TITLE OF THE INVENTION ENHANCED PROTEIN PRODUCTION IN HIGHER PLANTS BY N-TERMINAL FUSION OF A UBIQUITIN OR A CUCUMBER MOSAIC VIRUS COAT PROTEIN PEPTIDE BACKGROUND OF THE INVENTION Strategies for production of proteins in heterologous fusion form have been widely applied in biotechnology for many purposes, such as secretion of proteins from host cells (fused to signal peptides), easy detection or purification of protein products (fused to reporter enzymes for detection and to peptide tags for purification), searching for proteins with desired biological activities (e. g., in the phage display technique and the two-hybrid system). Enhanced expression of proteins of interest has also been achieved by N-terminal fusion of a small peptide to the target protein. Fusion of a ubiquitin gene together with a ubiquitin promoter to the 5'-end of a gene of interest is one of the systems which has been used to enhance protein expression. Ubiquitin exists in all eukaryotic cells and is the most highly conserved protein yet identified. It is abundant in cells and exhibits profound stability to heat and proteolytic degradation. Moreover, ubiquitin precursors, that is, polyubiquitin where ubiquitin monomers are linked up head to tail and ubiquitin extension proteins where a single ubiquitin is appended at its C-terminus to either of two small ribosomal proteins, undergo rapid processing by ubiquitin C-terminal hydrolases, which cleave C-terminal of the ubiquitin moieties and release the free ubiquitin monomer and the C-terminal extension proteins. All of these features have rendered ubiquitin as an excellent N-terminal fusion partner to augment target protein accumulation in genetic engineering.

The ubiquitin fusion approach was first developed by Butt et al (1989), who showed that fusion of ubiquitin to yeast metallothionein or to the a subunit of the adenoylate cyclase- stimulatory GTP-binding protein increased the yield of these otherwise unstable or poorly expressed proteins from undetectable levels to 20% of the total cellular proteins in E. coli. Ecker et al. (1989) demonstrated that in yeast. ubiquitin fusion resulted in enhanced expression of three mammalian proteins by up to 200-fold and all these ubiquitin fusion proteins were corrects processed by yeast ubiquitin-specific endopeptidase to release authentic functional proteins. A similar yeast ubiquitin fusion expression system was reported by Sabin ct al. (i989). in which ubiquitinhuman y-intcrferon and ubiquitin/al-proteinase inhibitor were lliglll expressed and quantitatively cleaved to yield y-IFN and a 1-PI with authentic amino termini.

Since these early reports, a wealth of studies on ubiquitin fusion expression of various proteins in E. coli and yeast have been described (Baker et al., 1994; Barr et al., 1991; Coggan etal., 1995; Gali and Board, 1995; Gehring et al., 1995; Han et al., 1994; Kiefer et al., 1992; Lu et al., 1990; Lytle et al., 1992; Mak et al., 1989; McDonnell et al., 1989; McDonnell et al., I 991; Pilon et al., 1996; Poletti et al., 1992; Rian et al., 1993; Tan and Board, 1996; Welch et al., 1995). Very often fusion to ubiquitin led to dramatic enhancement in yield of the fusion protein in bacteria, or of the cleaved product in yeast.

Enhanced expression of foreign proteins by ubiquitin fusion has also been observed in plants. In analysis of the promoter of the tobacco polyubiquitin gene, Ubi. U4, by driving transient expression of the GUS reporter in tobacco protoplasts, Genschik et al. (1994) found deletion of the intron sequence from the Ubi. U4 fragment spanning from-263 to the end of the first ubiquitin-coding unit had no detectable influence on the GUS activity, but further deletion of the ubiquitin-coding sequence diminished the GUS activity by 55%.

None of these studies has shown the direct enhancing function of the ubiquitin fusion from a heterologous promoter. Garbarino and Belknap (1994) observed that fusion of the promoter plus ubiquitin-coding region of the potato ubiquitin extension protein gene ubi 3 to the GUS reporter gene resulted in GUS activity 5-to 10-fold higher than the direct fusion of the ubi 3 promoter to the GUS gene did in transgenic potato. Again, the synergistic effect of the ubi 3 promoter and the ubiquitin-coding sequence on the enhanced GUS activity was not excluded.

