide sequence encoding RB47
precursor..].
.[.15. The nucleotide sequence of claim 12 from nucleotide position
197 to 1402 in FIGS. 1A-1B and SEQ ID NO 5..].
.[.16. The nucleotide sequence of claim 13 from nucleotide position
1 to 1269 in FIGS. 5A-5B and SEQ ID NO 14..].
.[.17. The nucleotide sequence of claim 14 shown in from nucleotide
position 197 to 2065 in FIGS. 1A-1C and SEQ ID NO 5..].
.[.18. An expression cassette comprising the nucleotide sequence of
claim 12, 13 or 14..].
.[.19. An isolated nucleotide sequence encoding RB60..].
.[.20. The nucleotide sequence of claim 18 from nucleotide position
16 to 1614 in FIGS. 2A-2B and SEQ ID NO 10..].
.[.21. An expression cassette comprising the nucleotide sequence of
claim 19..].
.[.22. An expression system comprising a cell transformed with the
expression cassette of claim 1..].
.[.23. The expression system of claim 22 wherein the cell is a
plant cell..].
.[.24. The expression system of claim 23 wherein the plant cell
endogenously expresses RB47..].
.[.25. The expression system of claim 23 wherein the plant cell
endogenously expresses RB60..].
.[.26. The expression system of claim 23 wherein the plant cell
endogenously expresses RB47 and RB60..].
.[.27. The expression system of claim 22 wherein the cell is a
eukaryotic cell..].
.[.28. The expression system of claim 22 wherein the cell is a
prokaryotic cell..].
.[.29. The expression system of claim 22 further comprising the
expression cassette of claim 21..].
.[.30. An expression system comprising a cell transformed with the
expression cassette of claim 8..].
.[.31. The expression system of claim 29 further comprising the
expression cassette of claim 18..].
.[.32. A cell stably transformed with the expression cassette of
claim 18..].
.[.33. A cell stably transformed with the expression cassette of
claim 21..].
.[.34. A cell stably transformed with the expression cassette of
claims 18 and 21..].
.[.35. The expression cassette of claim 1 further comprising an
inserted desired coding sequence..].
.[.36. An expression system comprising a cell transformed with the
expression cassette of claim 35, wherein the coding sequence is
expressed forming the desired molecule upon activation of the RB47
binding site with RB47..].
.[.37. The expression system of claim 36 wherein the cell is a
plant cell endogenously expressing RB47..].
.[.38. The expression system of claim 36 wherein the cell is stably
transformed with the expression cassette of claim 21..].
.[.39. An expression system comprising a cell transformed with an
expression cassette comprising a promoter sequence, a RB47 binding
site sequence, a desired coding sequence for a molecule, and a
nucleotide sequence for encoding a polypeptide which binds RB47
binding site, wherein all sequences are operably linked..].
.[.40. A method of preparing a desired recombinant molecule wherein
the method comprises cultivating the expression system of claim
36..].
.[.41. A method of preparing a desired recombinant molecule wherein
the method comprises cultivating the expression system of claim
39..].
.[.42. A method for expressing a desired coding sequence
comprising: a) forming an expression cassette by operably linking:
1) a promoter sequence; 2) a RB47 binding site sequence; 3) a
desired coding sequence; and 4) a nucleotide sequence encoding a
polypeptide which binds RB47 binding site; and b) introducing the
expression cassette into a cell..].
.[.43. The method of claim 42 wherein the cell is a plant cell
endogenously expressing RB47..].
.[.44. The method of claim 42 wherein the cell is a plant cell
endogenously expressing RB60..].
.[.45. The method of claim 42 further comprising inducing
expression with a promoter inducer molecule..].
.[.46. The method of claim 45 wherein the promoter inducer molecule
is IPTG..].
.[.47. The method of claim 42 wherein the cell is transformed with
the expression cassette of claim 21..].
.[.48. A method for expressing a desired coding sequence
comprising: a) forming an expression cassette by operably linking:
1) a promoter sequence; 2) a RB47 binding site sequence; and 3) a
desired coding sequence; and b) introducing the expression cassette
into a plant cell endogenously expressing RB47..].
.[.49. The method of claim 48 wherein the expression cassette
further comprises a nucleotide sequence encoding RB60..].
.[.50. A method for the regulated production of a recombinant
molecule from a desired coding sequence in a cell, wherein the cell
contains the expression cassette of claim 34, wherein expression of
the coding sequence is activated by RB47 binding to the RB47
binding site thereby producing the recombinant molecule..].
.[.51. A method of forming an expression cassette by operably
linking: a) a RB47 binding site sequence; b) a cloning site for
insertion of a desired coding sequence downstream of the RB47
binding site sequence; and c) a nucleotide sequence encoding a
polypeptide which binds the RB47 binding site..].
.[.52. The method of claim 51 further comprising a promoter
sequence operably linked upstream to the RB47 binding site
sequence..].
.[.53. The method of claim 51 further comprising a desired coding
sequence inserted into the insertion site..].
.[.54. An article of manufacture comprising a packaging material
and contained therein in a separate container the expression
cassette of claim 1, wherein the expression cassette is useful for
expression of a desired coding sequence, and wherein the packaging
material comprises a label which indicates that the expression
cassette can be used for expressing a desired coding sequence when
the RB47 binding site is activated by RB47..].
.[.55. The article of manufacture of claim 54 further comprising in
a separate container the expression cassette of claim 18..].
.[.56. The article of manufacture of claim 54 further comprising in
a separate container the expression cassette of claim 21..].
.[.57. An article of manufacture comprising a packaging material
and contained therein in a separate container the expression system
of claim 22, wherein the expression system is useful for expression
of a desired coding sequence, and wherein the packaging material
comprises a label which indicates that the expression system can be
used for expressing a desired coding sequence when the RB47 binding
site is activated by RB47..].
.[.58. An article of manufacture comprising a packaging material
and contained therein in a separate container the stably
transformed cell of claim 32, wherein the cell is useful as an
expression system, and wherein the packaging material comprises a
label which indicates that the expression system can be used for
expressing a desired coding sequence when the RB47 binding site is
activated by RB47..].
.[.59. An article of manufacture comprising a packaging material
and contained therein in a separate container the stably
transformed cell of claim 33, wherein the cell is useful as an
expression system, and wherein the packaging material comprises a
label which indicates that the expression system can be used for
expressing a desired coding sequence when the RB47 binding site is
activated by RB47 and regulated by RB60..].
.[.60. An article of manufacture comprising a packaging material
and contained therein in a separate container the stably
transformed cell of claim 34, wherein the cell is useful as an
expression system, and wherein the packaging material comprises a
label which indicates that the expression system can be used for
expressing a desired coding sequence when the RB47 binding site is
activated by RB47 and regulated by RB60..].
.[.61. An article of manufacture comprising a packaging material
and contained therein in a separate container the expression
cassette of claim 2, wherein the expression cassette is useful for
expression of a RNA transcript, and wherein the packaging material
comprises a label which indicates that the expression cassette can
be used for producing in vitro a RNA transcript when the RB47
binding site is activated by RB47..].
.[.62. The article of manufacture of claim 61 wherein the promoter
sequence is selected from the group consisting of T3 and T7
promoters..].
.[.63. The article of manufacture of claim 61 further comprising in
separate containers a polymerase, a buffer and each of four
ribonucleotides, reagents for in vitro RNA transcription..].
.Iadd.64. A chloroplast expression cassette comprising the
following components in the 5' to 3' direction of transcription: a)
a promoter functional in a chloroplast; b) a 5' leader sequence
comprising a 5' untranslated region (UTR), wherein the 5' UTR
comprises an RB47 binding site; and c) a DNA sequence encoding a
heterologous protein of interest..Iaddend.
.Iadd.65. The chloroplast expression cassette of claim 64, wherein
the DNA sequence encodes a vertebrate polypeptide..Iaddend.
.Iadd.66. The chloroplast cassette of claim 64, wherein the DNA
sequence encodes a mammalian polypeptide..Iaddend.
.Iadd.67. The chloroplast expression cassette of claim 64, wherein
the polypeptide is an antibody..Iaddend.
.Iadd.68. The chloroplast cassette of claim 67, wherein the
polypeptide is a single chain antibody..Iaddend.
.Iadd.69. The chloroplast cassette of claim 64, wherein the
chloroplast is a plant chloroplast..Iaddend.
.Iadd.70. The chloroplast cassette of claim 64, wherein the
chloroplast is an algal chloroplast..Iaddend.
.Iadd.71. The chloroplast expression cassette of claim 64, wherein
the 5' leader sequence is a 5' untranslated region
(UTR)..Iaddend.
.Iadd.72. A cell containing the chloroplast expression cassette of
claim 64..Iaddend.
.Iadd.73. An alga or plant comprising a cell of claim
72..Iaddend.
.Iadd.74. An algal chloroplast comprising the expression cassette
of claim 64..Iaddend.
.Iadd.75. A micro-algae containing a chloroplast of claim
74..Iaddend.
.Iadd.76. The micro-algae of claim 75, wherein the algae is
Chlamydomonas reinhardtii..Iaddend.
.Iadd.77. The chloroplast expression cassette of claim 64, further
comprising a 3' UTR..Iaddend.
.Iadd.78. The expression cassette of claim 64, wherein the DNA
sequence encodes a eukaryotic protein..Iaddend.
.Iadd.79. The chloroplast expression cassette of claim 77, wherein
the promoter and the 5' leader sequence and the 3' UTR are of a
length which allows for replacement of a homologous gene by genetic
recombination upon introduction into the chloroplast
genome..Iaddend.
.Iadd.80. The chloroplast expression cassette of claim 79, wherein
the homologous gene to be replaced is a psbA gene..Iaddend.
.Iadd.81. A method for producing a non-plant, non-plastid protein
in a chloroplast, comprising: a) transforming a chloroplast of a
cell with a cassette of claim 64, and b) growing the cell
comprising the transformed chloroplast under conditions wherein the
DNA sequence is expressed to produce the protein in the
chloroplast..Iaddend.
.Iadd.82. A eukaryotic cell comprising a transformed chloroplast
producing a protein according to the method of claim
81..Iaddend.
.Iadd.83. The chloroplast expression cassette of claim 79, wherein
the protein is an antibody..Iaddend.
.Iadd.84. A microalgal chloroplast transformed with an expression
cassette of claim 65..Iaddend.
.Iadd.85. The microalgal chloroplast of claim 84, wherein said
microalga is Chlamydomonas reinhardtii..Iaddend.
.Iadd.86. A method for producing a heterologous eukaryotic protein
in a microalgal chloroplast, comprising: a) transforming a
microalgal chloroplast of a cell with a cassette of claim 65, and
b) growing the cell comprising the transformed microalgal
chloroplast under conditions wherein the DNA sequence is expressed
to produce the protein in the microalgal chloroplast..Iaddend.
Description
TECHNICAL FIELD
The invention relates to expression systems and methods for
expression of desired genes and gene products in cells.
Particularly, the invention relates to a gene encoding a RNA
binding protein useful for regulating gene expression in cells, the
protein binding site, a gene encoding a regulating protein
disulfide isomerase and methods and systems for gene expression of
recombinant molecules.
BACKGROUND
Expression systems for expression of exogenous foreign genes in
eukaryotic and prokaryotic cells are basic components of
recombinant DNA technology. Despite the abundance of expression
systems and their wide-spread use, they all have characteristic
disadvantages. For example, while expression in E. coli is probably
the most popular as it is easy to grow and is well understood,
eukaryotic proteins expressed therein are not properly modified.
Moreover, those proteins tend to precipitate into insoluble
aggregates and are difficult to obtain in large amounts. Mammalian
expression systems, while practical on small-scale protein
production, are more difficult, time-consuming and expensive than
in E. coli.
A number of plant expression systems exist as well as summarized in
U.S. Pat. No. 5,234,834, the disclosures of which are hereby
incorporated by reference. One advantage of plants or algae in an
expression system is that they can be used to produce
pharmacologically important proteins and enzymes on a large scale
and in relatively pure form. In addition, micro-algae have several
unique characteristics that make them ideal organisms for the
production of proteins on a large scale. First, unlike most systems
presently used to produce transgenic proteins, algae can be grown
in minimal media (inorganic salts) using sunlight as the energy
source. These algae can be grown in contained fermentation vessels
or on large scale in monitored ponds. Ponds of up to several acres
are routinely used for the production of micro-algae. Second,
plants and algae have two distinct compartments, the cytoplasm and
the chloroplast, in which proteins can be expressed. The cytoplasm
of algae is similar to that of other eukaryotic organisms used for
protein expression, like yeast and insect cell cultures. The
chloroplast is unique to plants and algae and proteins expressed in
this environment are likely to have properties different from those
of cytoplasmically expressed proteins.
The present invention describes an expression system in which
exogenous molecules are readily expressed in either prokaryotic or
eukaryotic hosts and in either the cytoplasm or chloroplast. These
beneficial attributes are based on the discovery and cloning of
components of translation regulation in plants as described in the
present invention.
