TERRAMARK MARKENCREATION GMBH (DE)
KHABAR KHALID S ABU (SA)
US6762038B1 | 2004-07-13 | |||
US7129062B2 | 2006-10-31 |
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King Faisal Specialist Hospital and Research Centre Claims 1. Method for increasing the expression of a protein in cells, preferably in eukaryotic cells, said method comprising the step of reducing the number of RNase L cleavage sites in the nucleic acid sequence of said protein. 2. Method according to claim 1, wherein said number of RNase L cleavage sites is reduced by at least 10%, preferably at least 25%, more preferably at least 50%. 3. Method according to claim 1 or 2, wherein said cleavage sites are UU and/or UA dinucleotides. 4. Method according to any of the foregoing claims, wherein said step of reducing the number of RNase L cleavage sites reduces said number in the coding region of said nucleic acid sequence. 5. Method according to claim 4, wherein said step of reducing the number of RNase L cleavage sites in said nucleic acid sequence is performed without altering the amino acid sequence of said protein. 6. Method according to claim 5, wherein in said step of reducing the number of RNase L cleavage sites a codon comprising a UU and/or UA dinucleotide is exchanged for an alternative codon not comprising a UU and/or UA dinucleotide and coding for the same amino acid. 7. Method according to claim 5, wherein in said step of reducing the number of RNase L cleavage sites at least one codon of an adjacent pair of codons comprising a UU and/or UA dinucleotide is exchanged for an alternative codon coding for the same amino acid so that said adjacent pair of codons does no longer comprise a UU and/or UA dinucleotide. 8. Method according to claim 6 or 7, wherein said alternative codon is the more frequently used codon in said cells, preferably in said eukaryotic cells. 9. Method according to any of claims 1 to 3, wherein said step of reducing the number of RNase L cleavage sites reduces said number in the non-coding region of said nucleic acid sequence. 10. Method according to claim 9, wherein said non-coding region is a 5'UTR, a 3'UTR, or an intron. 1 1. Method according to claim 9 or 10, wherein said step of reducing the number of RNase L cleavage sites is performed by mutation, deletion, or insertion of nucleotides. 12. Method according to any of the foregoing claims further comprising the step of codon optimization prior to said step of reducing the number of RNase L cleavage sites. 13. Method according to any of the foregoing claims further comprising the step of transfecting said nucleic acid sequence of said protein into said cells, preferably into said eukaryotic cells in form of an expression active PCR product or contained in an expression vector after said step of reducing the number of RNase L cleavage sites. 14. Method according to claim 13 further comprising the step of translating said protein from said expression active PCR product or expression vector in said cells, preferably in said eukaryotic cells. 15. Method according to any of the foregoing claims, wherein said protein is selected from the group comprising reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs. 16. Nucleic acid sequence, wherein the number of RNase L cleavage sites is reduced by at least 10%, preferably at least 25%, more preferably at least 50%. 17. Nucleic acid sequence according to claim 16, wherein said nucleic acid sequence is the nucleic acid sequence of a protein selected from the group comprising reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs. 18. Nucleic acid sequence according to claim 16 having a sequence selected from SEQ ID NO: 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 24, 26, 28, 30-36. 19. Expression active PCR product or expression vector comprising the nucleic acid sequence according to any of claims 16 to 18. 20. Host cell of any organism containing the expression active PCR product or expression vector according to claim 19. 21. Protein produced by the method according to claim 14, wherein said protein is selected from the group comprising reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs. |
The present invention relates to a method for increasing the expression of a protein in cells, preferably in eukaryotic cells by reducing the number of RNase L cleavage sites in the coding and/or non-coding region of the nucleic acid sequence of said protein. Furthermore, it relates to nucleic acid sequences exhibiting a reduced number of RNase L cleavage sites as well as to the proteins translated from such sequences.
Transient response genes regulate critical biological responses that include cell proliferation, signal transduction events, and responses to exogenous agents, such as inflammatory stimuli, microbes, and radiation (Lai et al., 2006; LaI et al., 2004; Lopez de Silanes et al., 2005). They are controlled by both cis-acting factors such as specific sequence elements and trans-acting factors like certain RNA binding proteins. Sequence elements, mainly in the 3' untranslated region (3'UTR) and modulation by RNA binding proteins can affect messenger RNA (mRNA) stability and protein translation.
An appreciable number of genes harbor destabilizing sequence elements in the 3' UTR of their mRNA, mostly adenylate-uridylate (AU)-rich elements (AREs). These AREs comprise a heterogeneous group of sequence classes that can affect protein interactions with the mRNA, and therefore influence the mRNA decay characteristics (Bakheet et al., 2006; Barreau et al., 2005). The stabilization of cellular mRNAs can occur by the activity of mRNA stabilization- promoting proteins, such as the HuR protein, or by inactivation of RNA decay promoting proteins, such as the zinc finger protein tristetraprolin (TTP).
A different class of trans-acting factors that can affect cellular mRNAs is the endoribonuclease Ribonuclease L (RNase L), which is an ubiquitous intracellular enzyme that has previously been thought to be specific to viral mRNAs. However, recent studies showed that RNase L can also participate in the transient response of certain biological processes (Khabar et al., 2003a; Li et al., 2000). RNase L is considered to be a part of the interferon (IFN) system. IFN induces gene expression of the enzyme oligoadenylate synthetase (OAS) which, upon binding to viral double-stranded RNA intermediates, becomes activated and synthesizes short 2'-5'oligoadenylates (2-5A). These, in turn, activate RNase L, which potently degrades viral mRNAs. RNase L is activated by subnanomolar levels of 2-5A, resulting in the cleavage of single-stranded regions of viral RNA, preferentially after UU and UA dinucϊeotides in viral mRNAs (Wrechester et al., 1981; Han et al., 2004). At higher levels, RNase L may lead to broader effects such as cleavage of 18 S and 28 S ribosomal RNAs (Wreschner et al., 1981).
In recent years it has become widely accepted that RNase L participates in the degradation of selected cellular mRNAs (Bisbal et al., 2000; Chandrasekaran et al., 2004; Khabar et al., 2003b; Le Roy et al., 2001; Li et al., 2000). Specifically, RNase L has been shown to down- regulate PKR mRNA (Khabar et al., 2003b). During the IFN antiviral response in normal cells, PKR mRNA expression is transient, but in RNase L-null cells, extended kinetics of PKR mRNA expression is observed due to increased mRNA stability (Khabar et al., 2003b). The effect results in prolongation of the PKR-dependent phosphorylation of the subunit of eukaryotic translation initiation factor 2, eIF2α, a process that leads to inhibition of viral protein synthesis (Khabar et al., 2003b). Thus, RNase L contributes to the transient nature of the IFN response in order to ensure a brief translational arrest imparted by PKR. A similar role of RNase L negative regulation of the IFN response has also been suggested by the report that a novel IFN-stimulated gene encoding a 43-kDa ubiquitin-specific protease, designated ISG43, is down-regulated by RNase L (Li et al., 2000). This regulation occurs at the level of mRNA stability, since the ISG43 mRNA half life increases in RNase L-null cells (Li et al., 2000). RNase L can down-regulate another functionally important cellular mRNA, myoD, encoding an import transcription factor essential for muscle differentiation. RNase L and its inhibitor RLI are sequentially induced during C2 cell line myoblast differentiation to myotubes (Bisbal et al., 2000). Inhibition or over-expression of RNase L prolongs or decreases MyoD mRNA half life, respectively (Bisbal et al., 2000). Since a pool of RNase L molecules localizes to mitochondria and is increased following IFN-α treatment, a role of RNase L in down-regulating mitochondrial mRNAs, such as those of CYTB, ATPase 6 (ATP6), and cytochrome oxidase II (CO), has been proposed as a mechanism of the antiproliferative action of IFN (Le Roy et al., 2001). This was demonstrated by reducing RNase L activity through the introduction of an antisense construct or by directly activating RNase L activity by 2-5 A (Le Roy et al., 2001). It has further been shown that RNase L exhibits a preference for viral mRNA, for example encephalomyocarditits virus (EMCV), when compared to non- viral mRNAs, particularly in conditions where the levels of 2-5 A is limiting. (Li et al., 1998a). The effects of RNase L on cellular mRNAs appear to be highly restricted to specific mRNAs, since no global effects on cellular mRNAs are observed in the studies that have dealt with this topic. Furthermore, none of the above studies demonstrates a direct binding of RNasc L to ceiiuiar target mRNAs or a sequence specificity as shown for viral mRNAs. Thus, the mechanism of RNase L activity in connection with non-viral cellular mRNAs remains largely unclear.
Dominant negative forms of RNase L have previously been generated by either amino acid substitutions or by truncation of the full length protein. For example, a dominant negative RNase L, ZBl, inhibits the antiviral and antiproliferative action of wild type RNase L; it is a truncated form of murine RNase L, which lacks 89 carboxy-terminal amino acids (Hassel et al., 1993). Dong et al., 2001 describe other truncations and point mutations of RNase L, e.g. mutations in the nuclease domain (R667A).
U.S. patent 6,762,038 suggests the use of mutant embryonic fibroblasts cell lines (MEFs) generated from mice having a homozygous disruption in their RNase L gene (Zhou et al., 1997) for enhanced expression of transfected genes. However, the effect of enhanced expression is restricted to these particular cell lines. Furthermore, the creation of new RNase L-null cell lines is a very laborious process and not readily applicable to all mammalian and/or eukaryotic expression systems.
Mammalian expression systems have become an important means for therapeutic protein and antibody production and possess several advantages over prokaryotic expression systems, e.g. with respect to proper protein folding and posttranslational modifications, such as glycolysation. However, in many systems, low protein expression yields represent a costly technical obstacle. This is particularly problematic, if the desired protein is inherently difficult to express, e.g. membrane proteins, such as G-protein linked receptors (GPCRs), large proteins, antibodies, fusion proteins, protein complexes, vaccines, and blood plasma proteins. Reasons for this difficulty could be an inherent instability of the protein itself, an inherent instability of the mRNA, as it is the case for AU-rich elements containing mRNAs, or weak promoter activity. Common solutions to these problems focused on increasing the expression of proteins by providing strong promoters, such as the CMV promoter, and enhancer elements upstream or downstream of the promoter including specific types of introns, such as intron A of CMV. Other solutions involved chromatin Matrix Attachment R.egion (MAR) elements which are 300-3000 base pairs long DNA elements that are important in nuclear and chromosomal architecture. It was proposed that these elements prevent the neighbouring chromatin from affecting transgene expression, which leads to an increased probability of isolating a clone exhibiting the desired regulated expression (U.S. patent 7,129,062). This particular approach is potentially problematic as it may involve several different vector constructs to achieve the effect. Despite the availability of these approaches, there is still a need to further increase the protein expression of proteins that are difficult to express, such as those mentioned above.