In another study with a potato polyubiquitin gene, ubi 7, the same group (Garbarino et al., 1995) demonstrated that in transgenic potato plants GUS expression level from the fusion construct containing the ubi 7 promoter-5'untranslated sequence-intron-first ubiquitin coding unit was 10 times higher than that derived by only the ubi 7 promoter with the 5'untranslated sequence.

However, the effects of the intron and the ubiquitin protein fusion in increasing expression level of the GUS reporter were not clearly discriminated.

In addition to the above mentioned journal papers, a number of patents related to the ubiquitin fusion technology have been filed since 1989. They are shown in Table 1. The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience arc respectively grouped in the appended List of References.

Table 1 Patents related to the ubiquitin fusion technology Host Title Inventor Patent No. Filing Date cells Generating desired amino-MIT WO 8909829 10/19/1989 terminal residue in protein Regulation metabolic stability MIT US 5093242 3/3/1992 mammal, of a protein yeast Nucleic acid constructs, malaria Chiron WO 9208795 5/29/1992 yeast polypeptides and vaccines Production of a protein with a MIT US 5196321 3/23/1993 E. coli predetermined amino-terminal amino acid residue Yeast expression system for American EP 608532 8/3/1994 yeast retinoid-X receptor Cyanamid Recombinant DNA vectors Mascarenhas WO 9423040 10/13/1994 E. coli New heat-inducible N-degron Varshavsky, WO 9521269 8/10/1995 protein and nucleic acid Dohmen, encoding it Johnston, Wu Fusion proteins containing the Varshavsky, WO 9529195 11/2/1995 N-or C-terminal of ubiquitin Johnston New fusion protein of ubiquitin Carbarino, WO 9603519 2/8/1996 plant plant and lytic peptide Jaynes, Belknap Production of tissue factor Innis, WO 9604377 2/15/1996 yeast pathway-inhibitor in yeast cells Creasey Stable recombinant ubiquitin-J. Jaynes WO 9603522 2/8/1996 plant IN-tic peptide fusion protein Fusion protein encoded by a Bachmair, US 5496721 5/3/1990 mammal, gene construct Finley, yeast Varshavsky

SUMMARY OF THE INVENTION In accordance with the present invention a method for enhancing expression of proteins in plants or plant cells is achieved by the fusion of a ubiquitin monomer coding sequence to the 5'end of the coding sequence of the proteins. Expression of the ubiquitin fusion proteins is driven by a promoter other than promoters from polyubiquitin protein genes or ubiquitin extension protein genes. Thus enhancement of expression level of the proteins is due to the 5' terminal addition of the ubiquitin monomer coding sequence. The ubiquitin fusion proteins are cleaved at the carboxy-terminal glycine 76 residue of the ubiquitin, presumably by plant ubiquitin specific proteases, to produce proteins with desired biological properties. A second aspect of this invention is that the N-terminal peptide of 14 amino acid residues of cucumber mosaic virus coat protein (NP14) can be used as an N-terminal fusion partner to increase the expression level of target proteins in plants. The N-terminal fusion approaches described in this invention allow higher yield production of proteins in plants, either in the authentic forms in the ubiquitin fusion system or as the fusion protein in the NP14 fusion system.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the nucleotide sequence and deduced amino acid sequence of tobacco ubi. NC89. The nucleotide sequence is listed as SEQ ID NO: 1 and the amino acid sequence is SEQ ID NO: 2 in the Sequence Listing. The primers used in PCR are underlined and the mended 37-mer oligonucleotide is double-underlined.

Figure 2 shows the synthetic DNA coding for the 14 N-terminal amino acids of CMV CP (NP14). The nucleotide sequence is SEQ ID NO: 3 and the amino acid sequence is SEQ ID NO: 4.

Figure 3 illustrates the construction of the ubiquitin-GUS fusion protein expression vector pUG. The nucleotide sequence shown for pSKUBC I is SEQ ID NO: 5, the sequence shown for pub1221 is SEQ ID NO: 6, and the sequence shown for pUG is SEQ ID NO: 7.