Protein translation plays a key role in the regulation of gene
expression across the spectrum of organisms (Kozak, Ann. Rev. Cell
Biol., 8:197-225 (1992) and de Smit and Van Duin, Prog. Nucleic
Acid Res. Mol. Biol., 38:1-35 (1990)). The majority of regulatory
schemes characterized to date involve translational repression
often involving proteins binding to mRNA to limit ribosome
association (Winter et al., Proc. Natl. Acad. Sci., USA,
84:7822-7826 (1987) and Tang and Draper, Biochem., 29:4434-4439
(1990)). Translational activation has also been observed (Wulczyn
and Kahmann, Cell, 65:259-269 (1991)), but few of the underlying
molecular mechanisms for this type of regulation have been
identified. In plants, light activates the expression of many
genes. Light has been shown to activate expression of specific
chloroplast encoded mRNAs by increasing translation initiation
(Mayfield et al., Ann. Rev. Plant Physiol. Plant Mol. Biol.,
46:147-166 (1995) and Yohn et al., Mol. Cell Biol., 16:3560-3566
(1996)). Genetic evidence in higher plants and algae has shown that
nuclear encoded factors are required for translational activation
of specific chloroplast encoded mRNAs (Rochaix et al., Embo J.,
8:1013-1021 (1989), Kuchka et al., Cell, 58:869-876 (1989),
Girard-Bascou et al., Embo J., 13:3170-3181 (1994), Kim et al.,
Plant Mol. Biol., 127:1537-1545 (1994).
In the green algae Chlamydomonas reinhardtii, a number of nuclear
mutants have been identified that affect translation of single
specific mRNAs in the chloroplast, often acting at translation
initiation (Yohn et al., supra, (1996)). Mutational analysis of
chloroplast mRNAs has identified sequence elements within the 5'
untranslated region (UTR) of mRNAs that are required for
translational activation (Mayfield et al., supra, (1995), Mayfield
et al., J. Cell Biol., 127:1537-1545 (1994) and Rochaix, Ann. Rev.
Cell Biol., 8:1-28 (1992)), and the 5' UTR of a chloroplast mRNA
can confer a specific translation phenotype on a reporter gene in
vivo (Zerges and Rochaix, Mol. Cell Biol., 14:5268-5277 (1994) and
Staub and Maliga, Embo J., 12:601-606 (1993).
Putative translational activator proteins were identified by
purifying a complex of four proteins that binds with high affinity
and specificity to the 5' UTR of the chloroplast encoded psbA mRNA
[encoding the D1 protein, a major component of Photosystem II (PS
II)] (Danon and Mayfield, Embo J., 10:3993-4001 (1991)). Binding of
these proteins to the 5' UTR of psbA mRNA correlates with
translation of this mRNA under a variety of physiological (Danon
and Mayfield, id., (1991)) and biochemical conditions (Danon and
Mayfield, Science, 266:1717-1719 (1994) and Danon and Mayfield,
Embo J., 13:2227-2235 (1994)), and in different genetic backgrounds
(Yohn et al., supra, (1996)). The binding of this complex to the
psbA mRNA can be regulated in vitro in response to both redox
potential (Danon and Mayfield, Science, 266:1717-1719 (1994)) and
phosphorylation (Danon and Mayfield, Embo J., 13:2227-2235 (1994)),
both of which are thought to transduce the light signal to activate
translation of psbA mRNA. The 47 kDa member of the psbA RNA binding
complex (RB47) is in close contact with the RNA, and antisera
specific to this protein inhibits binding to the psbA mRNA in vitro
(Danon and Mayfield, supra, (1991)).
Although the translational control of psbA mRNA by RB47 has been
reported, the protein has not been extensively characterized and
the gene encoding RB47 has not been identified, cloned and
sequenced. In addition, the regulatory control of the activation of
RNA binding activity to the binding site by nuclear-encoded
trans-acting factors, such as RB60, have not been fully understood.
The present invention now describes the cloning and sequencing of
both RB47 and RB60. Based on the translation regulation mechanisms
of RB47 and RB60 with the RB47 binding site, the present invention
also describes a translation regulated expression system for use in
both prokaryotes and eukaryotes.
BRIEF DESCRIPTION OF THE INVENTION
The RB47 gene encoding the RB47 activator protein has now been
cloned and sequenced, and the target binding site for RB47 on
messenger RNA (mRNA) has now been identified. In addition, a
regulatory protein disulfide isomerase, a 60 kilodalton protein
referred to as RB60, has also been cloned, sequenced and
characterized. Thus, the present invention is directed to gene
expression systems in eukaryotic and prokaryotic cells based on
translational regulation by RB47 protein, its binding site and the
RB60 regulation of RB47 binding site activation.
More particularly, the present invention describes the use of the
RB47 binding site, i.e., a 5' untranslated region (UTR) of the
chloroplast psbA gene, in the context of an expression system for
regulating the expression of genes encoding a desired recombinant
molecule. Protein translation is effected by the combination of the
RB47 binding site and the RB47 binding protein in the presence of
protein translation components. Regulation can be further imposed
with the use of the RB60 regulatory protein disulfide isomerase.
Therefore, the present invention describes reagents and expression
cassettes for controlling gene expression by affecting translation
of a coding nucleic acid sequence in a cell expression system.
Thus, in one embodiment, the invention contemplates a RB47 binding
site sequence, i.e., a mRNA sequence, typically a mRNA leader
sequence, which contains the RB47 binding site. A preferred RB47
binding site is psbA mRNA. For use in expressing recombinant
molecules, the RB47 binding site is typically inserted 5' to the
coding region of the preselected molecule to be expressed. In a
preferred embodiment, the RB47 binding site is inserted into the 5'
untranslated region along with an upstream psbA promoter to drive
the expression of a preselected nucleic acid encoding a desired
molecule. In alternative embodiments, the RB47 binding site is
inserted into the regulatory region downstream of any suitable
promoter present in a eukaryotic or prokaryotic expression vector.
Preferably, the RB47 binding site is positioned within 100
nucleotides of the translation initiation site. In a further
aspect, 3' to the coding region is a 3' untranslated region (3'
UTR) necessary for transcription termination and RNA
processing.
Thus, in a preferred embodiment, the invention contemplates an
expression cassette or vector that contains a transcription unit
constructed for expression of a preselected nucleic acid or gene
such that upon transcription, the resulting mRNA contains the RB47
binding site for regulation of the translation of the preselected
gene transcript through the binding of the activating RB47 protein.
The RB47 protein is provided endogenously in a recipient cell
and/or is a recombinant protein expressed in that cell.
Thus, the invention also contemplates a nucleic acid molecule
containing the sequence of the RB47 gene. The nucleic acid molecule
is preferably in an expression vector capable of expressing the
gene in a cell for use in interacting with a RB47 binding site. The
invention therefore contemplates an expressed recombinant RB47
protein. In one embodiment, the RB47 binding site and RB47 encoding
nucleotide sequences are provided on the same genetic element. In
alternative embodiments, the RB47 binding site and RB47 encoding
nucleotide sequences are provided separately.
The invention further contemplates a nucleic acid molecule
containing the sequence encoding the 69 kilodalton precursor to
RB47. In alternative embodiments, the RB47 nucleic acid sequence
contains a sequence of nucleotides to encode a histidine tag. Thus,
the invention relates to the use of recombinant RB47, precursor
RB47, and histidine-modified RB47 for use in enhancing translation
of a desired nucleic acid.
The invention further contemplates a nucleic acid molecule
containing a nucleotide sequence of a polypeptide which regulates
the binding of RB47 to RB47 binding site. A preferred regulatory
molecule is the protein disulfide isomerase RB60. The RB60-encoding
nucleic acid molecule is preferably in an expression vector capable
of expressing the gene in a cell for use in regulating the
interaction of RB47 with a RB47 binding site. Thus, the invention
also contemplates an expressed recombinant RB60 protein. In one
embodiment, the RB47 binding site, RB47 encoding and RB60 encoding
nucleotide sequences are provided on the same genetic element. In
alternative embodiments, the expression control nucleotide
sequences are provided separately. In a further aspect, the RB60
gene and RB47 binding site sequence are provided on the same
construct.
The invention can therefore be a cell culture system, an in vitro
expression system or a whole tissue, preferably a plant, in which
the transcription unit is present that contains the RB47 binding
site and further includes a (1) transcription unit capable of
expressing RB47 protein or (2) the endogenous RB47 protein itself
for the purpose of enhancing translation of the preselected gene
having an RB47 binding site in the mRNA. Preferred cell culture
systems are eukaryotic and prokaryotic cells. Particularly
preferred cell culture systems include plants and more preferably
algae.
A further preferred embodiment includes (1) a separate
transcription unit capable of expressing a regulatory molecule,
preferably RB60 protein, or (2) the endogenous RB60 protein itself
for the purpose of regulating translation of the preselected gene
having an RB47 binding site in the mRNA. In an alternative
preferred embodiment, one transcription unit is capable of
expressing both the RB47 and RB60 proteins. In a further aspect,
the RB47 binding site sequence and RB60 sequence are provided on
the same construct.
In one aspect of the present invention, plant cells endogenously
containing RB47 and RB60 proteins are used for the expression of
recombinant molecules, such as proteins or polypeptides, through
activation of the RB47 binding in an exogenously supplied
expression cassette. Alternatively, stable plant cell lines
containing endogenous RB47 and RB60 are first generated in which
RB47 and/or RB60 proteins are overexpressed. Overexpression is
obtained preferably through the stable transformation of the plant
cell with one or more expression cassettes for encoding recombinant
RB47 and RB60. In a further embodiment, stable cell lines, such as
mammalian or bacterial cell lines, lacking endogenous RB47 and/or
RB60 proteins are created that express exogenous RB47 and/or
RB60.
Plants for use with the present invention can be a transgenic
plant, or a plant in which the genetic elements of the invention
have been introduced. Based on the property of controlled
translation provided by the combined use of the RB47 protein and
the RB47 binding site, translation can be regulated for any gene
product, and the system can be introduced into any plant species.
Similarly, the invention is useful for any prokaryotic or
eukaryotic cell system.
Methods for the preparation of expression vectors is well known in
the recombinant DNA arts, and for expression in plants is well
known in the transgenic plant arts. These particulars are not
essential to the practice of the invention, and therefore will not
be considered as limiting.
The invention allows for high level of protein synthesis in plant
chloroplasts and in the cytoplasm of both prokaryotic and
eukaryotic cells. Because the chloroplast is such a productive
plant organ, synthesis in chloroplasts is a preferred site of
translation by virtue of the large amounts of protein that can be
produced. This aspect provides for great advantages in agricultural
production of mass quantities of a preselected protein product.
The invention further provides for the ability to screen for
agonists or antagonists of the binding of RB47 to the RB47 binding
site using the expression systems as described herein. Antagonists
of the binding are useful in the prevention of plant
propagation.
Also contemplated by the present invention is a screening assay for
agonists or antagonists of RB60 in a manner analogous to that
described above for RB47. Such agonists or antagonists would be
useful in general to modify expression of RB60 as a way to regulate
cellular processes in a redox manner.
Kits containing expression cassettes and expression systems, along
with packaging materials comprising a label with instructions for
use, as described in the claimed embodiments are also contemplated
for use in practicing the methods of this invention.
Other uses will be apparent to one skilled in the art in light of
the present disclosures.
BRIEF DESCRIPTION OF DRAWINGS
In the figures forming a portion of this disclosure:
FIGS. 1A-1D show the complete protein amino acid residue sequence
of RB47 is shown from residues 1-623, together with the
corresponding nucleic acid sequence encoding the RB47 sequence,
from base 1 to base 2732. The nucleotide coding region is shown
from base 197-2065, the precursor form. The mature form is from
nucleotide position 197-1402. Also shown is the mRNA leader, bases
1-196, and poly A tail of the mRNA, bases 2066-2732. Both the
nucleotide and amino acid sequence are listed in SEQ ID NO 5.
FIGS. 2A-2B show the complete protein amino acid residue sequence
of RB60 is shown from residues 1-488, together with the
corresponding nucleic acid sequence from base 1 to base 2413, of
which bases 16-1614 encode the RB60 sequence. Both the nucleotide
and amino acid sequence are listed in SEQ ID NO 10.
FIGS. 3A-3C show the complete sequence of the psbA mRNA, showing
both encoded psbA protein amino acid residue sequence (residues
1-352) and the nucleic acid sequence as further described in
Example 3 is illustrated. Both the nucleotide and amino acid
sequence are listed in SEQ ID NO 13.
FIG. 4 is a schematic diagram of an expression cassette containing
on one transcription unit from 5' to 3', a promoter region derived
from the psbA gene for encoding the D1 protein from C. reinhardtii
further containing a transcription initiation site (TS), the RB47
binding site, a region for insertion of a foreign or heterologous
coding region, a RB47 coding region, a RB60 coding region, and the
3' flanking region containing transcription termination site (TS),
flanked by an origin of replication and selection marker.
Restriction endonuclease sites for facilitating insertion of the
independent genetic elements are indicated and further described in
Example 4A.
FIGS. 5A-5B show the nucleotide and amino acid sequence of the RB47
molecule containing a histidine tag, the sequences of which are
also listed in SEQ ID NO 14.
FIG. 6 is a schematic diagram of an expression cassette containing
on one transcription unit from 5' to 3', a promoter region derived
from the psbA gene for encoding the D1 protein from C. reinhardtii
further containing a transcription initiation site (TS), the RB47
binding site, a region for RB47 is also shown in FIGS. 1A-1D (SEQ
ID NO 5). As described in Section 2 above, the predicted protein
sequence from the cloned cDNA contained both the derived peptide
sequences of RB47 and is highly homologous to poly(A) binding
proteins (PABP) from a variety of eukaryotic organisms.
.Iadd.FIG. 7 diagrams a construct is essentially pD1/Nde including
a heterologous coding sequence having a 3' XbaI restriction site
for ligation with the 3' psbA gene..Iaddend.