Codon optimization (or codon usage optimization) is another method known in the art to boost protein production. It is based on the observation that, if a nucleic acid sequence encoding the protein to be expressed contains codons (triplets) that are rarely used by the host, its expression level will not be maximal. Codon optimization basically involves altering the rare codons in the target nucleic acid sequence, so that they more closely reflect the codon usage of the host. The information usually used for the optimization process is therefore the DNA or protein sequence to be optimized and a codon usage table of the respective host (see for example Table 1 for the human genome). The codon usage table lists the relative frequency of each possible codon for a particular amino acid in a given organism. A full list of codon usage bias in all organisms is found on the website: http://www.kazusa.or.ip/codon/. Several web-based programs are also available to optimize codons based on codon usage bias for a given host organism. Codon optimization may be successful in some situations where genes of non-human or non-mammalian origin are expressed in human or other mammalian host cells, and vice versa. However, codon usage is just one of many factors influencing the expression level of a protein, and the effect of codon optimization is often limited.
It was an object of the present invention to provide for a method to significantly improve the yield of endogenous and exogenous (recombinant) proteins expressed in cells of any organism, preferably in eukaryotic cells, including proteins that are inherently difficult to express. This method should be time and cost efficient and should allow for the large-scale production of proteins in cells of any type of organism including prokaryotic and eukaryotic cells. It was another object of the present invention to achieve this improvement in expression yields without changing regulatory approved features for the production of recombinant proteins, such as the cell lines used, recombinant protein characteristics, or the use of exogenous materials, in order to allow for the production of therapeutically used proteins.
The objects of the present invention are solved by a method for increasing the expression of a protein in cells, preferably eukaryotic cells, said method comprising the step of reducing the number of RNase L cleavage sites in the nucleic acid sequence of said protein. In one embodiment, said cells are prokaryotic cells; in another embodiment, said cells are eukaryotic cells.
The nucleic acid sequence of a protein comprises both coding and non-coding regions, i.e. regions that are translated into a sequence of amino acids (also referred to as exons) and regions that are not translated into a sequence of amino acids. Non-coding regions of the nucleic acid sequence are for example the 5' untranslated region (5'UTR), the 3' untranslated region (3'UTR), and introns. All of these elements (5'UTR, 3'UTR, introns, and the coding region) can control gene and protein expression, and are, thus, targets for the above method. According to the invention, said step of reducing the number of RNase L cleavage sites reduces said number either in the coding region or non-coding region, or in both.
In one embodiment said number of RNase L cleavage sites is reduced by at least 10%, preferably at least 25%, more preferably at least 50% (compared to the number of RNase L cleavage sites in the wild type nucleic acid sequence).
Preferably said cleavage sites are UU and/or UA dinucleotides.
RNase L is an endoribonuclease, and is, thus, only active on the RNA level (both primary RNA, i.e. unspliced, and mRNA, i.e. spliced). UU and UA dinucleotides only occur on the RNA level.
However, it is preferred that said step of reducing the number of RNase L cleavage sites in the nucleic acid sequence of said protein is performed on the DNA level: UU and UA dinucleotides in an RNA sequence correspond to TT and TA dinucleotides in a DNA sequence. Techniques that allow to specifically change a given DNA sequence are well know in the art and include, but are not limited to gene synthesis, site-directed mutagenesis, deletion mutations by restriction digestion, and mutation introduction by recombination. The technique of gene synthesis is particularly preferred according to the present invention.
In one embodiment of the present invention said step of reducing the number of RNase L cleavage sites reduces said number in the coding region of said nucleic acid sequence.
Preferably said step of reducing the number of RNase L cleavage sites in said nucleic acid sequence is performed without altering the amino acid sequence of said protein. The open reading frame (ORF) is, thus, not altered by said step.
In one embodiment in said step of reducing the number of RNase L cleavage sites a codon comprising a UU and/or UA dinucleotide is exchanged for an alternative codon not comprising a UU and/or UA dinucleotide and coding for the same amino acid.
In one embodiment in said step of reducing the number of RNase L cleavage sites at least one codon of an adjacent pair of codons comprising a UU and/or UA dinucleotide is exchanged for an alternative codon coding for the same amino acid so that said adjacent pair of codons does no longer comprise a UU and/or UA dinucleotide.
Preferably the first codon of said adjacent pair of codons comprising a UU and/or UA dinucleotide is exchanged.
In one embodiment said alternative codon is the more frequently used codon in said cells, preferably in said eukaryotic cells.
In one embodiment of the present invention said step of reducing the number of RNase L cleavage sites reduces said number in the non-coding region of said nucleic acid sequence.
Preferably said non-coding region is a 5'UTR, a 3'UTR, or an intron.
Examples for introns include, but are not limited to the CMV intron, SV40 intron, rabbit beta globin intron (RBTG), and synthetic introns. In one embodiment said step of reducing the number of RNase L cleavage sites in the non- coding region of said nucleic acid sequence is performed by mutation, deletion, or insertion of nucleotides.
Preferably said step of reducing the number of RNase L cleavage sites in the non-coding region of said nucieic acid sequence does not alter functionally important elements in the non- coding region, such as sequences in the 5'UTR that are close to the initiation codon (ATG), since they may harbor translation enhancing sequences (e.g. kozac), the poly A signal in the 3'UTR (e.g. AAUAAA or AUUAAA) or other necessary or accessory sequence elements used for polyadenylation, intron-exon junctions/boundaries, splicing branch points and exon donor/acceptor splice sites in introns, and the CT-rich area between the splice acceptor site to the end of the branch point. For example there is an U-rich 50 nucleotide region that is downstream of the strong poly A signal, which should not be altered when possible (Legendre and Gautheret, 2003).
Preferably said step of reducing the number of RNase L cleavage sites in the non-coding region does not (a) change the GC content of an intron to more than 80% and its length to less than 80%,
(b) change the GC content of a 5'UTR to more than 80%, and
(c) change the GC content of a 3'UTR to more than 80% and its length to less than 80%.
In one embodiment above method further comprises the step of codon optimization prior to said step of reducing the number of RNase L cleavage sites.
In one embodiment above method further comprises the step of transfecting said nucleic acid sequence of said protein into said cells, preferably into said eukaryotic cells in form of an expression active PCR product or contained in an expression vector after said step of reducing the number of RNase L cleavage sites.
The term "expression active PCR product" as used herein is meant to refer to a PCR product that is generated by PCR amplification using two primers complementary to sequences flanking the DNA sequence of interest, such as a cDNA, an open reading frame, or a gene that is contained in an expression vector, wherein the resulting PCR product contains a promoter, the DNA sequence of interest, and a termination sequence, and allows the expression of the DNA of interest, when transfected to a host cell (see also: Al-Zoghaibi et al., 2007).
According to the present invention any expression vector can be used, however, eukaryotic/mammalian expression vectors are preferable. Mammalian expression vectors are widespread tools to study the biological function of a protein, and various types (e.g. plasmid- based or viral-based vectors) are known in the art. Suitable expression vectors are not limited to a specific promoter, 5'UTR, 3'UTR, or intron. They can be constitutively expressed, inducible, repressed or regulatable.
According to the invention, it is preferred that the promoter is eukaryotic and the termination site is a poly A sequence containing a polyadenylation signal. Eukaryotic or mammalian promoters are known in the art and include, but are not limited to cytomegalovirus (CMV) immediate early promoter, SV40 promoter, elongation factor (EF) promoter, RSV promoter, and chicken β-actin promoter. Preferred eukaryotic polyadenylation signals are bovine growth factor (BGH) poly site, growth hormone poly A, SV40 poly A, and HSK poly A (Foecking and Hofstetter, 1986; Kobayashi et al., 1997). Examples of eukaryotic terminators are bovine growth factor (BGH) poly A site, SV40 poly A, HSK poly A, and synthetic poly A.
Methods for transiently or stably transfecting cells with DNA/vectors are well known in the art. These include, but are not limited to calcium phosphate co-precipitation, electroporation, cationic polymer transfection, and liposome-mediated transfection (e.g. lipofection). Reagents for liposome-mediated transfection are commercially available, e.g. lipofectamine (Invitrogen) and polyethylenimine (Sigma). Cells can also be transfected by viral transfection or via viral coat particles. Another preferred method for the transfection of cells according to the present invention is the in vivo microinjection of said expression active PCR product or said expression vector and selectively growing the cells containing said expression active PCR product or said expression vector with or without the help of a selection drug. Thus, the expression active PCR product or expression vector in accordance with the present invention may additionally comprise a selectable marker.
For the generation of stable cell lines, clones can be selected using various selectable markers, which include, but are not limited to neomycin, blasticidin, puromycin, zeocin, hygromycin, and dihydrofolate reductase (dhfr). Suitable eukaryotic/mammalian cells (host cells) for all of the above methods are also well known in the art and include, but are not limited to CHO, HEK 293, HeLa, and COS-7 cells.
In one embodiment the above method further comprises the step of translating said protein from said expression active PCR product or expression vector in said cells, preferably in said eukaryotic cells.
Preferably said protein is selected from the group comprising reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs.
The objects of the present invention are also solved by a nucleic acid sequence, wherein the number of RNase L cleavage sites is reduced by at least 10%, preferably at least 25%, more preferably at least 50% (compared to the number of RNase L cleavage sites in the wild type nucleic acid sequence).
Preferably said nucleic acid sequence is produced by the method as described above.
Preferably said nucleic acid sequence is the nucleic acid sequence of a protein selected from the group comprising reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs.
In one embodiment said nucleic acid sequence has a sequence selected from SEQ ID NO: 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 24, 26, 28, 30-36, preferably SEQ ID NO: 9, 15, 17, 23, 30-36.
The objects of the present invention are also solved by an expression active PCR product or an expression vector comprising the nucleic acid as described above.
The objects of the present invention are further solved by a host cell containing the above expression active PCR product or expression vector. Finally, the objects of the present invention are also solved by a protein produced by the method as described above, wherein said protein is selected from the group comprising reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs.
According to the present invention the term "therapeutic proteins" is meant to include proteins used in a pharmaceutical context, such as antibody fragments, immunoglobulin chains, heavy and light chains, Fab fragments, enzymes, growth factors, interferons, cytokines, lymphokines, adhesion molecules, receptors, as well as derivatives or fragments thereof.