Figure 4 illustrates the construction of the 'P 14-GUS fusion protein expression vector pCG. The nucleotide sequence shown for pUCG2 is SEQ ID N0: 8.

Figure 5 illustrates the construction of the ubiquitin-tuciferase fusion protein expression vector pUL. The arrow marked in the recognition sequence of Stu I in pBIubi indicates the end of the ubiquitin coding region and the cleavage site of the ubiquitin fusion protein. The upper

nucleotide sequence shown for pBIubi is SEQ ID NO: 9, the lower nucleotide sequence shown for pBIubi is SEQ ID NO: 10, and the nucleotide sequence shown for pUL is SEQ ID NO: 11.

Figure 6 illustrates the construction of the NP14-luciferase fusion protein expression vector. The nucleotide sequence shown for pCL is SEQ ID NO: 12.

Figure 7 illustrates the construction of ubiquitin-GUS fusion/LUC dual report binary vector pUGL121.

Figure 8 illustrates the construction of the NP14-GUS fusion/LUC dual reporter binary vector pCGL121.

Figure 9 illustrates the construction of the GUS/LUC dual reporter binary vector pBIL121.

Figure 10 illustrates the ubiquitin fusion cloning vector pBIubi. The upper nucleotide sequence is SEQ ID NO: 13 and the lower nucleotide sequence is SEQ ID NO: 14.

Figure I I illustrates the NP14 fusion cloning vector pBINP14.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to methods and constructs for enhancing protein production in plants. The methods comprise fusing an expression-enhancing nucleic acid at the 5'terminus of the gene for which enhanced expression is desired. In one aspect of the invention, a ubiquitin gene is inserted in front of the gene encoding the desired protein such that a fusion protein is produced wherein ubiquitin is directly fused to the amino terminus of the desired protein. Enzymes such as C-terminal hydrolases, will cleave at the C-terminus of the ubiquitin in the fusion protein thereby releasing the desired protein in its natural fonn as well as forming free ubiquitin. The presence of the ubiquitin gene in the resulting fusion protein results in enhanced expression of the gene thereby yielding a greater amount of the desired protein product than occurs in the absence of the ubiquitin gene. It is necessary to use only the coding portion <BR> <BR> <BR> <BR> of the ubiquitin gent.- (le ubiquitin promoter is unnecessary, and the ubiquitin gene fusion can be under the control of a hctero ! ogous promoter.

In a second aspect of the invention, enhanced protein production is seen when a nucleic acid encoding 14 amino acids of cucumber mosaic virus coat protein is placed in front of the gene encoding a desired protein such that a fusion protein is produced wherein the fusion protein

includes the 14 amino acids of the cucumber mosaic virus coat protein at the amino terminus of the fusion protein.

The aspects of the invention are set out in the following Examples which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.

Techniques such as transfection of protoplasts, preparation of transgenic tobacco plants, fluorometric GUS assays and luciferase assays are well known to those of skill in the art and are not described in detail herein.

EXAMPLE I DNA Sequences Coding for the Tobacco Ubiquitin and the N-tenminal Peptide of CMV Coat Protein The coding sequence of the ubiquitin monomer contains 228 base pairs. The 5'part of 191 base pairs was obtained by polymerase chain reaction (PCR) amplification on the total DNA of Nicotiana tobacum var. NC89 and the remaining 37 base pairs were prepared as a synthetic oligonucleotide. An SphI site encompassing the initiation codon ATG and an NcoI site following the last codon GGC were created to facilitate cloning. The tobacco ubiquitin coding sequence was then cloned into pGEM-5ZF and sequenced. Figure 1 shows the DNA sequence and the deduced amino acid sequence of the tobacco ubiquitin. The 76-amino acid sequence is identical to that derived from a tobacco polyubiquitin gene ubi. U4 (Genschik et al., 1994).

However, the nucleotide sequence of the region amplified from the tobacco DNA is different <BR> <BR> <BR> <BR> from the corresponding regions of all ubiquitin monomers found in ubi. U4. We have named this<BR> <BR> <BR> <BR> <BR> <BR> tobacco ubiquitin coding sequence as ubi. NC89.

