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.

Description

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.

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.

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.

FIG. 10 shows luciferase activity accumulated with the chloroplast.

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.

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 λ-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 (OD₆₀₀), 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 MgCl₂, 5 mM β-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 5×10⁶ cells/mL under constant light. Cells were harvested by centrifugation at 4° C. for 5 minutes at 4,000 g. Cells were either used immediately or frozen in liquid N₂ for storage at −70° 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⁺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 (OD₆₀₀), 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.

Claims

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;

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;

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.

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.

65. The chloroplast expression cassette of claim 64, wherein the dna sequence encodes a vertebrate polypeptide.

66. The chloroplast cassette of claim 64, wherein the dna sequence encodes a mammalian polypeptide.

67. The chloroplast expression cassette of claim 64, wherein the polypeptide is an antibody.

68. The chloroplast cassette of claim 67, wherein the polypeptide is a single chain antibody.

69. The chloroplast cassette of claim 64, wherein the chloroplast is a plant chloroplast.

70. The chloroplast cassette of claim 64, wherein the chloroplast is an algal chloroplast.

71. The chloroplast expression cassette of claim 64, wherein the 5′ leader sequence is a 5′ untranslated region (UTR).

72. A cell containing the chloroplast expression cassette of claim 64.

73. An alga or plant comprising a cell of claim 72.

74. An algal chloroplast comprising the expression cassette of claim 64.

75. A micro-algae containing a chloroplast of claim 74.

76. The micro-algae of claim 75, wherein the algae is Chlamydomonas reinhardtii.

77. The chloroplast expression cassette of claim 64, further comprising a 3′ UTR.

78. The expression cassette of claim 64, wherein the dna sequence encodes a eukaryotic protein.

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.

80. The chloroplast expression cassette of claim 79, wherein the homologous gene to be replaced is a psbA gene.

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.

82. A eukaryotic cell comprising a transformed chloroplast producing a protein according to the method of claim 81.

83. The chloroplast expression cassette of claim 79, wherein the protein is an antibody.

84. A microalgal chloroplast transformed with an expression cassette of claim 65.

85. The microalgal chloroplast of claim 84, wherein said microalga is Chlamydomonas reinhardtii.

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.