According to the present invention reporter proteins (either alone or fused to another protein) are particularly preferred "targets" for the above described method. There are many fluorescent and non-fluorescent reporter proteins including (without being limited to) green fluorescent proteins (GFP), red fluorescence proteins (RFP), yellow fluorescent proteins (YFP), blue and cyanine fluorescent proteins (CFP), luciferase, secreted alkaline phosphatase (SEAP), Chloramphenicol acetyltransferase (CAT), secreted hormone, secreted cytokine, β- galactosidase, and other fluorescent and bioluminescent proteins. The choice of reporter protein depends on the cell line used (endogenous activity), the nature of the experiment (e.g. dynamics of gene expression and transfection efficiency), and the adaptability of the assay to the chosen detection method (Naylor 1999). Several modifications of the reporter proteins themselves have been sought to improve the reporter performance, such as rapid response and magnitude of change, e.g. the use of destabilization elements (Li et al., 1998b; Zhao et al., 1995). Green fluorescent protein (GFP) and other fluorescent proteins are increasingly popular for the non-invasive monitoring of gene expression in living tissues, cells, and in laboratory animals (Naylor 1999). Thus, GFP (and its derivatives, such as EGFP), other fluorescent proteins (see above), as well as fusion proteins comprising a fluorescent protein are even more preferred "target proteins" for the method according to the present invention.
The inventor has surprisingly found that reducing the number of RNase L cleavage sites, preferably UU and UA dinucleotides, in the nucleic acid sequence of a protein to be expressed in a eukaryotic and/or mammalian expression system results in a significantly improved expression and yield of said protein. Without wishing to be bound to a certain theory, it is believed that the reduction of RNase L cleavage sites (i.e. reduced frequency of these sites) results in a nucleic acid sequence, namely an RNA - both primary RNA and mRNA — , that is less prone (i.e. more resistant) to attacks of endogenous RNase L in the host cells, preferably in the eukaryotic host cells, leading to increased mRNA stability and, thus increased protein expression.
Because of the universal concept involved, the present invention can be applied to any sequence. More specifically, the method according to the present invention is applicable to both endogenous and exogenous (recombinant) proteins/genes, to both stably integrated and transiently expressed genes, to proteins/genes that are difficult to express, and also to proteins/genes that are endogenously expressed in low-abundance. It can be applied to both prokaryotic and eukaryotic systems. The approach described herein will lead to a significant reduction of time and costs spent on protein production, and, thus, will allow a more efficient production of proteins/genes used as biopharmaceuticals, such as erythropoietin, growth factors, interferons, insulin, therapeutic and diagnostic antibodies, and protein- or peptide- based vaccines. It is especially useful for the expression of genes/proteins that have been proven to be very difficult to express/produce in large quantities. Examples include, but are not limited to membrane proteins, such as G-protein linked receptors (GPCR), large proteins, antibodies, fusion proteins, protein complexes, vaccines, and blood plasma proteins. It can also help to significantly improve the expression and performance of reporter proteins, e.g. fluorescent proteins, such as GFP or luciferase, and, thus, increase the sensitivity of methods using such reporter proteins (fluorescence or luminescence microscopy, fluorescence-based microarrays, cell-sorting, etc.).
Furthermore, the approach is particularly practical and simple, since it is applicable to any cell line, such as cell lines used in the biotechnology industry including (but not limited to) hamster CHOI and HEK293.
The following describes general principles for the step of reducing the number of RNase L cleavage sites according to the present invention:
Reduction of the number of UU and/or UA dinucleotides in a coding region
Table 2 and Table 3 show the changes that can be made. It is important to note that these modifications are entirely different from the codon usage frequency tables (see for comparison Table 1) that are used to optimize codons for protein expression on the basis of their codon bias (codon usage frequency) in a given organism. The present method is not directed at changing codons on the basis of the codon usage frequency, but in order to reduce the number of RNase L cleavage sites/targets (UU and UA dinucleotides). Moreover, the method according to the present invention is not "organism-dependent"; the number/frequency of UU and/or UA dinucleotides can be reduced in any gene (nucleic acid sequence) from any organism, as long as the corresponding amino acid sequence remains unaltered (see Tables 2 and 3). Furthermore, the reduction of the number of UU and/or UA dinucleotides can also be combined with the classical codon usage optimization for the desired organism (see for example Table 1), possibly resulting in an even more increased expression.
Table 2 shows the codons that harbor UU and/or UA dinucleotides as well as their non- UU/UA-harboring alternative(s). UUU coding for phenylalanine (Phe) can only be changed to UUC, since there is not other codon for phenylalanine that is totally devoid of UU or UA. This is also the case for tyrosine (UAU) that can only be changed to UAC. Once the changes according to Table 2 are performed, di-triplets that form a UU or UA, i.e. NNU UNN or NNU ANN (with N being any nucleotide) are changed according to Table 3. If there is more than one alternative, preference is given to the more frequently used codon in the respective organism or in highly expressed genes.
Steps for UU and/or UA dinucleotide reduction in coding regions:
1. Change codons according to Table 2.
2. If more than one alternative codon exist, use the more frequently used codon in the desired organism (optional).
3. Change the first codon of the di-triplets that form a UU or UA dinucleotide together (NNU UNN, NNU ANN) according to Table 3.
4. If more than one alternative codon exist, use the more frequently used codon or the strongest for expression in the desired organism (optional, see Tables 4 and 5).
5. Classical codon usage optimization (based on the host cell organism or on a list of codons most frequently used in high expression genes, can be performed optionally and preferably prior to the UU and UA reduction.
Reduction of the number of UU and/or UA dinucleotides in an intron Steps for UU and/or UA dinucleotide reduction in introns:
1. Mutate or delete one or two of the two nucleotides in UU/UA dinucleotides: UU or UA to UC, UG, GA, CA, or TA. Alternatively, insert one nucleotide.
2. The entire GC content of the intron should not be more than 80% and the length should not be changed to less than 80% of its original length.
3. Do not change exon-intron boundaries including exon donor and acceptor splice sites and branch points. Avoid disrupting the CT-rich area ranging from the splice acceptor site to the end of the branch point.
Reduction of the number of UU and/or UA dinucleotides in a 5'UTR
Steps for UU and/or UA dinucleotide reduction in 5'UTRs:
1. Mutate UU or UA to UC 5 UG, GA, CA, or TA.
2. The entire GC content of the 5'UTR should not be more than 80%.
3. Avoid context sequences near the initiation codon, ATG, since they may harbor translation enhancing sequences, such as kozac.
Reduction of the number of UU and/or UA dinucleotides in a 3'UTR
Steps for UU and/ UA dinucleotides reduction in 3' UTRs:
1. Mutate or delete one or two of the two nucleotides in UU/UA dinucleotides: UU or UA to UC, UG, GA, CA, or TA. Alternatively, insert one nucleotide.
2. The entire GC content of the 3'UTR should not be more than 80% and the length should not be changed to less than 80% of its original length.
3. Do not change polyA signals such as AAUAAA or AUUAAA and avoid to alter any necessary or accessory sequence elements used for polyadenylation, if found.
Possible changes in non-coding regions
1. Mutation: NUUN or NUAN to NUSN (where S is G or C, and N is any nucleotide)
2. Insertion: NUUN or NUAN to NUSUN or NUSA (where S is G or C, and N is any nucleotide)
3. Deletion: NUUN to NUS (where S is G or C, and N is any nucleotide)
4. Deletion: NUAN to NAN or NUS (where S is G or C, and N is any nucleotide) Tables
Table 1: Codon frequency in human genes fields: [triplet] [amino acid] [fraction] [frequency: per thousand] ([number])
UUU F 0.46 17.6 (714298) UCU S 0.19 15.2 (618711) UAU Y 0.44 12.2 (495699) UGU C 0.46 10.6 (430311)
UUC F 0.54 20.3 (824692) UCC S 0.22 17.7 (718892) UAC Y 0.56 15.3 (622407) UGC C 0.54 12.6 (513028)
UUA L 0.08 7.7 (311881) UCA S 0.15 12.2 (496448) UAA * 0.30 1.0 ( 40285) UGA * 0.47 1.6 ( 63237)
UUG L 0.13 12.9 (525688) UCG S 0.05 4.4 (179419) UAG * 0.24 0.8 ( 32109) UGG W 1.00 13.2 (535595)
CUU L 0.13 13.2 (536515) CCU P 0.29 17.5 (713233) CAU H 0.42 10.9 (441711) CGU R 0.08 4.5 (184609) CUC L 0.20 19.6 (796638) CCC P 0.32 19.8 (804620) CAC H 0.58 15.1 (613713) CGC R 0.18 10.4 (423516) CUA L 0.07 7.2 (290751) CCA P 0.28 16.9 (688038) CAA Q 0.27 12.3 (501911) CGA R 0.11 6.2 (250760) CUG L 0.4039.6 (1611801) CCG P 0.11 6.9 (281570) CAG Q 0.7334.2 (1391973) CGG R 0.20 11.4 (464485)
AUU I 0.36 16.0 (650473) ACU T 0.25 13.1 (533609) AAU N 0.47 17.0 (689701) AGU S 0.15 12.1 (493429) AUC I 0.47 20.8 (846466) ACC T 0.36 18.9 (768147) AAC N 0.53 19.1 (776603) AGC S 0.24 19.5 (791383) AUA I 0.17 7.5 (304565) ACA T 0.28 15.1 (614523) AAA K 0.43 24.4 (993621) AGA R 0.21 12.2 (494682) AUG M 1.00 22.0 (896005) ACG T 0.11 6.1 (246105) AAG K 0.57 31.9 (1295568) AGG R 0.21 12.0 (486463)
GUU V 0.18 11.0 (448607) GCU A 0.27 18.4 (750096) GAU D 0.46 21.8 (885429) GGU G 0.16 10.8 (437126) GUC V 0.24 14.5 (588138) GCC A 0.40 27.7 (1127679) GAC D 0.54 25.1 (1020595) GGC G 0.34 22.2 (903565) GUA V 0.12 7.1 (287712) GCA A 0.23 15.8 (643471) GAA E 0.42 29.0 (1177632) GGA G 0.25 16.5 (669873) GUG V 0.46 28.1 (1143534) GCG A 0.11 7.4 (299495) GAG E 0.58 39.6 (1609975) GGG G 0.25 16.5 (669768)
Source: http://www.kazusa.or.jp
Table 2: UU/UA-harboring codons and their non-UU/UA-harboring alternative(s)
Table 3: Di-Triplets forming UU/UA dinucleotides and the corresponding UU/UA-reduced di- triplet(s)
UNN: UUC, UCU, UCC, UCA, UCG,
ANN: AUC, AUG, ACU, ACC, ACA, ACG, AAU, AAC, AAA, AAG, AGU, AGC, AGA, AGG
Table 4: Combination of UU/UA dinucleotide reduction and classical codon optimization (for single codons / triplets)
*Human codon bias as an example of organism codon bias.
Table 5: Combination of UU/UA dinucleotide reduction and classical codon optimization (for codon pairs / di-triplets)
*Human codon bias as an example of organism codon bias.