The cucumber mosaic virus coat protein (CMV CP) is encoded by the viral subgenomic RNA 4 and comprises 218 amino acid residues. The CP gene of the strain CMV-SD was cloned by RT-PCR (Guo et al., 1993) and the cDNA sequence encoding the 14 N-terminal amino acids (NP 14) was either cut out of the CP gene by Ncol/Accl digestion or chemically synthesized. In the synthesized version of the NP14 coding sequence (Figure 2), overhanging adapters for BamHI and Astl sites were attached to the 5'-and 3'-ends, respectively, for easy cloning.

EXAMPLE 2 Translational Fusion Constructs for Transient Expression Assays A. Ubiquitin-GUS fusion construct pUG The ubi. NC89 sequence was taken from the plasmid pSKUBCI as an Xbal-Ncol (filled- in) fragment and inserted into the XbaI-BamHI (filled-in) site upstream of the GUS gene in pBI221 to construct pUG as shown in Figure 3.

B. NP14-GUS fusion construct pCG Plasmid pUCG2 is a derivative of pBI221, in which the ubi. NC89 sequence and the NP 14 sequence, linked as a read-through ORF, was inserted into the XbaI-SmaI sites in front of the GUS gene. The ubiquitin moiety was removed from pUCG2 by Xbal-SacIl digestion and pCG was formed by recircularizing. Figure 4 illustrates these steps clearly.

C. Ubiquitin-LUC fusion construct pUL An Ncol (filled-in)-SstI fragment containing the firefly luciferase (LUC) gene was inserted into the ubiquitin fusion vector pBIubi (see Figure 10) downstream of ubi.NC89 via the Stul-Sstl sites in the polylinker region, resulting in pUL as shown in Figure 5.

D. NP 14-LUC fusion construct pCL The Ncol (filled-in)-Sstl fragment containing the LUC gene was inserted into the NP 14 fusion vector pBINP 14 (see Figure 11) downstream of the NP 14 coding sequence via AccI (or Sall which is the equivalent site here) (filled-in)-Sstl sites, resulting in pCL as shown in Figure 6.

EXAMPLE 3 GUS/LUC Dual Reporter Constructs for Stable Transformation To examine the enhancing effects of the N-terminal addition of the ubiquitin or CMV CP NP 14 on GUS expression in stably transformed plants, a series of GUS/LUC (test/reference) dual reporter constructs were made. Essentially they are based on the fusion constructs used in transient expression assays, namely, pUG and pCG. The chimeric GUS expression cassettes were moved into the plant transformation intermediate plasmid pub1121, resulting in pUG 121 and

pCG121, respectively. The expression cassette of the reference reporter LUC, which was constructed by replacing the GUS gene in pBI221 with the LUC gene, was pre-made as a HindIII fragment (HindllI-35S/LUC/NOS-HindIII) and then inserted into the unique Hindi ! ! site of pUG121, pCG121 and pB ! 12 !, respectively. The resulting GUS/LUC dual reporter constructs, pUGL121, pCGL121 and pBIL 121 are shown in Figures 7,8 and 9, respectively.

EXAMPLE 4 Ubiquitin fusion enhances the expression of GUS and LUC in tobacco protoplasts The ubiquitin-GUS fusion construct pUG or the control plasmid pBI221 was introduced into tobacco protoplasts derived from tobacco BY-2 suspension cells, together with a reference plasmid FFO which contained LUC gene driven by the 35S promoter. GUS activities were determined and normalized by luciferase activities. In four independent transfection experiments, the normalized GUS activities (A GUS) from pUG were considerably higher than those from pB1221. The averaged increase fold due to the ubiquitin fusion is 6.0 (Table 2).

When using LUC as a reporter and GUS as an internal standard as expressed from pBI221, the normalized LUC activities from pUL were 1. 37 to 3.11 fold higher than those from the control plasmid p35SLUC (35S-LUC-NOS) in three independent transfection experiments, with the average increase fold about 2 (Table 3).

EXAMPLE 5 CMV CP NP 14 Is a More Efficient Fusion Partner than Ubiquitin The enhancing effects of the NP ! 4 fusion on GUS and LUC expression in tobacco protoplasts were examined in experiments parallel to the above mentioned ubiquitin fusion study.

