Grantee Research Project Results

Final Report: Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops

EPA Grant Number: R829479C024
Subproject: this is subproject number 024 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Human Models for Analysis of Pathways (H MAPs) Center
Center Director: Murphy, William L
Title: Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
Investigators: Vierstra, Richard D , Walker, Joseph M
Institution: University of Wisconsin - Madison
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2004 through September 30, 2007 (Extended to December 31, 2007)
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

Abstract

The genetic engineering of complex traits into crop plants will ultimately require strategies to co-express more than one protein at the same time. In this project, we developed a ubiquitin (Ub)-based expression method capable of generating multiple, separate proteins from a single transcript. The vector contains coding regions for the proteins of interest, separated in-frame by the coding region for the C-terminal end of Ub followed by a full-length Ub. Upon expression in plants, this polycistronic mRNA is translated to produce a chimeric protein that is rapidly processed by endogenous Ub-specific proteases (UBPs) to release the two proteins plus a Ub moiety in intact forms. Whereas the C-terminal protein is released without additional amino acids (aa), the N-terminal protein domain retains the short C-terminal end of the Ub sequence. Analysis of this vector system in yeast, tobacco, and Arabidopsis indicates that multiple proteins can be released by this method from a single large transcript. Quantitation of the products from a l uciferease (LUC)-Ub-β-glucuronidase (GUS) fusion indicates that stoichiometric accumulation often occurs. Deletion analysis revealed that the Ub moiety appended to the N-terminal protein can be reduced in length to just six amino acids and still promote cleavage by UBPs in both yeast and tobacco. From this work, it appears that Ub-based polyprotein vectors can provide a simple method for the coordinated and stoichiometric synthesis of two or more proteins in plants.

Introduction

The genetic engineering of crop plants has enormous potential to increase agricultural production while simultaneously minimizing chemical inputs and environmental impact. Possibilities include the development of plants with enhanced nutritional value and biomass ; greater resistance to herbicides, insects, and pathogens; or the ability to economically synthesize pharmaceuticals, vaccines, industrial proteins, and polymers. Current approaches typically involve the manipulation of simple traits that require only one transgenic protein. However, as more complex traits are attempted, methods to allow the introduction of multiple proteins will be needed. Examples include the use of plants to co-express the two subunits of antibodies, generate plants with both herbicide and insect resistance, or to introduce the entire enzymatic pathways needed to synthesize vitamin A or biodegradable plastics.

Current methods for introducing multiple proteins involve either transforming multiple genes simultaneously or using repeated rounds of transformation to individually introduce each gene. With both methods, achieving and maintaining balanced expression for each protein is a challenge given the genetic independence of the introduced loci. A more subtle complication can also arise in subsequent generations if the genes unpredictably vary their expression levels independent of the other gene(s). Transgene silencing, which can effectively suppress gene activity, may further complicate expression if the same promoter is used multiple times to drive transcription (Baulcombe, 1996). These problems are best illustrated by the time -consuming procedures needed to engineer Arabidopsis to produce polyhydroxybutyrate (PHB) plastics, tobacco to make antibodies, and rice seeds to synthesize Vitamin A (Hiatt, et al., 1989; During, et al., 1990; Poirier, et al., 1992; Poirier, et al., 1995; Ye, et al., 2000). Ultimately, the movement of multiple transgenes into elite cultivars will require expanded genotypic analyses that grow exponentially as the number of proteins needed to confer a particular trait(s) increases.

Obviously, new strategies are needed for expressing more than one transgenic protein in plants. One approach is the simultaneous expression of multiple proteins from a single gene. Here, the coding region for each protein is attached in-frame in a tandem array and expressed as a polyprotein, which is then proteolytically processed to release the individual polypeptides. Its advantages include the elimination of genetic independence and the ability to generate coordinated and equimolar expression through the use of one common promoter. The potential of this approach was first demonstrated by co-expressing two proteins separated by the NIa protease-recognition sequence from tobacco etch virus (TEV) along with the NI a protease (Marcos and Beachy, 1994, 1997; Bodman, et al., 1995; Dasgupta, et al., 1998). Halpin, et al. (1999) more recently reported the use of 2A sequences within mammalian aphtho- and cardioviruses as a way to release two polypeptides from a single transcript. Although first thought to work via self proteolytic cleavage, new studies indicate that 2A sequences promote ribosome skipping to create multiple products. That the proximal polypeptide is produced in far excess of the distal polypeptide precludes its use for stoichiometric expression.