.Iadd.FIG. 8 shows two of the transformants that contained the
single chain chimeric gene produced single chain antibodies at
approximately 1% of total protein levels..Iaddend.
.Iadd.FIG. 9 shows a construct, the bacterial LuxAB coding region
was ligated between the psbA 5' UTR and the psbA 3' end in an E.
coli plasmid..Iaddend.
.Iadd.FIG. 10 shows luciferase activity accumulated with the
chloroplast..Iaddend.
.Iadd.FIG. 11 shows a construct engineered so that the psbA
promoter and 5' UTR are used to drive the synthesis of the light
chain and heavy chains of an antibody, and the J chain normally
associated with IgA molecules..Iaddend.
2. Cloning of RB60
To clone the cDNA encoding the 60 kDa psbA mRNA binding protein
(RB60), the psbA-specific RNA binding proteins were purified from
light-grown C. reinhardtii cells using heparin-agarose
chromatography followed by psbA RNA affinity chromatography (RAC).
RAC-purified proteins were separated by two-dimensional
polyacrylamide gel electrophoresis. The region corresponding to
RB60 was isolated from the PVDF membrane. RB60 protein was then
digested with trypsin. Unambiguous amino acid sequences were
obtained from two peptide tryptic fragments (WFVDGELASDYNGPR (SEQ
ID NO 6) and (QLILWTTADDLKADAEIMTVFR (SEQ ID NO 7)) as described
above for RB47. The calculated molecular weights of the two tryptic
peptides used for further analysis precisely matched with the
molecular weights determine by mass spectrometry. The DNA sequence
corresponding to one peptide of 22 amino acid residues was
amplified by PCR using degenerate oligonucleotides, the forward
primer 5'CGCGGATCCGAYGCBGAGATYATGAC3' (SEQ ID NO 8) and the reverse
primer 5'CGCGAATTCGTCATRATCTCVGCRTC3' (SEQ ID NO 9), where R can be
A or G (the other IUPAC nucleotides have been previously defined
above). The amplified sequence was then used to screen a
.lamda.-gt10 cDNA library from C. reinhardtii. Three clones were
identified with the largest being 2.2 kb. Selection and sequencing
was performed as described for RB47 cDNA.
The resulting RB60 cDNA sequence is available via GenBank
(Accession Number AF027727). The nucleotide and encoded amino acid
sequence of RB60 is also shown in FIGS. 2A-2B (SEQ ID NO 10). The
protein coding sequence of 488 amino acid residues corresponds to
nucleotide positions 16-1614 of the 2413 base pair sequence. The
predicted amino acid sequence of the cloned cDNA contained the
complete amino acid sequences of the two tryptic peptides. The
amino acid sequence of the encoded protein revealed that it has
high sequence homology to both plant and mammalian protein
disulfide isomerase (PDI), and contains the highly conserved
thioredoxin-like domains with --CysGlyHisCys-- (--CGHC--) (SEQ ID
NO 11) catalytic sites in both the N-terminal and C-terminal
regions and the --LysAspGluLeu-- (--KDEL--) (SEQ ID NO 12)
endoplasmic reticulum (ER) retention signal at the C-terminus found
in all PDIs. PDI is a mutifunctional protein possessing enzymatic
activities for the formation, reduction, and isomerization of
disulfide bonds during protein folding, and is typically found in
the ER. The first 30 amino acid residues of RB60 were found to lack
sequence homology with the N-terminal signal sequence of PDI from
plants or mammalian cells. However, this region has characteristics
of chloroplast transit peptides of C. reinhardtii, which have
similarities with both mitochondrial and higher plant chloroplast
presequences. A transit peptide sequence should override the
function of the --KDEL-- ER retention signal and target the protein
to the chloroplast since the --KDEL-- signal acts only to retain
the transported protein in the ER.
3. Preparation of psbA Promoter Sequence and RB47 Binding Site
Nucleotide Sequence
The chloroplast psbA gene from the green unicellular alga C.
reinhardii was cloned and sequenced as described by Erickson et
al., Embo J., 3:2753-2762 (1984), the disclosure of which is hereby
incorporated by reference. The DNA sequence of the coding regions
and the 5' and 3' untranslated (UTR) flanking sequences of the C.
reinhardii psbA gene is shown in FIGS. 3A-3C. The psbA gene
sequence is also available through GenBank as further discussed in
Example 4. The nucleotide sequence is also listed as SEQ ID NO 13.
The deduced amino acid sequence (also listed in SEQ ID NO 13) of
the coding region is shown below each codon beginning with the
first methionine in the open reading frame. Indicated in the 5'
non-coding sequence are a putative Shine-Dalgarno sequence in the
dotted box, two putative transcription initiation sites determined
by S1 mapping (S1) and the Pribnow-10 sequence in the closed box.
Inverted repeats of eight or more base pairs are marked with arrows
and labeled A-D. A direct repeat of 31 base pairs with only two
mismatches is marked with arrows labeled 31. Indicated in the 3'
non-coding sequence is a large inverted repeat marked by a forward
arrow and the SI cleavage site marking the 3' end of the mRNA. Both
the 5' and 3' untranslated regions are used in preparing one of the
expression cassettes of this invention as further described
below.
The 5' UTR as previously discussed contains both the psbA promoter
and the RB47 binding site. The nucleotide sequence defining the
psbA promoter contains the region of the psbA DNA involved in
binding of RNA polymerase to initiate transcription. The -10
sequence component of the psbA promoter is indicated by the boxed
nucleotide sequence upstream of the first S1 while the -35 sequence
is located approximately 35 bases before the putative initiation
site. As shown in FIGS. 3A-3C, the -10 sequence is boxed, above
which is the nucleotide position (-100) from the first translated
codon. The -35 sequence is determined accordingly. A psbA promoter
for use in an expression cassette of this invention ends at the
first indicated S1 site (nucleotide position -92 as counting from
the first ATG) in FIGS. 3A-3C and extends to the 5' end (nucleotide
position -251 as shown in FIGS. 3A-3C). Thus, the promoter region
is 160 bases in length. A more preferred promoter region extends at
least 100 nucleotides to the 5' end from the S1 site. A most
preferred region contains nucleotide sequence ending at the s1 site
and extending 5' to include the -35 sequence, i.e., from -92 to
-130 as counted from the first encoded amino acid residue (39
bases).
The psbA RB47 binding site region begins at the first S1 site as
shown in FIGS. 3A-3C and extends to the first adenine base of the
first encoded methionine residue. Thus, a psbA RB47 binding site in
the psbA gene corresponds to the nucleotide positions from -91 to
-1 as shown in FIG. 3A-3C.
The above-identified regions are used to prepare expression
constructs as described below. The promoter and RB47 binding site
regions can be used separately; for example, the RB47 binding site
sequence can be isolated and used in a eukaryotic or prokaryotic
plasmid with a non-psbA promoter. Alternatively, the entire psbA 5'
UTR having 251 nucleotides as shown in FIGS. 3A-3C is used for the
regulatory region in an expression cassette containing both the
psbA promoter and RB47 binding site sequence as described
below.
4. Preparation of Expression Vectors and Expression of Coding
Sequences
A. Constructs Containing an psbA Promoter, an RB47 Binding Site
Nucleotide Sequence, a Desired Heterologous Coding Sequence, an
RB47-Encoding Sequence and an RB60-Encoding Sequence
Plasmid expression vector constructs, alternatively called
plasmids, vectors, constructs and the like, are constructed
containing various combinations of elements of the present
invention as described in the following examples. Variations of the
positioning and operably linking of the genetic elements described
in the present invention and in the examples below are contemplated
for use in practicing the methods of this invention. Methods for
manipulating DNA elements into operable expression cassettes are
well known in the art of molecular biology. Accordingly, variations
of control elements, such as constitutive or inducible promoters,
with respect to prokaryotic or eukaryotic expression systems as
described in Section C. are contemplated herein although not
enumerated. Moreover, the expression the various elements is not
limited to one transcript producing one mRNA; the invention
contemplates protein expression from more than one transcript if
desired.
As such, while the examples below recite one or two types of
expression cassettes, the genetic elements of RB47 binding site,
any desired coding sequence, in combination with RB47 and RB60
coding sequences along with a promoter are readily combined in a
number of operably linked permeations depending on the requirements
of the cell system selected for the expression. For example, for
expression in a chloroplast, endogenous RB47 protein is present
therefore an expression cassette having an RB47 binding site and a
desired coding sequence is minimally required along with an
operative promoter sequence. Overexpression of RB47 may be
preferable to enhance the translation of the coding sequence; in
that case, the chloroplast is further transformed with an
expression cassette containing an RB47-encoding sequence. Although
the examples herein and below utilize primarily the sequence
encoding the precursor form of RB47, any of the RB47-encoding
sequences described in the present invention, i.e., RB47 precursor,
mature RB47 and histidine-modified RB47 are contemplated for use in
any expression cassette and system as described herein. To regulate
the activation of translation, an RB60-encoding element is provided
to the expression system to provide the ability to regulate redox
potential in the cell as taught in Section B. These examples herein
and below represent a few of the possible permutations of genetic
elements for expression in the methods of this invention.
In one embodiment, a plasmid is constructed containing an RB47
binding site directly upstream of an inserted coding region for a
heterologous protein of interest, and the RB47 and RB60 coding
regions. Heterologous refers to the nature of the coding region
being dissimilar and not from the same gene as the regulatory
molecules in the plasmid, such as RB47 and RB60. Thus, all the
genetic elements of the present invention are produced in one
transcript from the IPTG-inducible psbA promoter. Alternative
promoters are similarly acceptable.
The final construct described herein for use in a prokaryotic
expression system makes a single mRNA from which all three proteins
are translated. The starting plasmid is any E. coli based plasmid
containing an origin of replication and selectable marker gene. For
this example, the Bluescript plasmid, pBS, commercially available
through Stratagene, Inc., La Jolla, Calif., which contains a
polylinker-cloning site and an ampicilin resistant marker is
selected for the vector.
The wild-type or native psbA gene (Erickson et al., Embo J.,
3:2753-2762 (1984), also shown in FIGS. 3A-3C, is cloned into pBS
at the EcoRI and BamHI sites of the polylinker. The nucleotide
sequence of the psbA gene is available on GenBank with the 5' UTR
and 3' UTR respectively listed in Accession Numbers X01424 and
X02350. The EcoRI site of psbA is 1.5 kb upstream of the psbA
initiation codon and the BamHI site is 2 kb downstream of the stop
codon. This plasmid is referred to as pDl.
Using site-directed PCR mutagenesis, well known to one of ordinary
skill in the art, an NdeI site is placed at the initiation codon of
psbA in the pDl plasmid so that the ATG of the NdeI restriction
site is the ATG initiation codon. This plasmid is referred to as
pDl/Nde. An Nde site is then placed at the initiation codon of the
gene encoding the heterologous protein of interest and an Xho I
site is placed directly downstream (within 10 nucleotides) of the
TAA stop codon of the heterologous protein coding sequence. Again
using site-directed mutagenesis, an XhoI site is placed within 10
nucleotides of the initiation codon of RB47, the preparation of
which is described in Example 2, and an NotI site is placed
directly downstream of the stop codon of RB47. The heterologous
coding region and the RB47 gene are then ligated into pDl/Nde so
that the heterologous protein gene is directly adjacent to the RB47
binding site and the RB47 coding region is downstream of the
heterologous coding region, using the Xho I site at the
heterologous stop codon and the Not I site of the pDl
polylinker.
These genetic manipulations result in a plasmid containing the 5'
end of the psbA gene including the promoter region and with the
RB47 binding site immediately upstream of a heterologous coding
region, and the RB47 coding region immediately downstream of the
heterologous coding region. The nucleotides between the stop codon
of the heterologous coding region and the initiation codon of the
RB47 coding region is preferably less than 20 nucleotides and
preferably does not contain any additional stop codons in any
reading frame. This plasmid is referred to as pD1/RB47.
Using site-directed mutagenesis, a NotI site is placed immediately
(within 10 nucleotides) upstream of the initiation codon of RB60,
the preparation of which is described in Example 2, and an Xba I
site is placed downstream of the RB60 stop codon. This DNA fragment
is then ligated to the 3' end of the psbA gene using the Xba I site
found in the 3' end of the psbA gene so that the psbA 3' end is
downstream of the RB60 coding region. This fragment is then ligated
into the pD1/RB47 plasmid using the NotI and BamHI sites so that
the RB60 coding region directly follows the RB47 coding region. The
resulting plasmid is designated pD1/RB47/RB60. Preferably there is
less then 20 nucleotides between the RB47 and RB60 coding regions
and preferably there are no stop codons in any reading frame in
that region. The final plasmid thus contains the following genetic
elements operably linked in the 5' to 3' direction: the 5' end of
the psbA gene with a promoter capable of directing transcription in
chloroplasts, an RB47 binding site, a desired heterologous coding
region, the RB47 coding region, the RB60 coding region, and the 3'
end of the psbA gene which contains a transcription termination and
mRNA processing site, and an E. coli origin of replication and
amplicillin resistance gene. A diagram of this plasmid with the
restriction sites is shown in FIG. 4.
Expression of pD1/RB47/RB60 in E. coli to produce recombinant RB47,
RB60 and the recombinant heterologous protein is performed as
described in Example 4B. The heterologous protein is then purified
as further described.