The NP 14-GUS fusion construct pCG produced an average I 1-fold higher GUS activity than did pB1221. These results are shown in Table 2. Fusion of NP14 to LUC increased the LUC activity by 2.87 times, calculated by comparing the normalized LUC activity of pCL to that of p35SLUC. These results are shown in Table 3. It is apparent that NP14 is a more efficient fusion partner than ubiquitin in augmenting GUS and LUC expression in tobacco cells.

Table 2 Normalized GUS activities and enhancing fold of the N-terminal fusion constructs

pUGpCGplasmidpBI221 #GUSE#GUSEactivitiesGUS 1 293. 3760.0 12.8 5743. 0 19.6 2 206. 7 584. 3 2. 8 940.8 4.6 3 856. 7 3733. 8 4. 4 6708.0 7.8 4 100. 0 408. 8 4. 1 1247.0 12.5 average E value 11.1~3.2 Notes: 1. The normalized GUS activity A GUS is calculated by the formula #GUSn=GUSnxLUC22@ LUC, where n represents a particular GUS fusion construct, 221 represents pBI221.

2. The enhancing fold E is calculated as AGUS, GUS,,, Table 3 Normalized LUC activities and enhancing fold of the N-terminal fusion constructs

Plasmid p35S LUC pUL pCL average average average activities ALUC ALUC ALUC ALUC E ALUC ALUC E 1 252 274 457 112 5183961.375294911.70290 1 169 556 701 2 2 ND 169 496 526 3.11 886 794 4.70 1 64 141 270 3 160 112 181 164 1.46 254 246 2.20 1702143ND 2.87~0.92Mean~SE1.98~0.56 Notes: 1. The normalized LUC activity ALUC is calculated by the formula LUCnxGUSp35SLUC#LUCn= GUS, where n represents a particular LUC fusion construct.

2. The enhancing fold E is calculated as ALUC, LUCp35SLUC.

EXAMPLE 6 Ubiquitin-and NP14-fusion Enhance GUS Expression in Transgenic Plants To examine the enhancing effects of the ubiquitin fusion and the NP 14 fusion on GUS expression in stably transformed plants, three GUS/LUC (test/reference) dual reporter constructs were made based on the binary vector pB1121. pUGL121, pCGL121 and pBIL121 contained expression cassettes ubiquitin-GUS, NP14-GUS and GUS only (control), respectively, and the reference LUC expression cassette was integrated in each plasmid (Figures 7-9). Tobacco plants transformed with each of the three constructs were prepared and analyzed for GUS and LUC activities. Each plant was analyzed twice in two independent experiments and only those plants displaying reasonable consistency of the relative GUS activities (GUS/LUC) in two experiments were included for comparison. As shown in Table 4, although variations in the relative GUS activities existed among different transformants from the same constructs, the average GUS expression level of 5 qualified plants containing the 35S-ubiquitin/GUS fusion construct was 4 times higher than that derived from 6 plants containing the 35S-GUS construct, confirming the enhancing effect of the ubiquitin fusion on GUS expression as previously observed in tobacco protoplasts. Again, the NP14 fusion displayed a higher enhancing effect on GUS expression than did the ubiquitin fusion. The average relative GUS activity of 14 pCGL plants was about 7 fold that derived from the pBIL 121 construct.

EXAMPLE 7 Ubiquitin fusion and NP14 fusion cloning vectors pBIubi (Figure 10) and pBINP14 (Figure 11) are two fusion protein expression vectors <BR> <BR> <BR> <BR> allowing for insertion of target genes downstream of the ubi. NC89 and the CMV CP NP14 I coding sequence, respectively. Both vectors are derivatives of pBI221, with the GUS gene being <BR> <BR> <BR> <BR> replace by the urbi. A'C89 or NP14 coding sequence. In pBIubi, a polylinker sequence was<BR> <BR> <BR> <BR> <BR> attache to the 3'end of the ubi. AC89 sequence and the penultimate codon of the ubi. NC89 was changed from GGT to GGA for creating a Stul site in the polylinker region. In pBINP 14, two cloning sites, Sall (here equivalent to an Accl site) and Sstl, are available for cloning the target genes downstream from the NP 14 sequence (the last 5 base pairs of the NP 14 sequence form part of the Sall recognition sequence). In order to use Accl instead of SalI for cleaving pBINP 14, the Accl site at-393 of the CaMV 35S promoter was eliminated.