Another more promising approach is to use Ub -based genes to simultaneously express two or more proteins. Their potential is derived from the fact that Ubs are naturally synthesized as protein fusions, either as poly-Ubs or as Ubs attached to the N-terminus of unrelated proteins (Callis, et al., 1995). Following translation, the fusions are rapidly and accurately processed in vivo by ubiquitin-specific proteases (or UBPs) to release Ubs and the attached proteins in functional forms (Figure 1). UBPs are Ub-specific and will remove almost any polypeptide appended to their C-termini, the exception being extensions bearing proline as the first residue (Varshavsky, 1992; Yang, et al., 1999). In fact, by exploiting UBPs, a variety of proteins have been expressed transgenically as Ub fusions, including those with N-terminal residues other than methionine (Butt, et al., 1989; Ecker, et al., 1989; Hondred, et al., 1999).

Figure 1. Ub-Based Expression System

With regard to expressing multiple proteins, Varshavksy and coworkers first tested the Ub-based approach in yeast using two proteins separated in frame by a single Ub moiety. While two proteins could be simultaneously expressed, it was unclear if the proximal protein retained the Ub moiety (Levy, et al., 1996; Suzuki and Varshavsky, 1999). In the completed project, we refined this strategy based on the molecular organization of several pathogenic biotypes of b ovine v iral d iarrhea v irus (BVDV). These biotypes contain a novel genetic arrangement whereby host sequence encoding poly-Ub became inserted within the viral gene encoding the nonstructural protein p125 (Meyers et al., 1991). In the BVDV strain CP14 for example, the p125 gene is interrupted by the C-terminal 14 amino acids of Ub followed by two complete Ub repeats (Figure 1). The Ub sequences were proteolytically removed from the p125 protein by three cleavage events to generate two intact ubiquitin monomers, the C-terminal half of p125 (p80), and the N-terminal half of p125 (p35) containing the 14 C-terminal aa of Ub (Tautz, et al., 1993). The success of the first cleavage suggested that the 14 C-terminal aa are sufficient for recognition by UBPs.

The overall goal of our work was to test this Ub-based approach in plants using the BVDV genomic organization as a frame work and create a general-use vector for its exploitation (Figure 1). As will be seen below, this strategy appears to be a useful method for the simultaneous and stoichiometric production of multiple proteins in plants.

Summary/Accomplishments (Outputs/Outcomes):

Results

In previous work funded by the Consortium for Plant Biotechnology Research (CPBR) and RhoBio, we created a general-use Ub-based vector for testing in yeast and plants. The vector was designed similar to the organization of Ub in the BVDV genome (Meyers et al., 1991), having the C-terminal 14 aa of Ub followed by a full Ub moiety (Figure 2). The Ub-coding regions were derived from the Arabidopsis UBQ11 polyUb gene (Callis et al., 1995) and engineered to contain a silent SacII site at the 3' end to facilitate addition and/or interchange of other coding regions (Hondred et al., 1999). Its a a sequence is 100 percent identical to the canonical Ub sequence present in all higher plants. The vector contained the coding sequence for LUC and GUS interrupted in-frame by the coding sequence for Ub. The vector also included: (1) the Cauliflower Mosaic Virus 35S promoter (CaMV 35S) to drive high level transcription (Odell, et al., 1985); (2) the untranslated 5'-leader from Alfalfa Mosaic Virus (AMV) to afford efficient translation (Gehrke et al., 198 3); and (3) the NOS 3'-terminator.

Figure 2. Expression of the Ub-Based Vector in Yeast and Tobacco. Crude extracts were subjected to SDS-PAGE and immunoblot analysis with anti-LUC and -GUS antibodies. Plants expressing non-fused LUC and GUS and non-transformed (NT) plants are included as controls.

To accelerate the analysis, the chimeric LUC/14aa/Ub/GUS gene was first tested in yeast. As can be seen in Figure 2, readily detectable levels of LUC and GUS accumulated. Immunoblot analysis with anti-GUS and anti-LUC antibodies revealed that both proteins were present in non fused forms with sizes substantially smaller than the expected size of the polyprotein precursor (~144 kDa). A single GUS protein of 74 kDa was observed that co-migrated with full-length GUS (Figure 2). In contrast, two LUC products were evident. The major species had the expected mass of LUC plus 14 aa of Ub (~62 kDa). The other species was approximately 8 kDa larger, consistent with a LUC protein bearing the full Ub moiety as well (~70 kDa). This minor product indicated that the cleavage between the two Ub sequences was incomplete in yeast and possibly in plants as well. Importantly, both proteins were shown to be enzymatically active by standard fluorometric assays (Ow, et al., 1986; Jefferson, et al., 1987).