Expression cassettes in which the sequences encoding RB47 and RB60
are similarly operably linked to a heterologous coding sequence
having the psbA RB47 binding site as described in Example 3 are
prepared with a different promoter for use in eukaryotic, such as
mammalian expression systems. In this aspect, the cassette is
similarly prepared as described above with the exception that
restriction cloning sites are dependent upon the available multiple
cloning sites in the recipient vector. Thus, the RB47 binding site
prepared in Example 3 is prepared for directed ligation into a
selected expression vector downstream of the promoter in that
vector. The RB47 and RB60 coding sequences are obtained from the
pD1/RB47/RB60 plasmid by digestion with XhoI and Xbal and inserted
into a similarly digested vector if the sites are present.
Alternatively, site-directed mutagenesis is utilized to create
appropriate linkers. A desired heterologous coding sequence is
similarly ligated into the vector for expression.
B. Constructs Containing RB47 Nucleotide Sequence
1) Purified Recombinant RB47 Protein
In one approach to obtain purified recombinant RB47 protein, the
full length RB47 cDNA prepared above was cloned into the E. coli
expression vector pET3A (Studier et al., Methods Enzymol.,
185:60-89 (1990)), also commercially available by Novagen, Inc.,
Madison, Wis. and transformed into BL21 E. coli cells. The cells
were grown to a density of 0.4 (OD.sub.600), then induced with 0.5
mM IPTG. Cells were then allowed to grow for an additional 4 hours,
at which point they were pelleted and frozen.
Confirmation of the identity of the cloned cDNA as encoding the
authentic RB47 protein was accomplished by examining protein
expressed from the cDNA by immunoblot analysis and by RNA binding
activity assay. The recombinant RB47 protein produced when the RB47
cDNA was expressed was recognized by antisera raised against the C.
reinhardtii RB47 protein. The E. coli expressed protein migrated at
80 kDa on SDS-PAGE, but the protein was actually 69 kDa, as
determined by mass spectrometry of the E. coli expressed protein.
This mass agrees with the mass predicted from the cDNA sequence. A
60 kDa product was also produced in E. coli, and recognized by the
antisera against the C. reinhardtii protein, which is most likely a
degradation or early termination product of the RB47 cDNA. The
recombinant RB47 protein expressed from the RB47 cDNA is recognized
by the antisera raised against the C. reinhardtii protein at levels
similar to the recognition of the authentic C. reinhardtii RB47
protein, demonstrating that the cloned cDNA produces a protein
product that is immunologically related to the naturally produced
RB47 protein. In order to generate a recombinant equivalent of the
endogenous native RB47, the location of the 47 kDa polypeptide was
mapped on the full-length recombinant protein by comparing mass
spectrometric data of tryptic digests of the C. reinhardtii 47 kDa
protein and the full-length recombinant protein. Thus, peptide
mapping by mass spectrometry has shown that the endogenous RB47
protein corresponds primarily to the RNA binding domains contained
within the N-terminal region of the predicted precursor protein,
suggesting that a cleavage event is necessary to produce the mature
47 kDa protein. Thus, full-length recombinant RB47 is 69 kDa and
contains a carboxy domain that is cleaved in vivo to generate the
endogenous mature form of RB47 that is 47 kDa.
To determine if the heterologously expressed RB47 protein was
capable of binding the psbA RNA, the E. coli expressed protein was
purified by heparin agarose chromatography. The recombinant RB47
protein expressed in E. coli was purified using a protocol similar
to that used previously for purification of RB47 from C.
reinhardtii. Approximately 5 g of E. coli cells grown as described
above were resuspended in low salt extraction buffer (10 mM Tris
[pH 7.5], 10 mM NaCl, 10 mM MgCl.sub.2, 5 mM
.beta.-mercaptoethanol) and disrupted by sonication. The soluble
cell extract was applied to a 5 mL Econo-Pac heparin cartridge
(Bio-Rad) which was washed prior to elution of the RB47 protein
(Danon and Mayfield, Embo J., 10:3993-4001 (1991)).
The E. coli expressed protein that bound to the heparin agarose
matrix was eluted from the column at the same salt concentration as
used to elute the authentic C. reinhardtii RB47 protein. This
protein fraction was used in in vitro binding assays with the psbA
5' UTR. Both the 69 and 60 kDa E. coli expressed proteins
crosslinked to the radiolabeled psbA 5' UTR at levels similar to
crosslinking of the endogenous RB47 protein, when the RNA/protein
complex is subjected to UV irradiation.
Heparin agarose purified proteins, both from the E. coli expressed
RB47 cDNA and from C. reinhardtii cells, were used in an RNA gel
mobility shift assay to determine the relative affinity and
specificity of these proteins for the 5' UTR of the psbA mRNA. The
E. coli expressed proteins bound to the psbA 5' UTR in vitro with
properties that are similar to those of the endogenous RB47 protein
purified from C. reinhardtii. RNA binding to both the E. coli
expressed and the endogenous RB47 protein was competed using either
200 fold excess of unlabeled psbA RNA or 200 fold excess of poly(A)
RNA. RNA binding to either of these proteins was poorly competed
using 200 fold excess of total RNA or 200 fold excess of the 5' UTR
of the psbD or psbC RNAs. Different forms of the RB47 protein (47
kDa endogenous protein vs. the 69 kDa E. coli expressed protein)
may account for the slight differences in mobility observed when
comparing the binding profiles of purified C. reinhardtii protein
to heterologously expressed RB47.
The mature form of RB47 is also produced in recombinant form by the
insertion by PCR of an artificial stop codon in the RB47 cDNA at
nucleotide positions 1403-1405 with a stop codon resulting in a
mature RB47 recombinant protein having 402 amino acids as shown in
FIGS. 1A-1D. An example of this is shown in FIGS. 5A-5B for the
production of a recombinant histidine-modified RB47 mature protein
as described below. The complete RB47 cDNA is inserted into an
expression vector, such as pET3A as described above, for expression
of the mature 47 kDa form of the RB47 protein. In the absence of
the inserted stop codon, the transcript reads through to nucleotide
position 2066-2068 at the TAA stop codon to produce the precursor
RB47 having the above-described molecular weight characteristics
and 623 amino acid residues.
Recombinant RB47 is also expressed and purified in plant cells. For
this aspect, C. reinhardtii strains were grown in complete media
(Tris-acetate-phosphate [TAP] (Harris, The Chlamydonas Sourcebook,
San Diego, Calif., Academic Press (1989)) to a density of
5.times.10.sup.6 cells/mL under constant light. Cells were
harvested by centrifugation at 4.degree. C. for 5 minutes at 4,000
g. Cells were either used immediately or frozen in liquid N.sub.2
for storage at -70.degree. C.
Recombinant RB47 protein was also produced as a modified RB47
protein with a histidine tag at the amino-terminus according to
well known expression methods using pET19-D vectors available from
Novagen, Inc., Madison, Wis. The nucleotide and amino acid sequence
of a recombinant histidine-modified RB47 of the mature 47 kDa form
is shown in FIGS. 5A-5B with the nucleotide and amino acid sequence
also listed in SEQ ID NO 14. Thus the nucleotide sequence of a
histidine-modified RB47 is 1269 bases in length. The precursor form
of the RB47 protein is similarly obtained in the expression system,
both of which are modified by the presence of a histidine tag that
allows for purification by metal affinity chromatography.
The recombinant histidine-modified RB47 purified through addition
of a poly-histidine tag followed by Ni.sup.+2 column chromatography
showed similar binding characteristics as that described for
recombinant precursor RB47 described above.
C. Constructs Containing RB60 Nucleotide Sequence
In one approach to obtain purified recombinant RB60 protein, the
full-length RB60 cDNA prepared above was cloned into the E. coli
expression vector pET3A (Studier et al., Methods Enzymol.,
185:60-89 (1990)), also commercially available by Novagen, Inc.,
Madison, Wis. and transformed into BL21 E. coli cells. The cells
were grown to a density of 0.4 (OD.sub.600), then induced with 0.5
mM IPTG. Cells were then allowed to grow for an additional 4 hours,
at which point they were pelleted and frozen.
Recombinant histidine-modified RB60 was also expressed with a
pET19-D vector as described above for RB47 that was similarly
modified. Purification of the recombinant RB60 proteins was
performed as described for RB47 thereby producing recombinant RB60
proteins for use in the present invention.
The RB60 coding sequence is also mutagenized for directional
ligation into an selected vector for expression in alternative
systems, such as mammalian expression systems.
D. Constructs Containing an RB47-Encoding Sequence and an
RB60-Encoding Sequence
To prepare an expression cassette for encoding both RB47 and RB60,
one approach is to digest plasmid pD1/RB47/RB60 prepared above with
XhoI and XbaI to isolate the fragment for both encoding sequences.
The fragment is then inserted into a similarly digested expression
vector if available or is further mutagenized to prepare
appropriate restriction sites.
Alternatively, the nucleotide sequences of RB47 and RB60, as
described in Example 2, are separately prepared for directional
ligation into a selected vector.
An additional embodiment of the present invention is to prepare an
expression cassette containing the RB47 binding site along with the
coding sequences for RB47 and RB60, the plasmid pD1/RB47/RB60
prepared above is digested with NdeI and XhoI to prepare an
expression cassette in which any desired coding sequence having
similarly restriction sites is directionally ligated. Expression
vectors containing both the RB47 and RB60 encoding sequences in
which the RB47 binding site sequence is utilized with a different
promoter are also prepared as described in Example 4A.
E. Constructs Containing an RB47 Binding Site Nucleotide Sequence,
Insertion Sites for a Desired Heterologous Coding Sequence, and an
RB47-Encoding Sequence
In another permutation, a plasmid or expression cassette is
constructed containing an RB47 binding site directly upstream of an
inserted coding region for a heterologous protein of interest, and
the RB47 coding region. The final construct described herein for
use in a prokaryotic expression system makes a single mRNA from
which both proteins are translated.
The plasmid referred to as pD1/RB47 is prepared as described above
in Example 4A. A diagram of this plasmid with the restriction sites
is shown in FIG. 6.
Expression of pD1/RB47 in E. coli to produce recombinant RB47 and
the recombinant heterologous protein is performed as described in
above. The heterologous protein is then purified as further
described.
To produce an expression cassette that allows for insertion of an
alternative desired coding sequence, the plasmid pD1/RB47 is
digested with NdeI and XhoI resulting in a vector having
restriction endonuclease sites for insertion of a desired coding
sequence operably linked to a RB47 binding site and RB47 coding
sequence on one transcriptional unit.
F. Constructs Containing an RB47 Binding Site Nucleotide Sequence,
Insertion Sites for a Desired Heterologous Coding Sequence, and an
RB47-Encoding Sequence
In another permutation, a plasmid or expression cassette is
constructed containing an RB47 binding site directly upstream of an
inserted coding region for a heterologous protein of interest, and
the RB60 coding region. The final construct described herein for
use in a prokaryotic expression system makes a single mRNA from
which both proteins are translated. In this embodiment, a separate
construct encoding recombinant RB47 as described in Example 4B is
co-transformed into the E. coli host cell for expression.
The plasmid referred to as pD1/RB60 is prepared as described above
for pD1/RB47 in Example 4A with the exception that XhoI and XbaI
sites are created on RB60 rather than RB47.
Expression of pD1/RB60 in E. coli to produce recombinant RB60 and
the recombinant heterologous protein is performed as described in
above with the combined expression of RB47 from a separate
expression cassette. The heterologous protein is then purified as
further described.
To produce an expression cassette that allows for insertion of an
alternative desired coding sequence, the plasmid pD1/RB60 is
digested with NdeI and XhoI resulting in a vector having
restriction endonuclease sites for insertion of a desired coding
sequence operably linked to a RB47 binding site and RB60 coding
sequence on one transcriptional unit.
G. Constructs Containing RB47 Binding Site Nucleotide Sequence and
Heterologous Coding Sequences
1) Expression of Recombinant Tetanus Toxin Single Chain
Antibody
The examples herein describe constructs that are variations of
those described above. The constructs described below contain an
RB47 binding site sequence and a heterologous coding sequence. The
activating protein RB47 was endogenously provided in the
chloroplast and or plant cell. In other aspects however as taught
by the methods of the present invention, the chloroplast is further
transformed with an RB47-expression construct as described above
for overexpression of RB47 to enhance translation capacities.
A strain of the green algae Chlamydomonas reinhardtii was designed
to allow expression of a single chain antibody gene in the
chloroplast. The transgenically expressed antibody was produced
from a chimeric gene containing the promoter and 5' untranslated
region (UTR) of the chloroplast psbA gene prepared as described
above, followed by the coding region of a single chain antibody
(encoding a tetanus toxin binding antibody), and then the 3' UTR of
the psbA gene also prepared as described above to provide for
transcription termination and RNA processing signals. This
construct is essentially pD1/Nde including a heterologous coding
sequence having a 3' XbaI restriction site for ligation with the 3'
psbA gene and is diagramed in FIG. 7.
The psbA-single chain construct was first transformed into C.
reinhardtii chloroplast and transformants were then screened for
single chain gene integration. Transformation of chloroplast was
performed via bolistic delivery as described in U.S. Pat. Nos.
5,545,818 and 5,553,878, the disclosures of which are hereby
incorporated by reference. Transformation is accomplished by
homologous recombination via the 5' and 3' UTR of the psbA
mRNA.