Table 4 Effects of ubiquitin-and NP 14-fusion on GUS expression in transgenic tobacco plants

Relative GUS activities: GUS/LUC (pmol MUmin-'lcpm x 10-3) pCGL121pBIL121PlantpUGL121 lines exp. 1 exp. 2 average exp. I exp 2 average exp. 1 exp. 2 average 1 12. 9 15.3 14.1 2.9 1.4 2.6 2 2 13 43 28 5.65 5.2 2.4 3.8 3 0.7 0.5 0.6 36.35 4.2 0.6 2.4 4 0.3 0.4 0.35 17.6 2.5 5.4 3.95 5 4.8 0.8 2.8 17. 8 22.2 20 0.4 0.38 0.39 6 2. 1 5 3.55 0.5 0.82 0.66 7 4. 6 5.8 5.2 8 58. 7 20.2 39.45 3.6915.6 9.6 10 17.2 4. 4 10.8 11 3 1.4 2.2 12 17. 9 24.2 21.05 13 20. 7 19.4 20.05 14 13. 7 25.3 19.5 Mean 9.17~5.34 15.28~3. 18 SE

While the invention has been disclosed by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.

LIST OF REFERENCES Baker RT, Smith SA, Marano R, McKee J, Board PG: Protein expression using cotranslational fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human pi class glutathione S-transferase. J. Biol. Chenu. 269: 25381-25386 (1994).

Barr PJ, Inselburg J, Green KM, Kansopon J, Hahm BK, Gibson HL, Lee-Ng CT, Bzik DJ, Li W, Bathurst IC: Immunogenicity of recombinant Plasmodiunifalciparuni SERA proteins in rodents. Mol. Biochem. Parasitol. 45: 159-170 (1991).

Butt TR, Jonnalagadda S, Monia BP, Stemberg EJ, Marsh JA, Stade @JM, Ecker DJ, Crooke ST: Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. Proc. Natl.

Acad. Sci. USA 86: 2540-2544 (1989).

Coggan M, Baker R, Miloszewski K, Woodfield G, Board P: Mutations causing coagulation factor XIII subunit A deficiency: characterization of the mutant proteins after expression in yeast. Blood 9 : 2455-2460 (1995).

Ecker DJ, Stadel JM, Butt TR, Marsh JA, Monia BP, Powers DA, Gorman JA, Clark PE, Warren F, Shatzman A, Crooke ST: Increasing gene expression in yeast by fusion to ubiquitin. J. Biolo.

Chent. 264: 7715-7719 (1989).

Gali RR, Board PG: Sequencing and expression of a cDNA for human glutathione synthetase.

Biochem. J. 310: 353-358 (1995).

Garbarino JE. Be) knap WR: Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants. Plant Mol. Biol. 24: 119-127 (1994).

Garbarino JE, Oosumi T, Belknap WR: Isolation of a polyubiquitin promoter and its expression in transgenic potato plants. Plant Physiol. 109: 1371-1378 (1995).

Gehring MR, Condon B, Margosiak SA, Kan CC: Characterization of the Phe-81 and Val-82 human fibroblast collagenase catalytic domain purified from Esc/erichia coli. J. Biol. Chenr.

270: 22507-22513 (1995).

Genschik P. Marbach J, Uze M. Feuerman M, Plesse B, Fleck J: Structure and promoter activity of a stress and developmental regulated polyubiquitin-encoding gene of Nicotiana tabacum.

Gene 148 : 195-202 (1994).

Guo DC. Qiao L, Fang RX, Mang KQ: Cloning of coat protein gene of cucumber mosaic virus (SD Strain) by PCR. Acta Microbiologica Sinica 33 : 233-235 (1993).

