Expression of phaseolin cDNA genes in yeast under control of natural plant DNA sequences

The seed storage protein P-phaseolin of the common bean (Pbaseolus vulgaris L.) was expressed i n the endosperm of transgenic rice (Oryza sativa L.) plants. The 5.1 -or 1.8-kb promoter fragment of the rice seed storage protein glutelin C t l gene was fused transcriptionally to either the genomic or cDNA coding sequence of the P-phaseolin gene. The highest quantity of phaseolin estimated by enzyme-linked immunosorbent assay was 4.0% of the total endosperm protein in the transgenic rice seeds. The phaseolin trait was segregated as a single dominant trait with a positive gene dosage effect and was stably inherited through three successive generations. 60th phaseolin genomic and cDNA coding sequences were used to synthesize four isoforms of mature phaseolin protein with apparent molecular masses of 51, 48, 47, and 45 kD. Enzyme deglycosylation experiments indicated that the 51 -kD form contains high-mannose N-glycans; the 48-and 47-kD forms have further modified N-glycans; and the 45-kD form is a nonglycosylated protein. lmmunolabeling studies using light and electron microscopy demonstrated that phaseolin accumulates primarily in the vacuolar type-I1 protein bodies located at the periphery of the endosperm near the aleurone layer. We discuss the implications of these results on nutritional improvement of rice grains.

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“…The plasmids AG-pPVPh3.8 and AG-pPVPh3.8/ cDNA were kindly provided by Dr. J.L. Slightom (Upjohn Co., Kalamazoo, MI) (Cramer et al, 1985).…”

Section: Construction Of Phaseolin Fusion Genesmentioning

confidence: 99%

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm

et al. 1995

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“…Quantitation of the immunoprecipitates showed that maximally 1-2% of the total protein synthesis was confined to the synthesis of virusspecific proteins. The levels of different soluble animal and plant proteins expressed in yeast from recombinant plasmids have been in the range of 0.01-5% of total protein (16,17,(27)(28)(29). So far, one animal virus envelope glycoprotein, the hemagglutinin of influenza virus, has been expressed in S. cerevisiae.…”

Section: Discussionmentioning

confidence: 99%

Expression of the RNA genome of an animal virus in Saccharomyces cerevisiae.

Makarow¹,

Nevalainen²,

Kääriäinen³

1986

Proc. Natl. Acad. Sci. U.S.A.

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The nucleocapsid of vesicular stomatitis virus (VSV) was introduced into the cytoplasm of Saccharomyces cerevisiae by low pH-dependent fusion of the viral envelope with the spheroplast plasma membrane. This led to de novo synthesis of the three major structural proteins of the virusthe G, N, and M proteins-as shown by immunoprecipitation of [35S]methionine-labeled spheroplast lysates. In NaDodSO4/ polyacrylamide gel electrophoresis, M and N proteins comigrated with those of the virion, whereas the yeast-made G protein migrated as two bands with apparent molecular sizes of 60 and 70 kDa. Both polypeptides appeared to be N-glycosylated, since only one polypeptide with the apparent molecular mass of -55 kDa was produced in the presence of tunicamycin. Phase separation into Triton X-114 suggested that the unglycosylated G protein was membrane bound. According to immunofluorescent surface staining of live spheroplasts, at least part of the G protein was transported to the plasma membrane. Spheroplasts expressing the VSV genes could be fused together by low pH to form polykaryons, indicating that G protein synthetized by yeast was fusogenic-i.e., biologically active.Yeast is an attractive host for production of foreign proteins by recombinant DNA technology, since it is able to glycosylate and secrete them. Intracellular transport and posttranslational modifications of soluble enzymes of Saccharomyces cerevisiae have been well-characterized (1), whereas much less is known about membrane proteins. Extensive glycosylation, characteristic of secretory enzymes and mannoproteins of yeast (2, 3), can be suspected to impair biological functions offoreign glycoproteins expressed in this organism. Studies of intracellular transport and glycosylation of heterologous proteins in yeast require laborious recombinant DNA constructions. We have used a different approach to study the direct expression of the proteins of an animal virus in S. cerevisiae, taking advantage of the activation of the particle-associated transcriptase (L protein) of vesicular stomatitis virus (VSV) (4, 5). The nucleocapsid of VSV was introduced into the yeast cytoplasm by fusion of the virus envelope with the spheroplast plasma membrane. The genes encoding the major structural proteins (G, M, and N) were expressed within 30-120 min. The G protein was transported to the plasma membrane where its fusogenic activity could be elicited in acid medium, resulting in polykaryon formation.MATERIALS AND METHODS Cells and Virus Preparations. The strain OL1 (6) of S. cerevisiae was grown in YPD medium (growth medium) containing 1% yeast extract (Oxoid, Basingstoke, U.K.), 2% Bacto-peptone (Difco), and 2% glucose (BDH), at 30°C in a shaker to densities of 4-15 x 107 cells per ml. Spheroplasts were prepared with zymolyase 60,000 (Seikagaku, Tokyo, Japan) as described (7). All media used for spheroplasts contained 1.2 M sorbitol to prevent osmotic lysis. Unlabeled and [35S]methionine-labeled VSV (Indiana serotype) were grown in baby hamster kidney cells and purifi...

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“…2). In S. cerevisiae, both a and , polypeptides are glycosylated, and the molecular weights of the products indicate that the phaseolin signal peptides have been removed (10). In earlier studies, cDNA copies of the genes were expressed after being spliced into the normal genomic phaseolin 5'-and 3'-flanking sequences, and the level of protein observed was quite low, only 0.01 to 0.03% of the total yeast protein.…”

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confidence: 99%

“…One of the major advantages of this sytem for the expression of heterologous eucaryotic proteins is the presence of well-characterized pathways for the posttranslational alteration and transport of proteins (26), offering the potential for correct modification and for intra-or extracellular targeting of foreign-gene products. Several plant proteins have been shown to undergo signal peptide cleavage, glycosylation, or both when synthesized in S. cerevisiae (10,11,24). However, the nature of the plant sequences required for correct protein modification in the yeast system has not been examined in detail.…”

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confidence: 99%

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Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae.

Cramer¹,

Lea²,

Schaber³

et al. 1987

Mol. Cell. Biol.

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We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PH05) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

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Expression of phaseolin cDNA genes in yeast under control of natural plant DNA sequences

Cited by 34 publications

References 36 publications

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm

Expression of the RNA genome of an animal virus in Saccharomyces cerevisiae.

Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae.

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