As shown in FIG. 8, two of the transformants that contained the
single chain chimeric gene produced single chain antibodies at
approximately 1% of total protein levels. The transgenic antibodies
were of the correct size and were completely soluble, as would be
expected of a correctly folded protein. Few degradation products
were detectable by this Western analysis, suggesting that the
proteins were fairly stable within the chloroplast. To identify if
the produced antibody retained the binding capacity for tetanus
toxin, ELISA assays were performed using a mouse-produced Fab, from
the original tetanus toxin antibody, as the control. The
chloroplast single chain antibody bound tetanus toxin at levels
similar to Fab, indicating that the single chain antibody produced
in C. reinhardtii is a fully functional antibody. These results
clearly demonstrate the ability of the chloroplast to synthesis and
accumulate function antibody molecules resulting from the
translational activation of an RB47 binding site in an expression
cassette by endogenous RB47 protein in the chloroplast.
2) Expression of Bacterial Luciferase Enzyme Having Two
Subunits
For the production of molecules that contain more than one subunit,
such as dIgA and bacterial luciferase enzyme, several proteins must
be produced in stoichiometric quantities within the chloroplast.
Chloroplast have an advantage for this type of production over
cytoplasmic protein synthesis in that translation of multiple
proteins can originate from a single mRNA. For example, a
dicistronic mRNA having 5' and 3' NdeI and XbaI restriction sites
and containing both the A and B chains of the bacterial luciferase
enzyme was inserted downstream of the psbA promoter and 5' UTR of
the pD1/Nde construct prepared in Example 4A above. In this
construct, the bacterial LuxAB coding region was ligated between
the psbA 5' UTR and the psbA 3' end in an E. coli plasmid that was
then transformed into Chlamydomonas reinhardtii cells as described
above for expression in the chloroplast. A schematic of the
construct is shown in FIG. 9. Single transformant colonies were
then isolated. A plate containing a single isolate was grown for 10
days on complete media and a drop of the luciferase substrate
n-Decyl Aldehyde was placed on the plate and the luciferase
visualized by video-photography in a dark chamber. Both proteins
were synthesized from this single mRNA and luciferase activity
accumulated within the chloroplast as shown in FIG. 10. Some mRNA
within plastids contained as many as 5 separate proteins encoded on
a single mRNA.
3) Expression of Dimeric IgA
To generate dimeric IgA, the construct shown in FIG. 11 is
engineered so that the psbA promoter and 5' UTR are used to drive
the synthesis of the light chain and heavy chains of an antibody,
and the J chain normally associated with IgA molecules. The nucleic
acid sequences for the dimeric IgA are inserted into the RB47
binding site construct prepared in Example 4A. The construct is
then transformed into C. reinhardtii cells as previously described
for expression of the recombinant dIgA.
Production of these three proteins within the plastid allows for
the self assembly of a dimeric IgA (dIgA). Production of this
complex is monitored in several ways. First, Southern analysis of
transgenic algae is used to identify strains containing the
polycistronic chimeric dIgA gene. Strains positive for integration
of the dIgA gene are screened by Northern analysis to ensure that
the chimeric mRNA is accumulating. Western blot analysis using
denaturing gels is used to monitor the accumulation of the
individual light, heavy and J chain proteins, and native gels
Western blot analysis will be used to monitor the accumulation of
the assembled dIgA molecule.
By using a single polycistronic mRNA in the context of RB47
regulated translation, two of the potential pitfalls in the
assembly of multimeric dIgA molecule are overcome. First, this
construct ensures approximately stoichiometric synthesis of the
subunits, as ribosomes reading through the first protein are likely
to continue to read through the second and third proteins as well.
Second, all of the subunits are synthesized in close physical
proximity to each other, which increases the probability of the
proteins self assembling into a multimeric molecule. Following the
production of a strain producing dIgA molecules, the production of
dIgA on an intermediate scale by growing algae in 300 liter
fermentors is then performed. Larger production scales are then
performed thereafter.
The foregoing specification, including the specific embodiments and
examples, is intended to be illustrative of the present invention
and is not to be taken as limiting. Numerous other variations and
modifications can be effected without departing from the true
spirit and scope of the invention.
SEQUENCE LISTINGS
1
18115PRTChlamydomonas reinhardtii 1Gln Tyr Gly Phe Val His Phe Glu
Asp Gln Ala Ala Ala Asp Arg1 5 10 15214PRTChlamydomonas reinhardtii
2Gly Phe Gly Phe Ile Asn Phe Lys Asp Ala Glu Ser Ala Ala1 5
10332DNAArtificial sequencePrimer 3cagtacggyt tcgtbcaytt cgaggaycag
gc 32440DNAArtificial sequencePrimer 4ggaattcggy ttcggyttca
tyaacttcaa ggaygcbgag 4052846DNAChlamydomonas
reinhardtiiCDS(197)..(2065) 5gaattcgcgg ccgctccgtg gttggtcctc
atggtgtctt tttgaagagg acctgagcct 60ttcacccaaa tatatcaaaa aacccgggca
accggccaaa aaaattgcaa aagcctctcg 120taggcacaaa agacctattc
tagccatcaa ctttgtatcc gacgctgccg tttagctgcg 180cgtcttgaag tcaagc
atg gcg act act gag tcc tcg gcc ccg gcg gcc acc 232 Met Ala Thr Thr
Glu Ser Ser Ala Pro Ala Ala Thr 1 5 10acc cag ccg gcc agc acc ccg
ctg gcg aac tcg tcg ctg tac gtc ggt 280Thr Gln Pro Ala Ser Thr Pro
Leu Ala Asn Ser Ser Leu Tyr Val Gly 15 20 25gac ctg gag aag gat gtc
acc gag gcc cag ctg ttc gag ctc ttc tcc 328Asp Leu Glu Lys Asp Val
Thr Glu Ala Gln Leu Phe Glu Leu Phe Ser 30 35 40tcg gtt ggc cct gtg
gcc tcc att cgc gtg tgc cgc gat gcc gtc acg 376Ser Val Gly Pro Val
Ala Ser Ile Arg Val Cys Arg Asp Ala Val Thr45 50 55 60cgc cgc tcg
ctg ggc tac gcc tac gtc aac tac aac agc gct ctg gac 424Arg Arg Ser
Leu Gly Tyr Ala Tyr Val Asn Tyr Asn Ser Ala Leu Asp 65 70 75ccc cag
gct gct gac cgc gcc atg gag acc ctg aac tac cat gtc gtg 472Pro Gln
Ala Ala Asp Arg Ala Met Glu Thr Leu Asn Tyr His Val Val 80 85 90aac
ggc aag cct atg cgc atc atg tgg tcg cac cgc gac cct tcg gcc 520Asn
Gly Lys Pro Met Arg Ile Met Trp Ser His Arg Asp Pro Ser Ala 95 100
105cgc aag tcg ggc gtc ggc aac atc ttc atc aag aac ctg gac aag acc
568Arg Lys Ser Gly Val Gly Asn Ile Phe Ile Lys Asn Leu Asp Lys Thr
110 115 120atc gac gcc aag gcc ctg cac gac acc ttc tcg gcc ttc ggc
aag att 616Ile Asp Ala Lys Ala Leu His Asp Thr Phe Ser Ala Phe Gly
Lys Ile125 130 135 140ctg tcc tgc aag gtt gcc act gac gcc aac ggc
gtg tcg aag ggc tac 664Leu Ser Cys Lys Val Ala Thr Asp Ala Asn Gly
Val Ser Lys Gly Tyr 145 150 155ggc ttc gtg cac ttc gag gac cag gcc
gct gcc gat cgc gcc att cag 712Gly Phe Val His Phe Glu Asp Gln Ala
Ala Ala Asp Arg Ala Ile Gln 160 165 170acc gtc aac cag aag aag att
gag ggc aag atc gtg tac gtg gcc ccc 760Thr Val Asn Gln Lys Lys Ile
Glu Gly Lys Ile Val Tyr Val Ala Pro 175 180 185ttc cag aag cgc gct
gac cgc ccc agg gca agg acg ttg tac acc aac 808Phe Gln Lys Arg Ala
Asp Arg Pro Arg Ala Arg Thr Leu Tyr Thr Asn 190 195 200gtg ttc gtc
aag aac ttg ccg gcc gac atc ggc gac gac gag ctg ggc 856Val Phe Val
Lys Asn Leu Pro Ala Asp Ile Gly Asp Asp Glu Leu Gly205 210 215
220aag atg gcc acc gag cac ggc gag atc acc agc gcg gtg gtc atg aag
904Lys Met Ala Thr Glu His Gly Glu Ile Thr Ser Ala Val Val Met Lys
225 230 235gac gac aag ggc ggc agc aag ggc ttc ggc ttc atc aac ttc
aag gac 952Asp Asp Lys Gly Gly Ser Lys Gly Phe Gly Phe Ile Asn Phe
Lys Asp 240 245 250gcc gag tcg gcg gcc aag tgc gtg gag tac ctg aac
gag cgc gag atg 1000Ala Glu Ser Ala Ala Lys Cys Val Glu Tyr Leu Asn
Glu Arg Glu Met 255 260 265agc ggc aag acc ctg tac gcc ggc cgc gcc
cag aag aag acc gag cgc 1048Ser Gly Lys Thr Leu Tyr Ala Gly Arg Ala
Gln Lys Lys Thr Glu Arg 270 275 280gag gcg atg ctg cgc cag aag gcc
gag gag agc aag cag gag cgt tac 1096Glu Ala Met Leu Arg Gln Lys Ala
Glu Glu Ser Lys Gln Glu Arg Tyr285 290 295 300ctg aag tac cag agc
atg aac ctg tac gtc aag aac ctg tcc gac gag 1144Leu Lys Tyr Gln Ser
Met Asn Leu Tyr Val Lys Asn Leu Ser Asp Glu 305 310 315gag gtc gac
gac gac gcc ctg cgt gag ctg ttc gcc aac tct ggc acc 1192Glu Val Asp
Asp Asp Ala Leu Arg Glu Leu Phe Ala Asn Ser Gly Thr 320 325 330atc
acc tcg tgc aag gtc atg aag gac ggc agc ggc aag tcc aag ggc 1240Ile
Thr Ser Cys Lys Val Met Lys Asp Gly Ser Gly Lys Ser Lys Gly 335 340
345ttc ggc ttc gtg tgc ttc acc agc cac gac gag gcc acc cgg ccg ccc
1288Phe Gly Phe Val Cys Phe Thr Ser His Asp Glu Ala Thr Arg Pro Pro