Han K. Hong J. I. im HC, Kim CH, Park Y, Cho JM: Tyrosinase production in recombinant E. <BR> <BR> coli containin y promoter and ubiquitin sequence. Ann. N. Y. Acad. Sci. 721: 30-42 (1994). Kiefer MC, Schmid C, Waldvogel M, Schlapfer 1, Futo E, Masiarz FR, Green K, Barr PJ, Zapf J: Characterization of recombinant human insulin-like growth factor binding proteins 4,5 and 6 produced in yeast. J. BioL Chem. 267: 12692-12699 (1992).

Lu C, Yang Y-F, Ohashi H, Walfish PG: In vivo expression of rat liver c-erbA beta thyroid hormone receptor in yeast (Saccharomyces cerevisiae). Biochem. Biophys. Res. Commun.

171: 138-142 (1990).

Lyttle CR. Damian-Matsumura P, Juul H, Butt TR: Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists. J. Sleroid Brochent. tlol Biol. 42: 677-685 (1992).

Mak P, McDonnell DP, Weigel NL, Schrader WT, O'Malley BW: Expression of functional chicken oviduct progesterone receptors in yeast (Saccharomyces cerevisiae). J. Biol. Chem.

264:21613-21618(1989).

McDonnell DP, Pike JW, Drutz DJ, Butt TR, O'Nlalley BW: Reconstitution of the vitamin-D- responsive osteocalcin transcription unit in Saccharomyces cerevisiae. Mol. Cell. Biol. 9:3517- 3523 (1989).

McDonnell DP, Nawaz Z, Densmore C, Weigel NL, Pham TA, Clark JH, O'Malley BW: High level expression of biologically active estrogen receptor in Saccharomyces cerevisiae. J. Steroid Biochem. Mol. Biol. 39 : 291-297 (1991).

Pilon AL. Yost P, Chase TE, Lohnas GL, Bentley WE: High-level expression and efficient recoverv of ubiquitin fusion proteins from Escherichia coli. Biotechnol. Prog. 12: 331-337 (1996).

Poletti A. Weigel NL, McDonnell DP, Schrader WT, O'Malley BW, Conneely OM: A novel, highly regulated, rapidly inducible system for the expression of chicken progesterone receptor, cPRA, in Saccharomyces cerevisiae. Gene 114: 51-58 (1992).

Rian E, Jemtland R, Olstad OK, Gordeladze JO, Gautvik KM: Synthesis of human parathyroid- hormone-related protein (1-141) in Saccharomyces cerevisiae. A correct amino-temiinal processing vital for the hormone's biological activity is obtained by an ubiquitin fusion protein approach. Etir. J. Biochenr. 213: 641-648 (1993).

Sabin EA. Lee-Ng CT, Shuster JR, Barr PJ: High-level expression and in vivo processing of chimeric ubiquitin fusion proteins in Saccharomyces cerevisiae. BiolTechnology 7: 705-709 (1989).

Tan KL. Board PG: Purification and characterization of a recombinant human theta-class glutathionc transferase (GSTT2-2). Biochem. J. 31S: 727-732 (1996). Welch AR, Holman CM, Browner MF, Gehring MR, Kan CC, Van-Wart HE: Purification of human matrilysin produced in Escherichia coli and characterization using a new optimized fluorogenic peptide substrate. Arch. Biochem. Biophs. S : 59-64 (1995).

U. S. Patent No. 5,093,242 U. S. Patent No. 5,196,321 U. S. Patent No. 5,496,721 PCT Publication No. WO 89/09829 PCT Publication No. WO 92/08795 PCT Publication No. WO 94/23040 PCT Publication No. WO 95/21269 PCT Publication No. WO 95/29195 PCT Publication No. WO 96/03519 PCT Publication No. WO 96/04377 PCT Publication No. WO 96/03522 EP 608532 SEQUENCE LISTING <110> Fang, Rong-Xiang <120> ENHANCED PROTEIN PRODUCTION IN HIGHER PLANTS BY N-TERMINAL FUSION OF A UBIQUITIN OR A CUCUMBER MOSAIC VIRUS COAT PROTEIN PEPTIDE <130> 2248-109 <140> <141> <160> 14 <170> PatentIn Ver. 2.0 <210> 1 <211> 235 <212> DNA <213> Nicotiana tabacum <220> <221> CDS <222> (3).. (230) <220> <223> Modified from wild-type to insert an SphI site in the region encompassing the initiation codon ATG and to insert an NcoI site following the last codon GGC.