UNN: UUC, UCU, UCC, UCA, UCG,
ANN: AUC, AUG, ACU, ACC, ACA, ACG, AAU, AAC, AAA, AAG, AGU, AGC, AGA, AGG
Reference is now made to the figures, wherein
Figure 1 is a graph showing the effect of the reduction of UU and UA dinucleotides on the expression of EGFP in a mammalian expression system in comparison to the wild type sequence,
Figure 2 is a graph showing the effect of the reduction of UU and UA dinucleotides on the expression of EGFP in a mammalian expression system in comparison to the wild type sequence with simultaneous over-expression of RNase L, and
Figure 3 is a graph showing the GFP fluorescence in mammalian cells transfected with PCR products harboring a modified UU/UA-reduced EGFP sequence or the wild type EGFP sequence. Figure 4 is a graph showing luciferase activity in Hek293 cells transfected with either a wild type or modified (i.e. UU/AG-reduced) firefly luciferase expression vector.
Figure 5 is a graph showing luciferase activity in Huh7 cells which have been transfected with different PCR products generated from wild type or UU/UA-reduced firefly luciferase expression vector,
Figure 6 is a graph showing expression of hepatitis B surface antigen in Hek293 cells transfected with a wild type or UU/UA-reduced hepatitis B surface antigen expression vector.
Figure 7 is a graph showing expression of various green fluorescent proteins in Hek293 cells transfected with MODC-destabilized wildtype and MODC-destabilized UU/UA-reduced green fluorescent proteins.
The invention is now further described by reference to the following examples, which are intended to illustrate, not to limit the scope of the invention.
EXAMPLE 1 : Reduction of UU and/or UA dinucleotides in introns
/ = splicing site
Underlined = consensus functional site
Bold underlined italics = branch point
SEO ID NO: 1 : Wild type rabbit beta globin intron 1 (RBTGl):
GGTGAGGCCGA/GTTTGGTAAGTATCCTTTTTACAGCACAACTT AATGAGACAGAT AGAAACTGACCGGTGGGAGTCTGCGGCCGCAGTCTTGTAGAAACAGAGTAGTCG CCTGCTTTTCTGCCAGGTGCTGACTTCTCTCCCCTTCTCTTTTTTCCTTTTCTCAG/GTT GGTGTCG
SEO ID NO: 2: UU/UA-reduced RBTGl (without UU/UA reduction in the CT-rich region):
GGTGAGGCCGAGTTTGGTAAGTGTCCTCTGAACAGCACAACTGAATGAGACAGA AGAAACTGACCGGTGGGAGTCTGCGGCCGCAGTCTGTAGAAACAGAGTAGTCGC CTGCTTΎΎCTGCCAGGΎGCTGACTTCTCTCCCCTTCTCTTTTTTCCTTTTCTCAG/G TT
GGTGTCG
SEO ID NO: 3: UU/UA-reduced RBTGl (with minimal UU/UA reduction in the CT-rich region):
GU 1 GAGGCCGAGTTTGGTAAGTGTCCTCTGAACAGCACAACTGAATGAGACAGA AGAAACTGACCGGTGGGAGTCTGCGGCCGCAGTCTGTAGAAACAGAGTAGTCGC CTGTCTTCTGCCAGGTGCTGACTCTCTCTCCCCTTCTCTCTCTTCCTCTTCTCAG/GTT GGTGTCG
EXAMPLE 2: Reduction of UU and/or UA dinucleotides in the 3'UTR
. = deletion
Underlined = mutation
Bold underlined = poly A signal
SEQ ID NO: 4: Wild type SV40 3'UTR
TGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA
AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTG
TGGTTTGTCCAAAC
SEQ ID NO: 5: UU/UA-reduced SV40 3'UTR
TGAATGC AAT.GT.GC.GTCAACT.GTCTGTCTGCAGCTCACAATGGTTACAAATAAA
GCAAT.GCATCACAAATCTCACAAATCAAGCATCTGT..CACTGCAT.CTAGT.GTG G
TCTGTCCAAAC
SEQ ID NO: 6: Wild type bovine growth hormone (BGH) 3'UTR
TCTAGAGATCTGTGTGTTGGTTTTTTGTGGATCTGCTGTGCCTTCTAGTTGCCAGC CATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCC ACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTC ATTCTATTCTGGGGGGTGGGGTGGGGCAGCACAGCAAGGGGGAGGATTGGGAAG ACAATAGCAGGCATGCTGCTTAAG
SEO ID NO: 7: UU/UA-reduced BGH 3'UTR
TCTAGAGATCTGTGTGTTGGTCTG.TGTGGATCTGCTGTGCCT.CTAGT.GCCAGCC A
TCTGT.GTCTGCCCCTCCCCCGTGCCT.CC l .GACCCTGGAAGGTGCCACTCCCACTG
TCCTGTCCTAATAAAATGAGGAAAT.GCATCGCAT.GTCTGAGT.GGTGTCATCTCT
ATCCTGGGGGGTGGGGTGGGGCAGCACAGCAAGGGGGAGGATCTGGGAAGACA
AT.GCAGGCATGCTGCTTAAG
EXAMPLE 3: Reduction of UU and/or UA dinucleotides in the coding region
SEO ID NO: 8: Wild type enhanced green fluorescent protein (EGFP)
ATGGCTAGCAAAGGAGAAGAACTCTTCACTGGAGTTGTCCCAATTCTTGTTGAAT
TAGATGGTGATGTTAACGGCCACAAGTTCTCTGTCAGTGGAGAGGGTGAAGGTGA
TGCAACATACGGAAAACTTACCCTGAAGTTCATCTGCACTACTGGCAAACTGCCT
GTTCCATGGCCAACACTAGTCACTACTCTGTGCTATGGTGTTCAATGCTTTTCAAG
ATACCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGT
TATGTACAGGAAAGGACCATCTTCTTCAAAGATGACGGCAACTACAAGACACGT
GCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTA
TTGACTTCAAGGAAGATGGCAACATTCTGGGACACAAATTGGAATACAACTATAA
CTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTGAA
CTTCAAGACCCGCCACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTAT
CAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACC
TGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGT
CCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTGTAC
AAC
SEO ID NO: 9: UU/UA-reduced EGFP ("SuperGFP")
ATGGCCAGCAAGGGCGAGGAACTGTTCACCGGCGTGGTGCCCATCCTGGTGGAG CTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAAGGC GACGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGC
CCGTGCCCTGGCCCACCCTGGTGACCACCCTGTGCTACGGCGTGCAGTGCTTCAG
CAGATACCCCGACCACATGAAGCGGCACGACTTCTTCAAGAGCGCCATGCCCGA
GGGCTACGTGCAGGAACGGACCATCTTCTTCAAGGACGACGGCAACTACAAGAC
CAGGGCCGAGGTGAAGTTCGAGGGCGACACACTGGTGAACCGGATCGAGCTGAA
GGGCATCGACTTCAAAGAGGACGGCAACATCCTGGGCCACAAGCTGGAATACAA
CTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAA
GGTCAACTTCAAGACCCGGCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGA
CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC
CACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGGGAC
CACATGGTGCTGCTGGAATTCGTGACAGCCGCCGGAATCACCCACGGCATGGACG
AGCTGTACAAC
SEO ID NO; 10
WILD TYPE GFP SEQUENCE FROM MONT ASTKEA CAVERNOSA
ATGGGCGTGATCAAGCCCGACATGAAGATCAAGCTGCGGATGGAGGGCGCCGTG
AACGGCCACAAATTCGTGATCGAGGGCGACGGGAAAGGCAAGCCCTTTGAGGGT
AAGCAGACTATGGACCTGACCGTGATCGAGGGCGCCCCCCTGCCCTTCGCTTATG
ACATTCTCACCACCGTGTTCGACTACGGTAACCGTGTCTTCGCCAAGTACCCCAA
GGACATCCCTGACTACTTCAAGCAGACCTTCCCCGAGGGCTACTCGTGGGAGCGA
AGCATGACATACGAGGACCAGGGAATCTGTATCGCTACAAACGACATCACCATG
ATGAAGGGTGTGGACGACTGCTTCGTGTACAAAATCCGCTTCGACGGGGTCAACT
TCCCTGCTAATGGCCCGGTGATGCAGCGCAAGACCCTAAAGTGGGAGCCCAGTAC
CGAGAAGATGTACGTGCGGGACGGCGTACTGAAGGGCGATGTTAATATGGCACT
GCTCTTGGAGGGAGGCGGCCACTACCGCTGCGACTTCAAGACCACCTACAAAGCC
AAGAAGGTGGTGCAGCTTCCCGACTACCACTTCGTGGACCACCGCATCGAGATCG
TGAGCCACGACAAGGACTACAACAAAGTCAAGCTGTACGAGCACGCCGAAGCCC
ACAGCGGACTACCCCGCCAGGCCGGCTAA
SEQ ID NO: 11
MONSTER-OM: MODIFIED TYPE OF SEQ ID NO: 10 BY REDUCING THE
NUMBER OF UU/UA DINUCLEOTIDES
ATGGGCGTGATCAAGCCCGACATGAAGATCAAGCTGCGGATGGAGGGCGCCGTG AACGGCCACAAATTCGTGATCGAGGGCGACGGGAAAGGCAAGCCCTTCGAGGGC AAGCAGACGATGGACCTGACCGTGATCGAGGGCGCCCCCCTGCCCTTCGCCTACG
ACATCCTGACCACCGTGTTCGACTACGGCAACCGTGTCTTCGCCAAGTACCCCAA
GGACATCCCTGACTACTTCAAGCAGACCTTCCCCGAGGGCTACTCGTGGGAGCGA
AGCATGACATACGAGGACCAGGGAATCTGCATCGCGACAAACGACATCACCATG
ATGAAC^GTGTGGACGACTGCTTCGTGTACA>JLATCCGCTTCGACGGGGTCAA CT
TCCCTGCCAATGGCCCGGTGATGCAGCGCAAGACCCTGAAGTGGGAGCCCAGCA
CCGAGAAGATGTACGTGCGGGACGGCGTCCTGAAGGGCGATGTGAACATGGCAC
TGCTCCTGGAGGGAGGCGGCCACTACCGCTGCGACTTCAAGACCACCTACAAAGC
CAAGAAGGTGGTGCAGCTGCCCGACTACCACTTCGTGGACCACCGCATCGAGATC
GTGAGCCACGACAAGGACTACAACAAAGTCAAGCTGTACGAGCACGCCGAAGCC
CACAGCGGACTGCCCCGCCAGGCCGGCTGAAGTCTCACGGCTTCCCACCCGAGGT
CGAGGAGCAGGATGATGGCACACTGCCCATGAGCTGTGCTCAGGAGTCTGGCAT
GGACAGACACCCCGCTGCCTGTGCCAGTGCCAGGATCAATGTG TGA
SEO ID NQ: 12
CLAVULARIA SPECIES - WILD TYPE SEQUENCE
ATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTAATCAAGCCCGACATGAAG
ATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGC
GAGGGCGAGGGCAAGCCCTACGACGGCACCAACACCATCAACCTGGAGGTGAAG
GAGGGAGCCCCCCTGCCCTTCTCCTACGACATTCTGACCACCGCGTTCAGTTACG
GCAACAGGGCCTTCACCAAGTACCCCGACGACATCCCCAACTACTTCAAGCAGTC
CTTCCCCGAGGGCTACTCTTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATC
GTGAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACGAGATAC
ACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATGCAGAAGGAGACCA
CCGGCTGGGACGCCTCCACCGAGAGGATGTACGTGCGCGACGGCGTGCTGAAGG
GCGACGTCAAGATGAAGCTGCTGCTGGAGGGCGGCGGCCACCACCGCGTTGACT
TCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTATCACTTTGT
GGACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGT
TTACGAGATCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTACAA
GTAA
SEO ID NO.: 13
CLAVULARIA SPECIES-OM: MODIFIED (UU/UA-reduced) ATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTGATCAAGCCCGACATGAAG
ATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGC
GAGGGCGAGGGCAAGCCCTACGACGGCACCAACACCATCAACCTGGAGGTGAAG
GAGGGAGCCCCCCTGCCCTTCTCCTACGACATCCTGACCACCGCGTTCAGCTACG
GCAACAGGGCCTTCACCAAGTACCCCGACGACATCCCC.AACTACTTCAAGCAGTC
CTTCCCCGAGGGCTACAGCTGGGAGCGCACCATGACCTTCGAGGACAAGGGCAT
CGTGAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACGAGATC
CACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATGCAGAAGGAGACC
ACCGGCTGGGACGCCTCCACCGAGAGGATGTACGTGCGCGACGGCGTGCTGAAG