350 355 360gtg acc gag atg aac ggc aag atg gtc aag ggc aag ccc ctg
tac gtg 1336Val Thr Glu Met Asn Gly Lys Met Val Lys Gly Lys Pro Leu
Tyr Val365 370 375 380gcc ctg gcg cag cgc aag gac gtg cgc cgt gcc
acc cag ctg gag gcc 1384Ala Leu Ala Gln Arg Lys Asp Val Arg Arg Ala
Thr Gln Leu Glu Ala 385 390 395aac atg cag gcg cgc atg ggc atg ggc
gcc atg agc cgc ccg ccg aac 1432Asn Met Gln Ala Arg Met Gly Met Gly
Ala Met Ser Arg Pro Pro Asn 400 405 410ccg atg gcc ggc atg agc ccc
tac ccc ggc gcc atg ccg ttc ttc gct 1480Pro Met Ala Gly Met Ser Pro
Tyr Pro Gly Ala Met Pro Phe Phe Ala 415 420 425ccc ggc ccc ggc ggc
atg gct gct ggc ccg cgc gct ccg ggc atg atg 1528Pro Gly Pro Gly Gly
Met Ala Ala Gly Pro Arg Ala Pro Gly Met Met 430 435 440tac ccg ccc
atg atg ccg ccg cgc ggc atg cct ggc ccc ggc cgc ggc 1576Tyr Pro Pro
Met Met Pro Pro Arg Gly Met Pro Gly Pro Gly Arg Gly445 450 455
460ccc cgc ggc ccc atg atg ccg ccc cag atg atg ggt ggc ccc atg atg
1624Pro Arg Gly Pro Met Met Pro Pro Gln Met Met Gly Gly Pro Met Met
465 470 475ggc ccg ccc atg ggc ccc ggg cgc ggc cgt ggc ggc cgc ggc
ccc tcc 1672Gly Pro Pro Met Gly Pro Gly Arg Gly Arg Gly Gly Arg Gly
Pro Ser 480 485 490ggc cgc ggc cag ggc cgc ggc aac aac gcc cct gcc
cag cag ccc aag 1720Gly Arg Gly Gln Gly Arg Gly Asn Asn Ala Pro Ala
Gln Gln Pro Lys 495 500 505ccc gcc gct gag ccg gcc gcc gcg ccc gcc
gcc gcc gcc ccc gct gcc 1768Pro Ala Ala Glu Pro Ala Ala Ala Pro Ala
Ala Ala Ala Pro Ala Ala 510 515 520gcg gcg cct gcc gcc gcg gcg gag
ccg gag gcc ccc gcc gcc cag cag 1816Ala Ala Pro Ala Ala Ala Ala Glu
Pro Glu Ala Pro Ala Ala Gln Gln525 530 535 540ccg ctg acc gcc tcc
gcg ctg gcc gcc gcc gcg ccg gag cag cag aag 1864Pro Leu Thr Ala Ser
Ala Leu Ala Ala Ala Ala Pro Glu Gln Gln Lys 545 550 555atg atg atc
ggc gag cgc ctg tac ccg cag gtg gcg gag ctg cag ccc 1912Met Met Ile
Gly Glu Arg Leu Tyr Pro Gln Val Ala Glu Leu Gln Pro 560 565 570gac
ctg gct ggc aag atc acc ggc atg ctg ctg gag atg gac aac gcc 1960Asp
Leu Ala Gly Lys Ile Thr Gly Met Leu Leu Glu Met Asp Asn Ala 575 580
585gag ctt ctg atg ctt ctg gag tcg cac gag gcg ctg gtg tcc aag gtg
2008Glu Leu Leu Met Leu Leu Glu Ser His Glu Ala Leu Val Ser Lys Val
590 595 600gac gag gcc atc gct gtg ctc aag cag cac aac gtg att gcc
gag gag 2056Asp Glu Ala Ile Ala Val Leu Lys Gln His Asn Val Ile Ala
Glu Glu605 610 615 620aac aag gct taaagcgcct gcacgcttgt gcgggctggt
ggcgccggcg 2105Asn Lys Alacgcgccggcg ctgcttgggc cgccggcagc
atgggcgcgg cggacgcggt gtgggagcag 2165tgcttgctgc ttctggccgc
cgtgaagccg cgccgaactg gggcggacgg caggctggcg 2225ttgacgccgg
cgcgccacaa cacaaagttg gtggcgtgaa agtctctggg cgtgctccgg
2285acggttgtaa ggttttaaga actggctttt ggccgggttg ccgcccaaag
gcggaacggc 2345ggtcttttca ggccaatcac atccggctgg aaaaattctt
accaaagcca acccctgcac 2405ccaaaaattt cgggttccga aagaacactc
cccttttttc cggcaacgcg ttctttcaag 2465gccaatcact ttccgggttg
gaagaaaatg ttacccggaa aaggcgggaa gccccctgca 2525cccggacaag
ttattcgggg tttcgccggg aatgagcaag cgttcgggct gttggccgta
2585tcgcgaacgc tgtcggggtg tcaggcgcca gaaggaagga tgacgttttg
gtgaaggggt 2645gcaaactgag cacacgagtt ttggcaatag acgtggagaa
agtccagtgc ggggtgaggc 2705ggatagcgga atcaagcgtg gcgggtccct
ggcgagacga gacgcttctg ttgttttgct 2765gagccctttg atggcacaat
cgcactgttt tgagcaggcg actgtaaagt gcccgacgct 2825aaaaaagcgg
ccgcgaattc c 28466623PRTChlamydomonas reinhardtii 6Met Ala Thr Thr
Glu Ser Ser Ala Pro Ala Ala Thr Thr Gln Pro Ala1 5 10 15Ser Thr Pro
Leu Ala Asn Ser Ser Leu Tyr Val Gly Asp Leu Glu Lys 20 25 30Asp Val
Thr Glu Ala Gln Leu Phe Glu Leu Phe Ser Ser Val Gly Pro 35 40 45Val
Ala Ser Ile Arg Val Cys Arg Asp Ala Val Thr Arg Arg Ser Leu 50 55
60Gly Tyr Ala Tyr Val Asn Tyr Asn Ser Ala Leu Asp Pro Gln Ala Ala65
70 75 80Asp Arg Ala Met Glu Thr Leu Asn Tyr His Val Val Asn Gly Lys
Pro 85 90 95Met Arg Ile Met Trp Ser His Arg Asp Pro Ser Ala Arg Lys
Ser Gly 100 105 110Val Gly Asn Ile Phe Ile Lys Asn Leu Asp Lys Thr
Ile Asp Ala Lys 115 120 125Ala Leu His Asp Thr Phe Ser Ala Phe Gly
Lys Ile Leu Ser Cys Lys 130 135 140Val Ala Thr Asp Ala Asn Gly Val
Ser Lys Gly Tyr Gly Phe Val His145 150 155 160Phe Glu Asp Gln Ala
Ala Ala Asp Arg Ala Ile Gln Thr Val Asn Gln 165 170 175Lys Lys Ile
Glu Gly Lys Ile Val Tyr Val Ala Pro Phe Gln Lys Arg 180 185 190Ala
Asp Arg Pro Arg Ala Arg Thr Leu Tyr Thr Asn Val Phe Val Lys 195 200
205Asn Leu Pro Ala Asp Ile Gly Asp Asp Glu Leu Gly Lys Met Ala Thr
210 215 220Glu His Gly Glu Ile Thr Ser Ala Val Val Met Lys Asp Asp
Lys Gly225 230 235 240Gly Ser Lys Gly Phe Gly Phe Ile Asn Phe Lys
Asp Ala Glu Ser Ala 245 250 255Ala Lys Cys Val Glu Tyr Leu Asn Glu
Arg Glu Met Ser Gly Lys Thr 260 265 270Leu Tyr Ala Gly Arg Ala Gln
Lys Lys Thr Glu Arg Glu Ala Met Leu 275 280 285Arg Gln Lys Ala Glu
Glu Ser Lys Gln Glu Arg Tyr Leu Lys Tyr Gln 290 295 300Ser Met Asn
Leu Tyr Val Lys Asn Leu Ser Asp Glu Glu Val Asp Asp305 310 315
320Asp Ala Leu Arg Glu Leu Phe Ala Asn Ser Gly Thr Ile Thr Ser Cys
325 330 335Lys Val Met Lys Asp Gly Ser Gly Lys Ser Lys Gly Phe Gly
Phe Val 340 345 350Cys Phe Thr Ser His Asp Glu Ala Thr Arg Pro Pro
Val Thr Glu Met 355 360 365Asn Gly Lys Met Val Lys Gly Lys Pro Leu
Tyr Val Ala Leu Ala Gln 370 375 380Arg Lys Asp Val Arg Arg Ala Thr
Gln Leu Glu Ala Asn Met Gln Ala385 390 395 400Arg Met Gly Met Gly
Ala Met Ser Arg Pro Pro Asn Pro Met Ala Gly 405 410 415Met Ser Pro
Tyr Pro Gly Ala Met Pro Phe Phe Ala Pro Gly Pro Gly 420 425 430Gly
Met Ala Ala Gly Pro Arg Ala Pro Gly Met Met Tyr Pro Pro Met 435 440
445Met Pro Pro Arg Gly Met Pro Gly Pro Gly Arg Gly Pro Arg Gly Pro
450 455 460Met Met Pro Pro Gln Met Met Gly Gly Pro Met Met Gly Pro
Pro Met465 470 475 480Gly Pro Gly Arg Gly Arg Gly Gly Arg Gly Pro
Ser Gly Arg Gly Gln 485 490 495Gly Arg Gly Asn Asn Ala Pro Ala Gln
Gln Pro Lys Pro Ala Ala Glu 500 505 510Pro Ala Ala Ala Pro Ala Ala
Ala Ala Pro Ala Ala Ala Ala Pro Ala 515 520 525Ala Ala Ala Glu Pro
Glu Ala Pro Ala Ala Gln Gln Pro Leu Thr Ala 530 535 540Ser Ala Leu
Ala Ala Ala Ala Pro Glu Gln Gln Lys Met Met Ile Gly545 550 555
560Glu Arg Leu Tyr Pro Gln Val Ala Glu Leu Gln Pro Asp Leu Ala Gly
565 570 575Lys Ile Thr Gly Met Leu Leu Glu Met Asp Asn Ala Glu Leu
Leu Met 580 585 590Leu Leu Glu Ser His Glu Ala Leu Val Ser Lys Val
Asp Glu Ala Ile 595 600 605Ala Val Leu Lys Gln His Asn Val Ile Ala
Glu Glu Asn Lys Ala 610 615 620715PRTChlamydomonas reinhardtii 7Trp
Phe Val Asp Gly Glu Leu Ala Ser Asp Tyr Asn Gly Pro Arg1 5 10
15822PRTChlamydomonas reinhardtii 8Gln Leu Ile Leu Trp Thr Thr Ala
Asp Asp Leu Lys Ala Asp Ala Glu1 5 10 15Ile Met Thr Val Phe Arg
20926DNAArtificial sequencePrimer 9cgcggatccg aygcbgagat yatgac
261026DNAArtificial sequencePrimer 10cgcgaattcg tcatratctc vgcrtc
26112413DNAChlamydomonas reinhardtiiCDS(16)..(1614) 11gagtacgttt
acgcc atg aac cgt tgg aac ctt ctt gcc ctt acc ctg ggg 51 Met Asn
Arg Trp Asn Leu Leu Ala Leu Thr Leu Gly 1 5 10ctg ctg ctg gtg gca
gcg ccc ttc acc aag cac cag ttt gct cat gct 99Leu Leu Leu Val Ala
Ala Pro Phe Thr Lys His Gln Phe Ala His Ala 15 20 25tcc gat gag tat
gag gac gac gag gag gac gat gcc ccc gcc gcc cct 147Ser Asp Glu Tyr
Glu Asp Asp Glu Glu Asp Asp Ala Pro Ala Ala Pro 30 35 40aag gac gac
gac gtc gac gtt act gtg gtg acc gtc aag aac tgg gat 195Lys Asp Asp
Asp Val Asp Val Thr Val Val Thr Val Lys Asn Trp Asp45 50 55 60gag
acc gtc aag aag tcc aag ttc gcg ctt gtg gag ttc tac gct cct 243Glu
Thr Val Lys Lys Ser Lys Phe Ala Leu Val Glu Phe Tyr Ala Pro 65 70
75tgg tgc ggc cac tgc aag acc ctc aag cct gag tac gct aag gct gcc
291Trp Cys Gly His Cys Lys Thr Leu Lys Pro Glu Tyr Ala Lys Ala Ala
80 85 90acc gcc ctg aag gct gct gct ccc gat gcc ctt atc gcc aag gtc
gac 339Thr Ala Leu Lys Ala Ala Ala Pro Asp Ala Leu Ile Ala Lys Val
Asp 95 100 105gcc acc cag gag gag tcc ctg gcc cag aag ttc ggc gtg
cag ggc tac 387Ala Thr Gln Glu Glu Ser Leu Ala Gln Lys Phe Gly Val
Gln Gly Tyr 110 115 120ccc acc ctc aag tgg ttc gtt gat ggc gag ctg
gct tct gac tac aac 435Pro Thr Leu Lys Trp Phe Val Asp Gly Glu Leu
Ala Ser Asp Tyr Asn125 130 135 140ggc ccc cgc gac gct gat ggc att
gtt ggc tgg gtg aag aag aag act 483Gly Pro Arg Asp Ala Asp Gly Ile
Val Gly Trp Val Lys Lys Lys Thr 145 150 155ggc ccc ccc gcc gtg acc
gtt gag gac gcc gac aag ctg aag tcc ctg 531Gly Pro Pro Ala Val Thr
Val Glu Asp Ala Asp Lys Leu Lys Ser Leu 160 165 170gag gcg gac gct
gag gtc gtt gtc gtc ggc tac ttc aag gcc ctg gag 579Glu Ala Asp Ala
Glu Val Val Val Val Gly Tyr Phe Lys Ala Leu Glu 175 180 185ggc gag
atc tac gac acc ttc aag tcc tac gcc gcc aag acc gag gac 627Gly Glu
Ile Tyr Asp Thr Phe Lys Ser Tyr Ala Ala Lys Thr Glu Asp 190 195
200gtg gtg ttc gtg cag acc acc agc gcc gac gtc gcc aag gcc gcc ggc
675Val Val Phe Val Gln Thr Thr Ser Ala Asp Val Ala Lys Ala Ala
Gly205 210 215 220ctg gac gcc gtg gac acc gtg tcc gtg gtc aag aac
ttc gcc ggt gag 723Leu Asp Ala Val Asp Thr Val Ser Val Val Lys Asn
