Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-07-10
2001-09-25
Ketter, James (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06294653
ABSTRACT:
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
., 1-3:2227-2235 (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 a
Fitting Thomas
Holmes Emily
Ketter James
The Scripps Research Institute
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