GGCGACGTCAAGATGAAGCTGCTGCTGGAGGGCGGCGGCCACCACCGCGTGGAC
TTCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTATCACTTCG
TGGACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCG
TGTACGAGATCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTACA
AGCTGA
SEO ID NO: 14
FIREFLY LUCIFERASE +:WILD TYPE SEQUENCE:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAA
GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT
CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTG
AGTACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAA
TACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCG
GTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATA
ATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGT
TTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATC
CAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGT
ACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCA
GAGTCCTTCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTA
CTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCG
CATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAG
TGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATAT
GTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAG
CCTTCAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCT
TCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGC TTCTGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTC
CATCTGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTC
TGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATT
TTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAA
AGAGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATC
CGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACA
TAGCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTCT
GATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCC
SEO ID NO: 15
LUC+DU (Superluciferase): (UU/UA-reduced)
ATGGAAGACGCCAAAAACATCAAGAAAGGCCCGGCGCCATTCTACCCGCTGGAA
GATGGAACCGCTGGAGAGCAACTGCACAAGGCCATGAAGAGATACGCCCTGGTG
CCTGGAACAATCGCGTTCACAGATGCACACATCGAGGTGGACATCACCTACGCTG
AGTACTTCGAAATGTCCGTCCGGCTGGCAGAAGCCATGAAACGATACGGGCTGA
ACACAAATCACAGAATCGTCGTGTGCAGTGAAAACTCTCTGCAATTCTTCATGCC
GGTGCTGGGCGCGCTGTTCATCGGAGTGGCAGTCGCGCCCGCGAACGACATCTAC
AATGAACGTGAACTCCTCAACAGCATGGGCATCTCGCAGCCCACCGTGGTGTTCG
TGTCCAAAAAGGGGCTGCAAAAAATCCTGAACGTGCAAAAAAAGCTCCCAATCA
TCCAAAAAATCATCATCATGGACAGCAAAACGGACTACCAGGGATTCCAGTCGA
TGTACACGTTCGTCACATCTCATCTGCCTCCCGGCTTCAATGAATACGACTTCGTG
CCAGAGTCCTTCGACAGGGACAAGACAATCGCACTGATCATGAACTCCTCTGGAA
GCACTGGTCTGCCCAAAGGTGTCGCTCTGCCTCACAGAACTGCCTGCGTGAGATT
CTCGCATGCCAGAGATCCCATCTTCGGCAATCAAATCATCCCGGACACTGCGATC
CTGAGTGTGGTCCCATTCCATCACGGCTTCGGAATGTTCACGACACTCGGATACC
TGATCTGTGGATTCCGAGTCGTCCTGATGTACAGATTCGAAGAAGAGCTGTTCCT
GAGGAGCCTCCAGGACTACAAGATCCAAAGTGCGCTGCTGGTGCCAACCCTGTTC
TCCTTCTTCGCCAAAAGCACTCTGATCGACAAATACGATCTCAGCAATCTGCACG
AAATCGCCTCTGGTGGCGCTCCCCTCTCCAAGGAAGTCGGGGAAGCGGTCGCCAA
GAGGTTCCATCTGCCAGGGATCAGGCAAGGATACGGGCTCACTGAGACGACATC
AGCCATCCTGATCACACCCGAGGGGGATGACAAACCGGGCGCGGTCGGGAAAGT
GGTCCCATTCTTCGAAGCGAAGGTTGTGGATCTGGACACCGGGAAAACGCTGGGC
GTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCCATGATCATGTCCGGCTACG
TGAACAATCCGGAAGCGACCAACGCCCTGATCGACAAGGATGGATGGCTCCACT CTGGAGACATCGCGTACTGGGACGAAGACGAACACTTCTTCATCGTGGACCGCCT
GAAGTCTCTGATCAAGTACAAAGGCTACCAGGTGGCTCCCGCTGAACTCGAATCC
ATCCTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTGCCCGACG
ATGACGCCGGTGAACTGCCCGCCGCCGTCGTGGTTCTGGAGCACGGAAAGACGA
TGACGGAAAAAGAGATCGTGGACTACGTCGCCAGTCAAGTAACAACCGCGAAAA
AGCTGCGCGGAGGAGTTGTGTTCGTGGACGAAGTGCCGAAAGGTCTGACCGGAA
AGATCGCCGTG
SEO ID NO: 16
Firefly LUC2 Iuciferase wildtype:
ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAA
GACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTG
CCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCG
AGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGA
ATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCC
CGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTAC
AACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTC
GTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATC
ATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGC
ATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGT
GCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGG
CAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGA
TTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTA
TCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTAC
TTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTT
GCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTT
AGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACG
AGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCA
AACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCA
GCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGG
TGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGG
TGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTAC
GTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCAC AGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGC
TGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGA
GCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGA
CGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAAC
CATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAA
GAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGC
AAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATT AAGGCCAAGAAGGGCGGC
AAGATCGCCGTGTAATAA
SEO ID NO: 17
LUC2OM: LUC2 modified SuperLuciferase2 fUU/UA-reduced)
ATGGAAGATGCCAAAAACATCAAGAAGGGCCCAGCGCCATTCTACCCACTCGAA
GACGGGACCGCAGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTG
CCCGGCACCATCGCCTTCACCGACGCACACATCGAGGTGGACATCACCTACGCCG
AGTACTTCGAGATGAGCGTGCGGCTGGCAGAAGCCATGAAGCGCTACGGGCTGA
ACACAAACCATCGGATCGTGGTGTGCAGCGAGAACAGCCTGCAGTTCTTCATGCC
CGTGCTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCCAACGACATCTAC
AACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTGTTC
GTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTGCCGATC
ATCCAAAAGATCATCATCATGGACAGCAAGACCGACTACCAGGGCTTCCAAAGC
ATGTACACCTTCGTGACCTCCCACCTGCCACCCGGCTTCAACGAGTACGACTTCGT
GCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGCAGTGG
CAGCACCGGACTGCCCAAGGGCGTGGCACTGCCGCACCGCACCGCCTGTGTCCGA
TTCAGTCATGCACGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCCA
TCCTCAGCGTGGTGCCATTCCACCACGGCTTCGGCATGTTCACCACGCTGGGCTA
CTGGATCTGCGGCTTCCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTGTTC
CTGCGCAGCCTGCAAGACTACAAGATCCAATCTGCCCTGCTGGTGCCCACACTGT
TCAGCTTCTTCGCCAAGAGCACTCTCATCGACAAGTACGACCTGAGCAACCTGCA
CGAGATCGCCAGCGGCGGAGCGCCGCTCAGCAAGGAGGTGGGTGAGGCCGTGGC
CAAACGCTTCCACCTGCCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAAC
CAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTGGGCAA
GGTGGTGCCCTTCTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAGACACTG
GGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCT
ACGTGAACAACCCCGAGGCCACAAACGCTCTCATCGACAAGGACGGCTGGCTGC ACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCG
GCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTGGCCCCAGCCGAACTGGA
GAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGAGTCGCCGGACTGCCA
GACGACGATGCCGGCGAGCTGCCCGCAGCAGTCGTCGTGCTGGAACACGGCAAA
ACCATGACCGAGAAGGAGATCGTGGACTACGTGGCCAGCCAGGTGACAACCGCC
AAGAAGCTGCGCGGTGGTGTGGTGTTCGTGGACGAGGTGCCCAAAGGACTGACC
GGCAAGCTGGACGCCCGCAAGATCCGCGAGATCCTCATCAAGGCCAAGAAGGGC
GGCAAGATCGCCGTGTGA
SEO ID NO; 18
Puntellina Plumate (GFP) wild type;
ATGGAGAGCGACGAGAGCGGCCTGCCCGCCATGGAGATCGAGTGCCGCATCACC
GGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGCACCCCC
GAGCAGGGCCGCATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTC
AGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCT
ACCCCAGCGGCTACGAGAACCCCTTCCTGCACGCCATCAACAACGGCGGCTACAC
CAACACCCGCATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAG
CTACCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGATGGGCACCGG
CTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCCGCAGCAACGCCACC
GTGGAGCACCTGCACCCCATGGGCGATAACGATCTGGATGGCAGCTTCACCCGCA
CCTTCAGCCTGCGCGACGGCGGCTACTACAGCTCCGTGGTGGACAGCCACATGCA
CTTCAAGAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCC
TTCCGCCGCGTGGAGGAGGATCACAGCAACACCGAGCTGGGCATCGTGGAGTAC
CAGCACGCCTTCAAGACCCCGGATGCAGATGCCGGTGAAGAAA
SEO ID NO: 19
Puntellina Plumate (GFP) : modified sequence (UU/UA-reduced)
ATGGAGAGCGACGAGAGCGGCCTGCCCGCCATGGAGATCGAGTGCCGCATCACC
GGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGCACCCCC
GAGCAGGGCCGCATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTC
AGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCT
ACCCCAGCGGCTACGAGAACCCCTTCCTGCACGCCATCAACAACGGCGGCTACAC
CAACACCCGCATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAG
CTACCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGATGGGCACCGG CTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCCGCAGCAACGCCACC
GTGGAGCACCTGCACCCCATGGGCGACAACGACCTGGATGGCAGCTTCACCCGC
ACCTTCAGCCTGCGCGACGGCGGCTACTACAGCTCCGTGGTGGACAGCCACATGC
ACTTCAAGAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGC
CTTCCGCCGCGTGGAGGAGGATCACAGCAACACCGAGCTGGGCATCGTGGAGTA
CCAGCACGCCTTCAAGACCCCGGATGCAGATGCCGGTGAAGAACTGA
SEO ID NO: 20
Red Fluorescent protein from Discosoma wild type sequence atgagcgagctgatcaaggagaacatgcacatgaagctgtacatggagggcaccgtgaac aaccaccacttcaagtgcacatccgag ggcgaaggcaagccctacgagggcacccagaccatgaagatcaaggtggtcgagggcggc cctctccccttcgccttcgacatcct ggctaccagcttcatgtacggcagcaaagccttcatcaaccacacccagggcatccccga cttctttaagcagtccttccctgagggctt cacatgggagagaatcaccacatacgaagacgggggcgtgctgaccgctacccaggacac cagcttccagaacggctgcatcatct acaacgtcaagatcaacggggtgaacttcccatccaacggccctgtgatgcagaagaaaa cacgcggctgggaggccaacaccga gatgctgtaccccgctgacggcggcctgagaggccacagccagatggccctgaagctcgt gggcgggggctacctgcactgctcct tcaagaccacatacagatccaagaaacccgctaagaacctcaagatgcccggcttccact tcgtggaccacagactggaaagaatca aggaggccgacaaagagacctacgtcgagcagcacgagatggctgtggccaagtactgcg acctccctagcaaactggggcacag agatga
SEO ID NO: 21
Red Fluorescent protein modified sequence
ATGAGCGAGCTGATCAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACC
GTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAG
GGCACCCAGACCATGAAGATCAAGGTGGTCGAGGGCGGCCCACTCCCCTTCGCCT
TCGACATCCTGGCCACCAGCTTCATGTACGGCAGCAAAGCCTTCATCAACCACAC
CCAGGGCATCCCCGACTTCTTCAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAG
AGAATCACCACATACGAAGACGGGGGCGTGCTGACCGCCACCCAGGACACCAGC
TTCCAGAACGGCTGCATCATCTACAACGTCAAGATCAACGGGGTGAACTTCCCAT
CCAACGGCCCTGTGATGCAGAAGAAAACACGCGGCTGGGAGGCCAACACCGAGA
TGCTGTACCCCGCTGACGGCGGCCTGAGAGGCCACAGCCAGATGGCCCTGAAGCT
CGTGGGCGGGGGCTACCTGCACTGCTCCTTCAAGACCACATACAGATCCAAGAAA
CCCGCCAAGAACCTCAAGATGCCCGGCTTCCACTTCGTGGACCACAGACTGGAAA
GAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGATGGCTGTGG
CCAAGTACTGCGACCTCCCAAGCAAACTGGGGCACAGAC SEO ID NO: 22
Hepatitis B surface antigen wild type, adr hepatitis B virus strain:
ATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGT
TTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGAC
TTCTCTCAATTTTCTAGGGGGAGCACCCACGTGTCCTGGCCCAAATTCGCAGTCCC
CAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTGG
ATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTC
TTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGnTGTCCTCTACTTCCAGG
AACATCAACTACCAGCACGGGACCATGCAAGACCTGCACGATTCCTGCTCAAGG
AACCTCTATGTTTCCCTCCTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTT
GTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCA
GTCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCT
TTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTC
TGTACAACATCTTGAGTCCCTTTTTACCTCTATTACCAATTTTCTTTTGTCTTTGGG
TATACATTTGA
SEO ID NO: 23
HBSAGOM: Hepatitis B surface antigen modified sequence:
ATGGAGAACACCACCAGCGGCTTCCTGGGCCCTCTGCTGGTGCTGCAGGCCGGCT
TCTTCCTGCTGACCCGCATCCTGACCATCCCCCAGAGCCTGGACAGCTGGTGGAC
CAGCCTGAACTTCCTGGGCGGAGCCCCAACCTGTCCCGGCCCCAACAGCCAGAGC
CCCACCAGCAACCACAGCCCAACCAGCTGCCCACCCATCTGTCCCGGCTACCGGT
GGATGTGCCTGCGGCGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATC
TTCCTCCTGGTGCTCCTGGACTACCAGGGCATGCTGCCCGTGTGTCCTCTGCTGCC
TGGCACCAGCACCACCTCCACCGGCCCCTGCAAGACCTGCACAATCCCCGCCCAG
GGAACCAGCATGTTCCCAAGCTGCTGCTGCACCAAGCCCAGCGACGGCAACTGC
ACCTGCATCCCCATCCCAAGCAGCTGGGCCTTCGCCAGATTCCTGTGGGAGTGGG
CCTCCGTGAGATTCAGCTGGCTGTCACTGCTGGTGCCCTTCGTGCAGTGGTTCGTG
GGCCTGAGCCCAACAGTGTGGCTGAGCGTGATCTGGATGATGTGGTACTGGGGAC
CCAGCCTGTACAACATCCTGAGCCCCTTCCTGCCCCTGCTGCCCATCTTCTTCTGC
CTGTGGGTGTACATCTGA
SEO ID NO: 24 HBSAGM: HBSAGQM: Hepatitis B surface antigen modified sequence 2
ATGGAGAACACAACATCAGGATTCCTCGGACCCCTGCTCGTGCTGCAGGCGGGGT
TCTTCCTGCTCACAAGAATCCTCACAATCCCACAGAGTCTGGACTCGTGGTGGAC
GTCTCTCAACTTCCTCGGGGGAGCACCCACGTGTCCTGGCCCAAACTCGCAGTCC
CCAACCTCCAATCACTCACCAACCTCGTGTCCTCCAATCTGTCCTGGCTACCGCTG
GATGTGTCTGCGGCGCTTCATCATCTTCCTCTTCATCCTGCTGCTGTGCCTCATCTT
CCTGCTCGTCCTCCTGGACTACCAAGGGATGCTGCCCGTCTGTCCTCTGCTGCCAG
GAACATCAACCACCAGCACGGGACCATGCAAGACCTGCACGATCCCTGCTCAAG
GAACCAGCATGTTCCCCTCCTGCTGCTGCACAAAACCATCGGACGGAAACTGCAC
CTGCATCCCCATCCCATCATCCTGGGCCTTCGCAAGATTCCTCTGGGAGTGGGCCT
CAGTCCGGTTCTCCTGGCTCAGTCTCCTGGTGCCATTCGTGCAGTGGTTCGTCGGG
CTGTCCCCCACTGTGTGGCTGTCAGTGATCTGGATGATGTGGTACTGGGGGCCAA
GTCTGTACAACATCCTCAGTCCCTTCCTGCCTCTGCTGCCAATCTTCTTCTGTCTGT
GGGTGTACATCTGA
This sequence includes both an UU/UA reduction as well as a humanization.
SEQ ID NO: 25 IFN-ALPHA, HUMAN
ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGC TGCT
CTGTGGGCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGC TCC
TGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTG GAT
TTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCC ATG
AGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGG ATG
AGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAG CCT
GTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTC TG
GCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGC CCT
TGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAAC TTGC
AAGAAAGTTTAAGAAGTAAGGAATGA
SEO ID NO: 26
IFN-ALPHAOM: modified sequence
ATGGCCCTGACCTTCGCCCTGCTGGTGGCTCTGCTGGTGCTGAGCTGCAAGAGCA
GCTGCAGCGTGGGCTGCGATCTGCCTCAGACCCACAGCCTGGGCAGCAGACGGA
CACTGATGCTGCTGGCCCAGATGCGGCGGATCAGCCTGTTCAGCTGCCTGAAGGA
CCGGCACGACTTCGGCTTCCCCCAGGAAGAGTTCGGCAACCAGTTCCAGAAGGCC
GAGACAATCCCCGTGCTGCACGAGATGATCCAGCAGATCTTCAACCTGTTCAGCA CCAAGGACAGCAGCGCCGCCTGGGACGAGACACTGCTGGACAAGTTCTACACCG AGCTGTACCAGCAGCTGAACGACCTGGAAGCCTGCGTGATCCAGGGCGTGGGCG TGACCGAGACACCCCTGATGAAGGAAGACAGCATCCTGGCCGTGCGGAAGTACT TCCAGCGGATCACCCTGTACCTGAAAGAGAAGAAGTACAGCCCCTGCGCCTGGG AAGTGGTCCGGGCCGAGATCATGCGGAGCTTCAGCCTGAGCACCAACCTGCAGG AAAGCCTGCGGAGCAAAGAGTGA
SEQ ID NO: 27
CSF3M Colony stimulating factor wild type sequence, human:
ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGC
TGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAG
CTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAG
GGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCAC
CCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGA
GCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAG
CGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAG
TTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCA
TCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGG
GTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTAGT
TGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTG
CCCAGCCC
SEO ID NO: 28
CSF3M Colony stimulating factor modified sequence,:
ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGC
TGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAG
CTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCCTGGAGCAAGTGAGGAAGATCCAG
GGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCAC
CCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGA
GCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCCTGAGCCAACTCCACAG
CGGCCTCTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAG
CTGGGTCCCACCCTGGACACACTGCAGCTGGACGTCGCCGACTTCGCCACCACCA
TCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGG
GTGCCATGCCGGCCTTCGCCTCTGCCTTCCAGCGCCGGGCAGGAGGGGTCCTGGT GGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTGCTCCGCCACCTC GCCCAGCCC
SEQ ID NO: 29
MODC wild type sequence (mouse ornithine decarboxylase)
CAGAGCCATGGCTTCCCGCCGGAGGTGGAGGAGCAGGATGATGGCACGCTGCCCATG TC
TTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGAT CAA
TGTG
SEO ID NO: 30
MODC modified sequence:
AGTCTCACGGCTTCCCACCCGAGGTCGAGGAGCAGGATGATGGCACACTGCCCATGA GCT GTGCTCAGGAGTCTGGCATGGACAGACACCCCGCTGCCTGTGCCAGTGCCAGGATCAATG TG TGA
Destabilized and sequence modified reporters:
EXAMPLES OF destabilized and modified REPORTER SEQUENCES:
SEO ID NO: 31 MONTASTRAEA CAVERNOSA
ATGGGCGTGATCAAGCCCGACATGAAGATCAAGCTGCGGATGGAGGGCGCCGTGAAC GG
CCACAAATTCGTGATCGAGGGCGACGGGAAAGGCAAGCCCTTCGAGGGCAAGCAGAC GA
TGGACCTGACCGTGATCGAGGGCGCCCCCCTGCCCTTCGCCTACGACATCCTGACCA CCG
TGTTCGACTACGGCAACCGTGTCTTCGCCAAGTACCCCAAGGACATCCCTGACTACT TCA
AGCAGACCTTCCCCGAGGGCTACTCGTGGGAGCGAAGCATGACATACGAGGACCAGG GA
ATCTGCATCGCGACAAACGACATCACCATGATGAAGGGTGTGGACGACTGCTTCGTG TAC
AAAATCCGCTTCGACGGGGTCAACTTCCCTGCCAATGGCCCGGTGATGCAGCGCAAG ACC
CTGAAGTGGGAGCCCAGCACCGAGAAGATGTACGTGCGGGACGGCGTCCTGAAGGGC GA
TGTGAACATGGCACTGCTCCTGGAGGGAGGCGGCCACTACCGCTGCGACTTCAAGAC CAC
CTACAAAGCCAAGAAGGTGGTGCAGCTGCCCGACTACCACTTCGTGGACCACCGCAT CGA
GATCGTGAGCCACGACAAGGACTACAACAAAGTCAAGCTGTACGAGCACGCCGAAGC CC
ACAGCGGACTGCCCCGCCAGGCCGGCAGTCTCACGGCTTCCCACCCGAGGTCGAGGA
GCAGGATGATGGCACACTGCCCATGAGCTGTGCTCAGGAGTCTGGCATGGACAGAC
ACCCCGCTGCCTGTGCCAGTGCCAGGATCAATGTGTGA
Bold in MODC seqeuence.