Phe Ala Gly Glu 225 230 235gac cgc gcc acc gcc gtc ctg gcc acg gac
atc gac act gac tcc ctg 771Asp Arg Ala Thr Ala Val Leu Ala Thr Asp
Ile Asp Thr Asp Ser Leu 240 245 250acc gcg ttc gtc aag tcg gag aag
atg ccc ccc acc att gag ttc aac 819Thr Ala Phe Val Lys Ser Glu Lys
Met Pro Pro Thr Ile Glu Phe Asn 255 260 265cag aag aac tct gac aag
atc ttc aac agc ggc atc aac aag cag ctg 867Gln Lys Asn Ser Asp Lys
Ile Phe Asn Ser Gly Ile Asn Lys Gln Leu 270 275 280att ctg tgg acc
acc gcc gac gac ctg aag gcc gac gcc gag atc atg 915Ile Leu Trp Thr
Thr Ala Asp Asp Leu Lys Ala Asp Ala Glu Ile Met285 290 295 300act
gtg ttc cgc gag gcc agc aag aag ttc aag ggc cag ctg gtg ttc 963Thr
Val Phe Arg Glu Ala Ser Lys Lys Phe Lys Gly Gln Leu Val Phe 305 310
315gtg acc gtc aac aac gag ggc gac ggc gcc gac ccc gtc acc aac
ttc
1011Val Thr Val Asn Asn Glu Gly Asp Gly Ala Asp Pro Val Thr Asn Phe
320 325 330ttc ggc ctc aag ggc gcc acc tcg cct gtg ctg ctg ggc ttc
ttc atg 1059Phe Gly Leu Lys Gly Ala Thr Ser Pro Val Leu Leu Gly Phe
Phe Met 335 340 345gag aag aac aag aag ttc cgc atg gag ggc gag ttc
acg gct gac aac 1107Glu Lys Asn Lys Lys Phe Arg Met Glu Gly Glu Phe
Thr Ala Asp Asn 350 355 360gtg gct aag ttc gcc gag agc gtg gtg gac
ggc acc gcg cag gcc gtg 1155Val Ala Lys Phe Ala Glu Ser Val Val Asp
Gly Thr Ala Gln Ala Val365 370 375 380ctc aag tcg gag gcc atc ccc
gag gac ccc tat gag gat ggc gtc tac 1203Leu Lys Ser Glu Ala Ile Pro
Glu Asp Pro Tyr Glu Asp Gly Val Tyr 385 390 395aag att gtg ggc aag
acc gtg gag tct gtg gtt ctg gac gag acc aag 1251Lys Ile Val Gly Lys
Thr Val Glu Ser Val Val Leu Asp Glu Thr Lys 400 405 410gac gtg ctg
ctg gag gtg tac gcc ccc tgg tgc ggc cac tgc aag aag 1299Asp Val Leu
Leu Glu Val Tyr Ala Pro Trp Cys Gly His Cys Lys Lys 415 420 425ctg
gag ccc atc tac aag aag ctg gcc aag cgc ttt aag aag gtg gat 1347Leu
Glu Pro Ile Tyr Lys Lys Leu Ala Lys Arg Phe Lys Lys Val Asp 430 435
440tcc gtc atc atc gcc aag atg gat ggc act gag aac gag cac ccc gag
1395Ser Val Ile Ile Ala Lys Met Asp Gly Thr Glu Asn Glu His Pro
Glu445 450 455 460atc gag gtc aag ggc ttc cct acc atc ctg ttc tat
ccc gcc ggc agc 1443Ile Glu Val Lys Gly Phe Pro Thr Ile Leu Phe Tyr
Pro Ala Gly Ser 465 470 475gac cgc acc ccc atc gtg ttc gag ggc ggc
gac cgc tcg ctc aag tcc 1491Asp Arg Thr Pro Ile Val Phe Glu Gly Gly
Asp Arg Ser Leu Lys Ser 480 485 490ctg acc aag ttc atc aag acc aac
gcc aag atc ccg tac gag ctg ccc 1539Leu Thr Lys Phe Ile Lys Thr Asn
Ala Lys Ile Pro Tyr Glu Leu Pro 495 500 505aag aag ggc tcc gac ggc
gac gag ggc acc tcg gac gac aag gac aag 1587Lys Lys Gly Ser Asp Gly
Asp Glu Gly Thr Ser Asp Asp Lys Asp Lys 510 515 520ccc gcg tcc gac
aag gac gag ctg taa gcggctatct gaactacccc 1634Pro Ala Ser Asp Lys
Asp Glu Leu525 530aggtttggag cgtctgcttg cgcgcttgcg cttgcacact
gtgcatggat gggagttaag 1694gaggagacgg agcacggagg ctgcgctcgg
ttggtggctt ggagcaccgg cagcgcgtga 1754tccgtcctgg cagcagcaac
ggcggagcgg gcgcatattg gcgcgagctg gcgagcggct 1814gttgctggag
aggatatgct gccgggcggg aggaagggct aggggcagag atgagagcgt
1874tacgggctgg catgcgggcg cccgtgcctc tccctgcggt gcagtccttg
ctaggagacg 1934cacggttttg ccaaagaggg acgctgtcca cagccctgcg
actggaagtt ttttaggccc 1994tgcggtggta gtggtgttgg tacggttgtg
tgcataagat gaacaacgtt tctctcaaga 2054cgagactact agtatgctga
cggtgtgtgt atgtggtgga tggattgtgc cccgaccatg 2114aagagtgctg
tgttgcctcg gcgcttctgt cgccctggat gtgcgtggtt ccgaacgctg
2174gagtcatctg ttgaggagcg agggtgttgt cgggtccgcc cggcacggcc
gcgtgatgtc 2234cggatgggga ttgcgagcga gggcaaccgc agcgcagata
gcgccgcagc ggatcgagct 2294agcgcaggat gatgagagcc gggccttcgc
ggcgtgggat cagggaggag ccaaggcgga 2354gtgcatgcga ggaaaacagt
gtgcggcaaa gaacgggctg caagaacgcc ttgcgcaaa
241312532PRTChlamydomonas reinhardtii 12Met Asn Arg Trp Asn Leu Leu
Ala Leu Thr Leu Gly Leu Leu Leu Val1 5 10 15Ala Ala Pro Phe Thr Lys
His Gln Phe Ala His Ala Ser Asp Glu Tyr 20 25 30Glu Asp Asp Glu Glu
Asp Asp Ala Pro Ala Ala Pro Lys Asp Asp Asp 35 40 45Val Asp Val Thr
Val Val Thr Val Lys Asn Trp Asp Glu Thr Val Lys 50 55 60Lys Ser Lys
Phe Ala Leu Val Glu Phe Tyr Ala Pro Trp Cys Gly His65 70 75 80Cys
Lys Thr Leu Lys Pro Glu Tyr Ala Lys Ala Ala Thr Ala Leu Lys 85 90
95Ala Ala Ala Pro Asp Ala Leu Ile Ala Lys Val Asp Ala Thr Gln Glu
100 105 110Glu Ser Leu Ala Gln Lys Phe Gly Val Gln Gly Tyr Pro Thr
Leu Lys 115 120 125Trp Phe Val Asp Gly Glu Leu Ala Ser Asp Tyr Asn
Gly Pro Arg Asp 130 135 140Ala Asp Gly Ile Val Gly Trp Val Lys Lys
Lys Thr Gly Pro Pro Ala145 150 155 160Val Thr Val Glu Asp Ala Asp
Lys Leu Lys Ser Leu Glu Ala Asp Ala 165 170 175Glu Val Val Val Val
Gly Tyr Phe Lys Ala Leu Glu Gly Glu Ile Tyr 180 185 190Asp Thr Phe
Lys Ser Tyr Ala Ala Lys Thr Glu Asp Val Val Phe Val 195 200 205Gln
Thr Thr Ser Ala Asp Val Ala Lys Ala Ala Gly Leu Asp Ala Val 210 215
220Asp Thr Val Ser Val Val Lys Asn Phe Ala Gly Glu Asp Arg Ala
Thr225 230 235 240Ala Val Leu Ala Thr Asp Ile Asp Thr Asp Ser Leu
Thr Ala Phe Val 245 250 255Lys Ser Glu Lys Met Pro Pro Thr Ile Glu
Phe Asn Gln Lys Asn Ser 260 265 270Asp Lys Ile Phe Asn Ser Gly Ile
Asn Lys Gln Leu Ile Leu Trp Thr 275 280 285Thr Ala Asp Asp Leu Lys
Ala Asp Ala Glu Ile Met Thr Val Phe Arg 290 295 300Glu Ala Ser Lys
Lys Phe Lys Gly Gln Leu Val Phe Val Thr Val Asn305 310 315 320Asn
Glu Gly Asp Gly Ala Asp Pro Val Thr Asn Phe Phe Gly Leu Lys 325 330
335Gly Ala Thr Ser Pro Val Leu Leu Gly Phe Phe Met Glu Lys Asn Lys
340 345 350Lys Phe Arg Met Glu Gly Glu Phe Thr Ala Asp Asn Val Ala
Lys Phe 355 360 365Ala Glu Ser Val Val Asp Gly Thr Ala Gln Ala Val
Leu Lys Ser Glu 370 375 380Ala Ile Pro Glu Asp Pro Tyr Glu Asp Gly
Val Tyr Lys Ile Val Gly385 390 395 400Lys Thr Val Glu Ser Val Val
Leu Asp Glu Thr Lys Asp Val Leu Leu 405 410 415Glu Val Tyr Ala Pro
Trp Cys Gly His Cys Lys Lys Leu Glu Pro Ile 420 425 430Tyr Lys Lys
Leu Ala Lys Arg Phe Lys Lys Val Asp Ser Val Ile Ile 435 440 445Ala
Lys Met Asp Gly Thr Glu Asn Glu His Pro Glu Ile Glu Val Lys 450 455
460Gly Phe Pro Thr Ile Leu Phe Tyr Pro Ala Gly Ser Asp Arg Thr
Pro465 470 475 480Ile Val Phe Glu Gly Gly Asp Arg Ser Leu Lys Ser
Leu Thr Lys Phe 485 490 495Ile Lys Thr Asn Ala Lys Ile Pro Tyr Glu
Leu Pro Lys Lys Gly Ser 500 505 510Asp Gly Asp Glu Gly Thr Ser Asp
Asp Lys Asp Lys Pro Ala Ser Asp 515 520 525Lys Asp Glu Leu
530134PRTChlamydomonas reinhardtii 13Cys Gly His
Cys1144PRTChlamydomonas reinhardtii 14Lys Asp Glu
Leu1151424DNAChlamydomonas
reinhardtiiCDS(252)..(1310)misc_feature(279)..(279)Codon also can
encode Ser 15cgtcctattt taatactccg aaggaggcag ttggcaggca actgccactg
acgtcccgta 60agggtaaggg gacgtccact ggcgtcccgt aaggggaagg ggacgtaggt
acataaatgt 120gctaggtaac taacgtttga ttttttgtgg tataatatat
gtaccatgct tttaatagaa 180gcttgaattt ataaattaaa atatttttac
aatattttac ggagaaatta aaactttaaa 240aaaattaaca t atg aca gca att
tta gaa cgt cgt gaa aat tct agc cta 290 Met Thr Ala Ile Leu Glu Arg
Arg Glu Asn Ser Ser Leu 1 5 10tgg gct cgt ttt tgt gag tgg atc act
tca act gaa aac cgt tta tac 338Trp Ala Arg Phe Cys Glu Trp Ile Thr
Ser Thr Glu Asn Arg Leu Tyr 15 20 25atc ggt tgg ttc ggt gta atc atg
atc cca tgt ctt ctt act gca aca 386Ile Gly Trp Phe Gly Val Ile Met
Ile Pro Cys Leu Leu Thr Ala Thr30 35 40 45tca gta ttc atc atc gct
ttc atc gct gct ccg cca gta gac atc gat 434Ser Val Phe Ile Ile Ala
Phe Ile Ala Ala Pro Pro Val Asp Ile Asp 50 55 60ggt atc cgt gaa cca
gtt tca ggt tct ctt ctt tac ggt aac aac atc 482Gly Ile Arg Glu Pro
Val Ser Gly Ser Leu Leu Tyr Gly Asn Asn Ile 65 70 75att aca ggt gct
gta atc cca act tct aac gca atc ggt ctt cac ttc 530Ile Thr Gly Ala
Val Ile Pro Thr Ser Asn Ala Ile Gly Leu His Phe 80 85 90tac cca att
tgg gaa gct gct tct cta gac gag tgg tta tac aac ggt 578Tyr Pro Ile
Trp Glu Ala Ala Ser Leu Asp Glu Trp Leu Tyr Asn Gly 95 100 105ggt
cct tac caa ctt atc gtt tgt cac ttc ctt cta ggt gta tac tgc 626Gly
Pro Tyr Gln Leu Ile Val Cys His Phe Leu Leu Gly Val Tyr Cys110 115
120 125tac atg ggt cgt gag tgg gaa tta tct ttc cgt tta ggt atg cgt
cca 674Tyr Met Gly Arg Glu Trp Glu Leu Ser Phe Arg Leu Gly Met Arg
Pro 130 135 140tgg atc gct gta gct tac tca gct cca gta gct gca gct
tca gct gta 722Trp Ile Ala Val Ala Tyr Ser Ala Pro Val Ala Ala Ala
Ser Ala Val 145 150 155ttc tta gtt tac cct atc ggc caa ggt tca ttc
tct gac ggt atg cct 770Phe Leu Val Tyr Pro Ile Gly Gln Gly Ser Phe
Ser Asp Gly Met Pro 160 165 170tta ggt atc tct ggt act ttc aac ttc
atg atc gta ttc caa gca gaa 818Leu Gly Ile Ser Gly Thr Phe Asn Phe
Met Ile Val Phe Gln Ala Glu 175 180 185cac aac atc ctt atg cac cca
ttc cac atg tta ggt gtt gct ggt gta 866His Asn Ile Leu Met His Pro
Phe His Met Leu Gly Val Ala Gly Val190 195 200 205ttc ggt ggt tca
tta ttc tca gct atg cac ggt tct tta gtt act tca 914Phe Gly Gly Ser
Leu Phe Ser Ala Met His Gly Ser Leu Val Thr Ser 210 215 220tct tta
atc cgt gaa aca act gaa aac gaa tca gct aac gaa ggt tac 962Ser Leu
Ile Arg Glu Thr Thr Glu Asn Glu Ser Ala Asn Glu Gly Tyr 225 230