<400> 1 gc atg cag atc ttc gta aag acc ctg acg ggg aag act att acc tta 47 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu 1 5 10 15 gag gta gag tca tcg gac acc att gac aat gtt aag gct aag att cag 95 Glu Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ala Lys Ile Gln 20 25 30 gac aag gaa ggc att cca ccg gac cag cag cgg ttg att ttc gca ggt 143 Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly 35 40 45 aag cag ctt gag gat ggc cga aca cta gct gac tac aac atc cag aag 191 Lys Gln Leu Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys 50 55 60 gag tcc act ctc cat ctc gtc tta aga ctc cgc ggt ggc catgg 235 Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 65 70 75 <210> 2 <211> 76 <212> PRT <213> Nicotiana tabacum <400> 2 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 65 70 75 <210> 3 <211> 53 <212> DNA <213> cucumber mosaic virus <220> <221> CDS <222> (6).. (47) <400> 3 gatcc atg gac aaa tct gaa tca acc agt gct ggt cgt aac cgt cga 47 Met Asp Lys Ser Glu Ser Thr Ser Ala Gly Arg Asn Arg Arg 1 5 10 cgagct 53 <210> 4 <211> 14 <212> PRT <213> cucumber mosaic virus <400> 4 Met Asp Lys Ser Glu Ser Thr Ser Ala Gly Arg Asn Arg Arg 1 5 10 <210> 5 <211> 13 <212> DNA <213> Plasmid pSKUBCl <220> <221>misc feature <222>()..) <223> Joining region of fusion of two genes.

<400> 5 ggccatggac aaa 13 <210> 6 <211> 33 <212> DNA <213> Plasmid pBI221 <220> <221>misc feature <222> (1).. (33) <223> Joining region between 35S promoter and GUS gene.

<400> 6 tctagaggat ccccgggtgg tcagtccctt atg 33 <210> 7 <211> 18 <212> DNA <213> Plasmid pUG <220> <221>misc feature <222> (1).. (18) <223> Joining region of fusion of genes.

<400> 7 ggccatggat ccccgggt 18 <210> 8 <211> 18 <212>DNA <213> Plasmid pUCG2 <220> <221>misc feature <222> (1).. (18) <223> Joining region of fusion of genes.

<400> 8 ctccgcggtg gcatggac 18 <210> 9 <211> 29 <212> DNA <213> Plasmid pBIubi <220> <221>misc feature <222> (1).. (29) <223> Joining region between promoter and fused gene.

<400> 9 tctagaacta gtggatccct ggcatgcag 29 <210> 10 <211> 35 <212> DNA <213> Plasmid pBIubi <220> <221>misc feature <222> (1).. (35) <223> Final 2 codons of the ubiquitin gene followed by polylinker sequence.

<400> 10 ggaggcctgt cgactcgagc ccgggtaccg agctc 35 <210> 11 <211> 12 <212> DNA <213> Plasmid pUL <220> <221>misc feature <222> (1).. (12) <223> Joining region between fusion of genes.

<400> 11 ggaggcatgg aa 12 <210> 12 <211> 12 <212> DNA <213> Plasmid pCL <220> <221>misc feature <222> (1).. (12) <223> Joining region between fusion of genes.

<400> 12 cgtcgcatgg aa 12 <210> 13 <211> 29 <212> DNA <213> Plasmid pBIubi <220> <221>misc feature <222> (1).. (29) <223> Joining region of fusion of promoter and gene.

<400> 13 tctagaacta gtggatccct ggcatgcag 29 <210> 14 <211> 35 <212> DNA <213> Plasmid pBIubi <220> <221>misc feature <222> (1).. (35) <223> Joining region with multicloning sequence between fusion of gene and terminator.

<400> 14 ggaggcctgt cgactcgagc ccgggtaccg agctc 35