SEO ID NO: 32
Clavulariidae Clavularia -OM: MODIFIED
ATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTGATCAAGCCCGACATGAAGATC AA
GCTGAAGATGGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGCGAGGGCGA GG
GCAAGCCCTACGACGGCACCAACACCATCAACCTGGAGGTGAAGGAGGGAGCCCCCC TG
CCCTTCTCCTACGACATCCTGACCACCGCGTTCAGCTACGGCAACAGGGCCTTCACC AAG
TACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAGGGCTACAGCTGG GAG
CGCACCATGACCTTCGAGGACAAGGGCATCGTGAAGGTGAAGTCCGACATCTCCATG GA
GGAGGACTCCTTCATCTACGAGATCCACCTCAAGGGCGAGAACTTCCCCCCCAACGG CCC
CGTGATGCAGAAGGAGACCACCGGCTGGGACGCCTCCACCGAGAGGATGTACGTGCG CG
ACGGCGTGCTGAAGGGCGACGTCAAGATGAAGCTGCTGCTGGAGGGCGGCGGCCACC AC
CGCGTGGACTTCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTAT CA CTTCGTGGACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCG TGTACGAGATCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTACAAGC AGTCTCACGGCTTCCCACCCGAGGTCGAGGAGCAGGATGATGGCACACTGCCCATG AGCTGTGCTCAGGAGTCTGGCATGGACAGACACCCCGCTGCCTGTGCCAGTGCCAG GATCAATGTG TGA
SEO ID NO: 33
Firefly LUC+DU (Superluciferase):
ATGGAAGACGCCAAAAACATCAAGAAAGGCCCGGCGCCATTCTACCCGCTGGAAGAT GG
TCGCGTTCACAGATGCACACATCGAGGTGGACATCACCTACGCTGAGTACTTCGAAA TGT
CCGTCCGGCTGGCAGAAGCCATGAAACGATACGGGCTGAACACAAATCACAGAATCG TC
GTGTGCAGTGAAAACTCTCTGCAATTCTTCATGCCGGTGCTGGGCGCGCTGTTCATC GGA
GTGGCAGTCGCGCCCGCGAACGACATCTACAATGAACGTGAACTCCTCAACAGCATG GG
CATCTCGCAGCCCACCGTGGTGTTCGTGTCCAAAAAGGGGCTGCAAAAAATCCTGAA CGT
GCAAAAAAAGCTCCCAATCATCCAAAAAATCATCATCATGGACAGCAAAACGGACTA CC
AGGGATTCCAGTCGATGTACACGTTCGTCACATCTCATCTGCCTCCCGGCTTCAATG AATA
CGACTTCGTGCCAGAGTCCTTCGACAGGGACAAGACAATCGCACTGATCATGAACTC CTC
TGGAAGCACTGGTCTGCCCAAAGGTGTCGCTCTGCCTCACAGAACTGCCTGCGTGAG ATT
CTCGCATGCCAGAGATCCCATCTTCGGCAATCAAATCATCCCGGACACTGCGATCCT GAG
TGTGGTCCCATTCCATCACGGCTTCGGAATGTTCACGACACTCGGATACCTGATCTG TGGA
TTCCGAGTCGTCCTGATGTACAGATTCGAAGAAGAGCTGTTCCTGAGGAGCCTCCAG GAC
TACAAGATCCAAAGTGCGCTGCTGGTGCCAACCCTGTTCTCCTTCTTCGCCAAAAGC ACTC
TGATCGACAAATACGATCTCAGCAATCTGCACGAAATCGCCTCTGGTGGCGCTCCCC TCT
CCAAGGAAGTCGGGGAAGCGGTCGCCAAGAGGTTCCATCTGCCAGGGATCAGGCAAG GA
TACGGGCTCACTGAGACGACATCAGCCATCCTGATCACACCCGAGGGGGATGACAAA CC
GGGCGCGGTCGGGAAAGTGGTCCCATTCTTCGAAGCGAAGGTTGTGGATCTGGACAC CG
GGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCCATGATCA TG
TCCGGCTACGTGAACAATCCGGAAGCGACCAACGCCCTGATCGACAAGGATGGATGG CT
CCACTCTGGAGACATCGCGTACTGGGACGAAGACGAACACTTCTTCATCGTGGACCG CCT
GAAGTCTCTGATCAAGTACAAAGGCTACCAGGTGGCTCCCGCTGAACTCGAATCCAT CCT
GCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTGCCCGACGATGACGC CGG
TGAACTGCCCGCCGCCGTCGTGGTTCTGGAGCACGGAAAGACGATGACGGAAAAAGA GA
TCGTGGACTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGCTGCGCGGAGGAGTTG TG
TTCGTGGACGAAGTGCCGAAAGGTCTGACCGGAAAACTCGACGCAAGAAAAATCAGA GA
GATCCTCATCAAGGCCAAGAAGGGCGGAAAGATCGCCGTGAGTCTCACGGCTTCCCA C
CCGAGGTCGAGGAGCAGGATGATGGCACACTGCCCATGAGCTGTGCTCAGGAGTCT
GGCATGGACAGACACCCCGCTGCCTGTGCCAGTGCCAGGATCAATGTG TGA
SEO ED NO: 34
Firefly LUC2OM: LUC2 modified SuyerLuciferase2
ATGGAAGATGCCAAAAACATCAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGAC GG
GACCGCAGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCAC CA
TCGCCTTCACCGACGCACACATCGAGGTGGACATCACCTACGCCGAGTACTTCGAGA TGA
GCGTGCGGCTGGCAGAAGCCATGAAGCGCTACGGGCTGAACACAAACCATCGGATCG TG
GTGTGCAGCGAGAACAGCCTGCAGTTCTTCATGCCCGTGCTGGGTGCCCTGTTCATC GGT
GTGGCTGTGGCCCCAGCCAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATG GG
CATCAGCCAGCCCACCGTCGTGTTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAA CGT
GCAAAAGAAGCTGCCGATCATCCAAAAGATCATCATCATGGACAGCAAGACCGACTA CC
AGGGCTTCCAAAGCATGTACACCTTCGTGACCTCCCACCTGCCACCCGGCTTCAACG AGT
ACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACA GCA
GTGGCAGCACCGGACTGCCCAAGGGCGTGGCACTGCCGCACCGCACCGCCTGTGTCC GAT
TCAGTCATGCACGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCCATCC TCA GCGTGGTGCCATTCCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTGGATCTGCG
GCTTCCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTGTTCCTGCGCAGCCTGC AAG
ACTACAAGATCCAATCTGCCCTGCTGGTGCCCACACTGTTCAGCTTCTTCGCCAAGA GCA
CTCTCATCGACAAGTACGACCTGAGCAACCTGCACGAGATCGCCAGCGGCGGAGCGC CG
CTCAGCAAGGAGGTGGGTGAGGCCGTGGCCAAACGCTTCCACCTGCCAGGCATCCGC CA
GGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGA CA
AGCCTGGCGCAGTGGGCAAGGTGGTGCCCTTCTTCGAGGCCAAGGTGGTGGACCTGG AC
ACCGGCAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATG AT
CATGAGCGGCTACGTGAACAACCCCGAGGCCACAAACGCTCTCATCGACAAGGACGG CT
GGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGG ACC
ATCCTGCTGCAACACCCCAACATCTTCGACGCCGGAGTCGCCGGACTGCCAGACGAC GAT
GCCGGCGAGCTGCCCGCAGCAGTCGTCGTGCTGGAACACGGCAAAACCATGACCGAG AA
GGAGATCGTGGACTACGTGGCCAGCCAGGTGACAACCGCCAAGAAGCTGCGCGGTGG TG
TGGTGTTCGTGGACGAGGTGCCCAAAGGACTGACCGGCAAGCTGGACGCCCGCAAGA TC
CGCGAGATCCTCATCAAGGCCAAGAAGGGCGGCAAGATCGCCGTGAGTCTCACGGCT T
CCCACCCGAGGTCGAGGAGCAGGATGATGGCACACTGCCCATGAGCTGTGCTCAGG
AGTCTGGCATGGACAGACACCCCGCTGCCTGTGCCAGTGCCAGGATCAATGTG TGA
SEQ ID NO: 35
Puntellina Plumate GFP : modified sequences
ATGGAGAGCGACGAGAGCGGCCTGCCCGCCATGGAGATCGAGTGCCGCATCACCGGC AC
CCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGCACCCCCGAGCAGGG CC
GCATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGC TG
AGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACGAG AAC
CCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCCGCATCGAGAAGTAC GAG
GACGGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTACGAGGCCGGCCGCGTGATC GGC
GACTTCAAGGTGATGGGCACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAG ATC
ATCCGCAGCAACGCCACCGTGGAGCACCTGCACCCCATGGGCGACAACGACCTGGAT GG
CAGCTTCACCCGCACCTTCAGCCTGCGCGACGGCGGCTACTACAGCTCCGTGGTGGA CAG
CCACATGCACTTCAAGAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGCCCCAT GTT
CGCCTTCCGCCGCGTGGAGGAGGATCACAGCAACACCGAGCTGGGCATCGTGGAGTA CC
AGCACGCCTTCAAGACCCCGGATGCAGATGCCGGTGAAGAACAGTCTCACGGCTTCC C
ACCCGAGGTCGAGGAGCAGGATGATGGCACACTGCCCATGAGCTGTGCTCAGGAGT
CTGGCATGGACAGACACCCCGCTGCCTGTGCCAGTGCCAGGATCAATGTG TGA
SEO ID NO: 36
Red Fluorescent protein modified sequence
ATGAGCGAGCTGATCAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTG AA
CAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCA GA
CCATGAAGATCAAGGTGGTCGAGGGCGGCCCACTCCCCTTCGCCTTCGACATCCTGG CCA
CCAGCTTCATGTACGGCAGCAAAGCCTTCATCAACCACACCCAGGGCATCCCCGACT TCT
TCAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAATCACCACATACGAAGACG GG
GGCGTGCTGACCGCCACCCAGGACACCAGCTTCCAGAACGGCTGCATCATCTACAAC GTC
AAGATCAACGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACGC GG
CTGGGAGGCCAACACCGAGATGCTGTACCCCGCTGACGGCGGCCTGAGAGGCCACAG CC
AGATGGCCCTGAAGCTCGTGGGCGGGGGCTACCTGCACTGCTCCTTCAAGACCACAT ACA
GATCCAAGAAACCCGCCAAGAACCTCAAGATGCCCGGCTTCCACTTCGTGGACCACA GAC
TGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGATGGCTG TG
GCCAAGTACTGCGACCTCCCAAGCAAACTGGGGCACAGACAGTCTCACGGCTTCCCA C
CCGAGGTCGAGGAGCAGGATGATGGCACACTGCCCATGAGCTGTGCTCAGGAGTCT
GGCATGGACAGACACCCCGCTGCCTGTGCCAGTGCCAGGATCAATGTG TGA EXAMPLE 4: In vivo analysis of UU/UA-reduced EGFP
(1) The modified EGFP sequence was custom synthesized by a gene synthesis company and supplied contained in a pUC19 vector with flanking Sail and BamHI sites. 10 μg of the vector were digested with 10 units of Sail in a buffer containing 0.1 μg/ml BSA for 1 hr at 37 0 C, followed by digestion with BamHI in BamHI buffer for an additional hour at 37°C. The digested DNA was extracted using the phenol-chloroform method, followed by ethanol precipitation. The synthetic EGFP -coding region was ligated into an expression vector, which had a CMV promoter and a BGH 3'UTR and had been digested with the same restriction enzymes (Sail and Xbal) and purified by phenol-chloroform extraction, followed by ethanol precipitation. Cloning of the EGFP-DNA into the expression vector was performed using the following ligation reaction: 30 μg of digested vector DNA were mixed with 90 μg of digested EGFP-DNA in a 10 μl reaction containing T4 DNA ligase. The ligated products were used to transform DH5α competent E. coli cells followed by expansion of the resulting colonies in a bacterial culture medium. The recombinant DNA was extracted using a Qiagen plasmid purification kit (Qiagen, Germany). The sizes of the vectors harboring the inserts were verified using gel electrophoresis. The resultant expression vector with the modified UU/UA- reduced coding region along with a vector containing the wild type DNA were used for functional studies to confirm the expression of the encoded protein. HEK293 cells were grown at standard culture conditions (37°C, 5% CO 2 ) in DMEM medium supplemented with 10% FBS and antibiotics (Invitrogen). 2.5 x 10 4 cells per well in 96-well plates were transfected with 100 ng of the vector with the modified UU/UA-reduced coding region of EGFP or the vector containing the wild-type EGFP-DNA. Transfections were performed in serum-free medium using Lipofectamine 2000 (Invitrogen) for 5 h, followed by replacing the medium with serum-supplemented medium. After approximately 24 or 48 hours, the plates were imaged and quantified using a BD high-content imager. Quantification was performed with a Proxcell imaging algorithm.