235cgt ttc ggt caa gaa gaa gaa act tac aac att gta gct gct cat ggt
1010Arg Phe Gly Gln Glu Glu Glu Thr Tyr Asn Ile Val Ala Ala His Gly
240 245 250tac ttt ggt cgt cta atc ttc caa tac gct tct ttc aac aac
tct cgt 1058Tyr Phe Gly Arg Leu Ile Phe Gln Tyr Ala Ser Phe Asn Asn
Ser Arg 255 260 265tca tta cac ttc ttc tta gct gct tgg ccg gta atc
ggt att tgg ttc 1106Ser Leu His Phe Phe Leu Ala Ala Trp Pro Val Ile
Gly Ile Trp Phe270 275 280 285act gct tta ggt tta tca act atg gca
ttc aac tta aac ggt ttc aac 1154Thr Ala Leu Gly Leu Ser Thr Met Ala
Phe Asn Leu Asn Gly Phe Asn 290 295 300ttc aac caa tca gta gta gac
tca caa ggt cgt gta cta aac act tgg 1202Phe Asn Gln Ser Val Val Asp
Ser Gln Gly Arg Val Leu Asn Thr Trp 305 310 315gca gac atc atc aac
cgt gct aac tta ggt atg gaa gta atg cac gag 1250Ala Asp Ile Ile Asn
Arg Ala Asn Leu Gly Met Glu Val Met His Glu 320 325 330cgt aac gct
cac aac ttc cct cta gac tta gct tca act aac tct agc 1298Arg Asn Ala
His Asn Phe Pro Leu Asp Leu Ala Ser Thr Asn Ser Ser 335 340 345tca
aac aac taa ttttttttta aactaaaata aatctggtta accataccta 1350Ser Asn
Asn350gtttatttta gtttatacac acttttcata tatatatact taatagctac
cataggcagt 1410tggcaggacg tccc 142416352PRTChlamydomonas
reinhardtii 16Met Thr Ala Ile Leu Glu Arg Arg Glu Asn Ser Ser Leu
Trp Ala Arg1 5 10 15Phe Cys Glu Trp Ile Thr Ser Thr Glu Asn Arg Leu
Tyr Ile Gly Trp 20 25 30Phe Gly Val Ile Met Ile Pro Cys Leu Leu Thr
Ala Thr Ser Val Phe 35 40 45Ile Ile Ala Phe Ile Ala Ala Pro Pro Val
Asp Ile Asp Gly Ile Arg 50 55 60Glu Pro Val Ser Gly Ser Leu Leu Tyr
Gly Asn Asn Ile Ile Thr Gly65 70 75 80Ala Val Ile Pro Thr Ser Asn
Ala Ile Gly Leu His Phe Tyr Pro Ile 85 90 95Trp Glu Ala Ala Ser Leu
Asp Glu Trp Leu Tyr Asn Gly Gly Pro Tyr 100 105 110Gln Leu Ile Val
Cys His Phe Leu Leu Gly Val Tyr Cys Tyr Met Gly 115 120 125Arg Glu
Trp Glu Leu Ser Phe Arg Leu Gly Met Arg Pro Trp Ile Ala 130 135
140Val Ala Tyr Ser Ala Pro Val Ala Ala Ala Ser Ala Val Phe Leu
Val145 150 155 160Tyr Pro Ile Gly Gln Gly Ser Phe Ser Asp Gly Met
Pro Leu Gly Ile 165 170 175Ser Gly Thr Phe Asn Phe Met Ile Val Phe
Gln Ala Glu His Asn Ile 180 185 190Leu Met His Pro Phe His Met Leu
Gly Val Ala Gly Val Phe Gly Gly 195 200 205Ser Leu Phe Ser Ala Met
His Gly Ser Leu Val Thr Ser Ser Leu Ile 210 215 220Arg Glu Thr Thr
Glu Asn Glu Ser Ala Asn Glu Gly Tyr Arg Phe Gly225 230 235 240Gln
Glu Glu Glu Thr Tyr Asn Ile Val Ala Ala His Gly Tyr Phe Gly 245 250
255Arg Leu Ile Phe Gln Tyr Ala Ser Phe Asn Asn Ser Arg Ser Leu His
260 265 270Phe Phe Leu Ala Ala Trp Pro Val Ile Gly Ile Trp Phe Thr
Ala Leu 275 280 285Gly Leu Ser Thr Met Ala Phe Asn Leu Asn Gly Phe
Asn Phe Asn Gln 290 295 300Ser Val Val Asp Ser Gln Gly Arg Val Leu
Asn Thr Trp Ala Asp Ile305 310 315 320Ile Asn Arg Ala Asn Leu Gly
Met Glu Val Met His Glu Arg Asn Ala 325 330 335His Asn Phe Pro Leu
Asp Leu Ala Ser Thr Asn Ser Ser Ser Asn Asn 340 345
350171278DNAChlamydomonas reinhardtiiCDS(1)..(1272) 17atg ggc cat
cat cat cat cat cat cat cat cat cac agc agc ggc cat 48Met Gly His
His His His His His His His His His Ser Ser Gly His1 5 10 15atc gaa
ggt cgt cat atg gcg act act gag tcc tcg gcc ccg gcg gcc 96Ile Glu
Gly Arg His Met Ala Thr Thr Glu Ser Ser Ala Pro Ala Ala 20 25 30acc
acc cag ccg gcc agc acc ccg ctg gcg aac tcg tcg ctg tac gtc 144Thr
Thr Gln Pro Ala Ser Thr Pro Leu Ala Asn Ser Ser Leu Tyr Val 35 40
45ggt gac ctg gag aag gat gtc acc gag gcc cag ctg ttc gag ctc ttc
192Gly Asp Leu Glu Lys Asp Val Thr Glu Ala Gln Leu Phe Glu Leu Phe
50 55 60tcc tcg gtt ggc cct gtg gcc tcc att cgc gtg tgc cgc gat gcc
gtc 240Ser Ser Val Gly Pro Val Ala Ser Ile Arg Val Cys Arg Asp Ala
Val65 70 75 80acg cgc cgc tcg ctg ggc tac gcc tac gtc aac tac aac
agc gct ctg 288Thr Arg Arg Ser Leu Gly Tyr Ala Tyr Val Asn Tyr Asn
Ser Ala Leu 85 90 95gac ccc cag gct gct gac cgc gcc atg gag acc ctg
aac tac cat gtc 336Asp Pro Gln Ala Ala Asp Arg Ala Met Glu Thr Leu
Asn Tyr His Val 100 105 110gtg aac ggc aag cct atg cgc atc atg tgg
tcg cac cgc gac cct tcg 384Val Asn Gly Lys Pro Met Arg Ile Met Trp
Ser His Arg Asp Pro Ser 115 120 125gcc cgc aag tcg ggc gtc ggc aac
atc ttc atc aag aac ctg gac aag 432Ala Arg Lys Ser Gly Val Gly Asn
Ile Phe Ile Lys Asn Leu Asp Lys 130 135 140acc atc gac gcc aag gcc
ctg cac gac acc ttc tcg gcc ttc ggc aag 480Thr Ile Asp Ala Lys Ala
Leu His Asp Thr Phe Ser Ala Phe Gly Lys145 150 155 160att ctg tcc
tgc aag gtt gcc act gac gcc aac ggc gtg tcg aag ggc 528Ile Leu Ser
Cys Lys Val Ala Thr Asp Ala Asn Gly Val Ser Lys Gly 165 170 175tac
ggc ttc gtg cac ttc gag gac cag gcc gct gcc gat cgc gcc att 576Tyr
Gly Phe Val His Phe Glu Asp Gln Ala Ala Ala Asp Arg Ala Ile 180 185
190cag acc gtc aac cag aag aag att gag ggc aag atc gtg tac gtg gcc
624Gln Thr Val Asn Gln Lys Lys Ile Glu Gly Lys Ile Val Tyr Val Ala
195 200 205ccc ttc cag aag cgc gct gac cgc ccc agg gca agg acg ttg
tac acc 672Pro Phe Gln Lys Arg Ala Asp Arg Pro Arg Ala Arg Thr Leu
Tyr Thr 210 215 220aac gtg ttc gtc aag aac ttg ccg gcc gac atc ggc
gac gac gag ctg 720Asn Val Phe Val Lys Asn Leu Pro Ala Asp Ile Gly
Asp Asp Glu Leu225 230 235 240ggc aag atg gcc acc gag cac ggc gag
atc acc agc gcg gtg gtc atg 768Gly Lys Met Ala Thr Glu His Gly Glu
Ile Thr Ser Ala Val Val Met 245 250 255aag gac gac aag ggc ggc agc
aag ggc ttc ggc ttc atc aac ttc aag 816Lys Asp Asp Lys Gly Gly Ser
Lys Gly Phe Gly Phe Ile Asn Phe Lys 260 265 270gac gcc gag tcg gcg
gcc
aag tgc gtg gag tac ctg aac gag cgc gag 864Asp Ala Glu Ser Ala Ala
Lys Cys Val Glu Tyr Leu Asn Glu Arg Glu 275 280 285atg agc ggc aag
acc ctg tac gcc ggc cgc gcc cag aag aag acc gag 912Met Ser Gly Lys
Thr Leu Tyr Ala Gly Arg Ala Gln Lys Lys Thr Glu 290 295 300cgc gag
gcg atg ctg cgc cag aag gcc gag gag agc aag cag gag cgt 960Arg Glu
Ala Met Leu Arg Gln Lys Ala Glu Glu Ser Lys Gln Glu Arg305 310 315
320tac ctg aag tac cag agc atg aac ctg tac gtc aag aac ctg tcc gac
1008Tyr Leu Lys Tyr Gln Ser Met Asn Leu Tyr Val Lys Asn Leu Ser Asp
325 330 335gag gag gtc gac gac gac gcc ctg cgt gag ctg ttc gcc aac
tct ggc 1056Glu Glu Val Asp Asp Asp Ala Leu Arg Glu Leu Phe Ala Asn
Ser Gly 340 345 350acc atc acc tcg tgc aag gtc atg aag gac ggc agc
ggc aag tcc aag 1104Thr Ile Thr Ser Cys Lys Val Met Lys Asp Gly Ser
Gly Lys Ser Lys 355 360 365ggc ttc ggc ttc gtg tgc ttc acc agc cac
gac gag gcc acc cgg ccg 1152Gly Phe Gly Phe Val Cys Phe Thr Ser His
Asp Glu Ala Thr Arg Pro 370 375 380ccc gtg acc gag atg aac ggc aag
atg gtc aag ggc aag ccc ctg tac 1200Pro Val Thr Glu Met Asn Gly Lys
Met Val Lys Gly Lys Pro Leu Tyr385 390 395 400gtg gcc ctg gcg cag
cgc aag gac gtg cgc cgt gcc acc cag ctg gag 1248Val Ala Leu Ala Gln
Arg Lys Asp Val Arg Arg Ala Thr Gln Leu Glu 405 410 415gcc aac atg
cag gcg cgc atg taa ggatcc 1278Ala Asn Met Gln Ala Arg Met
42018423PRTChlamydomonas reinhardtii 18Met Gly His His His His His
His His His His His Ser Ser Gly His1 5 10 15Ile Glu Gly Arg His Met
Ala Thr Thr Glu Ser Ser Ala Pro Ala Ala 20 25 30Thr Thr Gln Pro Ala
Ser Thr Pro Leu Ala Asn Ser Ser Leu Tyr Val 35 40 45Gly Asp Leu Glu
Lys Asp Val Thr Glu Ala Gln Leu Phe Glu Leu Phe 50 55 60Ser Ser Val
Gly Pro Val Ala Ser Ile Arg Val Cys Arg Asp Ala Val65 70 75 80Thr
Arg Arg Ser Leu Gly Tyr Ala Tyr Val Asn Tyr Asn Ser Ala Leu 85 90
95Asp Pro Gln Ala Ala Asp Arg Ala Met Glu Thr Leu Asn Tyr His Val
100 105 110Val Asn Gly Lys Pro Met Arg Ile Met Trp Ser His Arg Asp
Pro Ser 115 120 125Ala Arg Lys Ser Gly Val Gly Asn Ile Phe Ile Lys
Asn Leu Asp Lys 130 135 140Thr Ile Asp Ala Lys Ala Leu His Asp Thr
Phe Ser Ala Phe Gly Lys145 150 155 160Ile Leu Ser Cys Lys Val Ala
Thr Asp Ala Asn Gly Val Ser Lys Gly 165 170 175Tyr Gly Phe Val His
Phe Glu Asp Gln Ala Ala Ala Asp Arg Ala Ile 180 185 190Gln Thr Val
Asn Gln Lys Lys Ile Glu Gly Lys Ile Val Tyr Val Ala 195 200 205Pro
Phe Gln Lys Arg Ala Asp Arg Pro Arg Ala Arg Thr Leu Tyr Thr 210 215
220Asn Val Phe Val Lys Asn Leu Pro Ala Asp Ile Gly Asp Asp Glu
Leu225 230 235 240Gly Lys Met Ala Thr Glu His Gly Glu Ile Thr Ser
Ala Val Val Met 245 250 255Lys Asp Asp Lys Gly Gly Ser Lys Gly Phe
Gly Phe Ile Asn Phe Lys 260 265 270Asp Ala Glu Ser Ala Ala Lys Cys
Val Glu Tyr Leu Asn Glu Arg Glu 275 280 285Met Ser Gly Lys Thr Leu
Tyr Ala Gly Arg Ala Gln Lys Lys Thr Glu 290 295 300Arg Glu Ala Met
Leu Arg Gln Lys Ala Glu Glu Ser Lys Gln Glu Arg305 310 315 320Tyr
Leu Lys Tyr Gln Ser Met Asn Leu Tyr Val Lys Asn Leu Ser Asp 325 330
335Glu Glu Val Asp Asp Asp Ala Leu Arg Glu Leu Phe Ala Asn Ser Gly
340 345 350Thr Ile Thr Ser Cys Lys Val Met Lys Asp Gly Ser Gly Lys
Ser Lys 355 360 365Gly Phe Gly Phe Val Cys Phe Thr Ser His Asp Glu
Ala Thr Arg Pro 370 375 380Pro Val Thr Glu Met Asn Gly Lys Met Val
Lys Gly Lys Pro Leu Tyr385 390 395 400Val Ala Leu Ala Gln Arg Lys
Asp Val Arg Arg Ala Thr Gln Leu Glu 405 410 415Ala Asn Met Gln Ala
Arg Met 420
* * * * *