In an attempt to improve processing, we engineered and tested in yeast a series of LUC/??aa/ Ub/GUS constructions modified to increase or decrease the length of the N-terminal Ub fragment. Controls for the various expected cleavage products were also introduced that were used as molecular mass markers. Shortening the 14-aa stretch by two-residue increments did little to affect processing in yeast (Figure 3). It was only until most of the domain was removed ( six residues remaining) that we saw a significant further reduction in cleavage, detected by an increase in the partially processed 70-kDa form of LUC. Increasing the length of the Ub fragment beyond 14 aa did little to improve processing. A vector bearing the last 25 residues of Ub still generated a significant amount of partially processed LUC (Figure 3). From this, we concluded that such incomplete processing may be intrinsic to the expression of partial Ub moieties in yeast.

Figure 3. Minimal Length of the 5’ Ub Sequence for Efficient UBP Processing. Length of the 5’ sequence is shown above each lane. Crude extracts from yeast and tobacco were subjected to SDS-PAGE and immunoblot analysis with anti-LUC antibodies. Unprocessed LUC14aa-Ub (70 kDa) was synthesized in Escherichia coli. LUC (62 kDa), non fused LUC. NT, non-transformed cells.

To test how the polyprotein Ub vector would perform in plants, we created transgenic tobacco expressing the chimeric LUC/14aa/Ub/GUS gene (Figure 2). As with yeast, enzymatically active LUC and GUS were readily detected. Immunoblots of crude leaf extracts revealed that a single GUS of the correct size accumulated. However, unlike yeast, only the fully processed LUC product of 62 kDa was evident even after prolonged development of the immunoblots. The absence of the incompletely processed 70-kDa form of LUC suggested that cleavage of the polyprotein by UBPs is more efficient in plants than in yeast.

We then tested the possibility that the 5’ Ub fragment could be shortened further from 14 aa and still promote efficient processing. Tobacco was transformed with the various Ub14-4 aa truncations and the molecular mass of the LUC product was determined. As can be seen in Figure 3, shortening the 14-aa stretch to as little as 6 aa still provided effective cleave, which appeared even better than our observations with yeast.

By comparing the relative amounts of enzymatically active GUS and LUC, it appeared that the two enzymes were coordinately expressed (i.e., all plants expressing high levels of LUC had high levels of GUS and all plants that had low levels of LUC had low levels of GUS) (Figure 4). The amounts of LUC and GUS obtained (determined by enzyme assays) with this first version ranged from one -half to one -tenth that obtained by expressing the two proteins from separate genes. Based on these results, we concluded that the Ub-based vectors can coordinately express two separate proteins from one mRNA.

Figure 4. Stoichiometric Accumulation of Two Proteins by the Ub Polyprotein Vector in Tobacco. LUC and GUS were simultaneously expressed as a polyprotein with an N-terminal Ub domain and the LUC and GUS domains separated by the six aa of Ub and a full-length Ub. LUC and GUS activities for 24 independent transfomants were plotted relative to the mean activities.

Supplemental Keywords:

Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Genetics, Technology, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, agrobacterium, bioengineering, transgenic plants, anti-pest proteins, biomass crop plants, plant genes, biotechnology, plant biotechnology, cloning

Progress and Final Reports:

Original Abstract

2005

2006

2007

Main Center Abstract and Reports:

R829479    Human Models for Analysis of Pathways (H MAPs) Center

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829479C001 Plant Genes and Agrobacterium T-DNA Integration
R829479C002 Designing Promoters for Precision Targeting of Gene Expression
R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
R829479C005 Development of Novel Plastics From Agricultural Oils
R829479C006 Conversion of Paper Sludge to Ethanol
R829479C007 Enhanced Production of Biodegradable Plastics in Plants
R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
R829479C025 Chemical Induction of Disease Resistance in Trees
R829479C026 Development of Herbicide-Tolerant Hardwoods
R829479C027 Environmentally Superior Soybean Genome Development
R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
R829479C030 Engineered Plant Virus Proteins for Biotechnology