The data clearly shows that the use of the UU/UA-reduced coding region of EGFP allows a significantly (2.5 to 3-fold) higher expression of EGFP in eukaryotic cells than that of the wild type EGFP-DNA (Figure 1). £2) HEK293 cells were grown at standard culture conditions (37 0 C, 5% CO 2 ) in DMEM medium supplemented with 10% FBS and antibiotics (Invitrogen). 2.5 x 10 4 cells per well in 96- well plates were transfected with 100 ng of the vector with the modified UU/UA-reduced coding region of EGFP or the vector containing the wild-type EGFP-DNA. The cells were also co-transfected with either an empty control vector (pcDNA 3.1) or a RNase L vector (pcDNA 3.1). Transfections were performed in sεrurn=frεe medium using Lipofcctarnine 2000 (Invitrogen) for 5 h, followed by replacing the medium with serum-supplemented medium. After approximately 24 or 48 hours, the plates were imaged and quantified using a BD high- content imager. Quantification was performed with a Proxcell imaging algorithm.
The data shows that - in comparison/contrast to the wild type EGFP sequence - the use of the modified EGFP sequence resulted in higher expression, which was not constrained by the co- expression of RNase L (Figure 2).
(3) Expression active PCR products were generated by using primers in which the forward (5') primers were complementary to the beginning of the CMV promoter region or a sequence upstream of the CMV promoter, while the reverse (3') primers were complementary to the BGH poly A site or a sequence downstream of this site. The PCR was carried out using a mixture of Taq and Pfu polymerase in a 100 μl reaction with the following cycle conditions: - 95°C for 12 min (to activate hot start polymerases), - 32 cycles of: 94°C, 1 min; 52°C, 1 min; 72°C, 4 min, and a final extension at 72°C for 7 min. The PCR products were purified using Qiagen PCR purification columns to eliminate the primers, small PCR products, buffer, and enzymes, and eluted in sterile water. HEK293 cells were grown at standard culture conditions (37°C, 5% CO 2 ) in DMEM medium supplemented with 10% FBS and antibiotics (Invitrogen). 2.5 x 10 4 cells per well in 96-well plates were transfected with 100 ng of purified PCR products generated from the EGFP expression vector with the wild type or with the modified sequence. Transfections were performed in serum-free medium using Lipofectamine 2000 (Invitrogen) for 5 h, followed by replacing the medium with serum-supplemented medium. After approximately 24 or 48 hours, the plates were imaged and quantified using a BD high-content imager. Quantification was performed with a Proxcell imaging algorithm. The data shows that PCR products harboring the UU/UA-reduced coding region of EGFP led to higher expression of EGFP (5 to 10-fold increase) than those harboring the wild type sequence (Figure 3).
(4) Using the same methodology as described in (2) and (3), Hek293 cells were transfected with wild type or UU/UA-reduced firefly luciferase expression vector ("Superluciferase", SEQ ID NO: 15). The lucifεrasc activity levels were quantified by a luminometer. The data show that, within two independent experiments, there was an approximately 5- and approximately 100-fold difference (Figure 4).
Likewise, Huh7 cells were transfected with different PCR products generated in accordance with the methodology outlined in (3) above, from the wild type or modified firefly luciferase expression vector ("Superluciferase"; SEQ ID NO: 15). The luciferase activity levels were, again, quantified by a luminometer. The data show that PCR products harbouring the UU/UA- reduced coding region of superluciferase led to a substantially higher expression of luciferase (20- 100-fold increase) than those harbouring the wildtype sequence (Figure 5). This demonstrates the method in accordance with the present invention works with a verity of variety of reporter proteins.
Likewise, Hek293 cells were transfected, using the same methodology as in (2), above, with a wild type or UU/UA-reduced hepatitis B surface (SEQ ID NO: 23) antigen expression vector. The expressed protein was quantified as mlU/ml. There was approximately a 4-fold difference (Figure 6), but it is likely that this may be even higher in independent experiments.
This is an example that the method in accordance with the present invention also works with therapeutic proteins, antibodies and vaccines which have been modified, i.e. their coding sequence has been UU/UA-reduced, and this leads to a substantial increase in expression.
(5) In order to reflect the transcriptional changes and the subsequent effects on reported protein levels, protein-destabilizing amino acid regions that include a PEST sequence (= peptide sequence which is rich in proline, glutamic acid, serine and threonine) have been used to reduce the half-life of various reporter proteins. PEST sequences are associated with proteins that have a short intracellular half-life. Li et al. (J. Biol. Chem., 1998, 273, pp. 34970-34975) describe the use of a PEST sequence of MODC to destabilize the EGFP 3 and Leclerc et al. (Biotechniques, 2000, 29, pp. 590-591, pp. 594-596 used a PEST sequence to reduce the protein half-life of firefly luciferase.
Using such MODC-domain (mouse ornithine decarboxylase), more specifically, amino acids 422-461 of the degradation domain of the highly unstable MODC, the present inventor rendered a number of reporter genes unstable by fusing them with the afore-mentioned MODC domain. Moreover, the present inventor modified such fusions by reducing the number of UU/UA dinucleotides in both the reporter gene and the MODC domain in accordance with the present invention with respect to EGFP from Aequorea Victoria, Montastrea Cavernosa, Clavularia and Puntelina Plumate. The number of UU/UA dinucleotides waas reduced both in the EGFP-part and the MODC part of the fusion.
The MODC domain was amplified from genomic DNA of mouse fibroblasts using specific primers that contain EcoRI and BamHI sites in the forward and reverse primer, respectively. The amplified cDNA was cloned in frame with the GFP coding region using the same restriction sites. Hek293 cells were transfected with destabilized GFPs as indicated in figure 7. The fluorescence intensity was quantitated by imaging apparatus and software. Compared to destabilized wildtype EGFP from Aequorea Victoria (i.e. the point of reference was wildtype EGFP, SEQ ID NO: 8, fused to wildtype MODC, SEQ ID NO: 29), there was a two-fold increase in fluorescence from modified Aequorea Victoria, an 8-fold increase from modified Montastrea Cavernosa green fluorescent protein, a 4-fold increase from modified Clavularia green fluorescent protein and a 7-fold increase from modified Puntelina Plumate green fluorescent protein (see figure 7). The term "modified" here means "UUAJA reduced and fused with MODC which itself has also been UU/UA reduced".
Consequently, this shows that the present invention also works in situations where expression signals normally are weaker, and improves the fold-increase in expression in such situations.
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