Final Report: Coordinated Expression of Multiple Anti-Pest Proteins

EPA Grant Number: R829479C015
Subproject: this is subproject number 015 , 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: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Coordinated Expression of Multiple Anti-Pest Proteins
Investigators: Spalding, Martin H.
Institution: Iowa State University
EPA Project Officer: Lasat, Mitch
Project Period: July 1, 2002 through December 31, 2003
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


The overall objective of this research project was to develop a single-gene system for the coordinated expression of multiple, secreted, anti-pest proteins in transgenic biomass crop plants.The specific objectives were to:

  1. link potential target proteins in pairs with the carbonic anhydrase (pCA1) spacer peptide and expression of the combined polypeptides in tobacco;
  2. and characterize elements necessary for pCA1 spacer processing in tobacco.

Summary/Accomplishments (Outputs/Outcomes):

The goal of this project was to investigate and exploit the unique post-translational processing characteristics of the Chlamydomonas gene, carbonic anhydrase 1 (Cah1), for application of this processing technology toward simultaneous multiple foreign gene expression in crops with agronomical value. The open reading frame of Cah1 contains sequences for the large subunit, small subunit, and a connecting spacer region between the large and small subunits that are proteolytically removed post-translation. Exploitation of the spacer proteolytic processing will involve construction of a unique chimeric plasmid comprised of four essential components: an endoplasmic reticulum (ER) signal sequence, an antibody heavy chain open reading frame (ORF), the Cah1 spacer sequence, and an antibody light chain ORF.

Investigation of pCA1 Processing in Arabidopsis

The demonstration of pCA1 expression and activity in maize indicated that the basic processing mechanism may be conserved across species. To investigate the basic processing and the potential for applied technologies, the model plant system, Arabidopsis has been chosen for future work. This research project utilizes Arabidopsis thaliana as a model system for investigating the conserved DNA sequences required for proper proteolytic processing of the spacer region in Cah1. The expression vector construct, pCB302-3:::AtL-Cah1, developed for the maize project served as a template for mutational analysis. Previously, I have described the development of the construct, the transformation of Agrobacterium tumefaciens and Arabidopsis, and demonstrated the presence of functional Cah1 through the use of western and activity assays. I also have described the construction of site-directed mutants designed with modifications at the 5’ and 3’ terminal cleavage sites of the Cah1 spacer.

Site Directed Mutations in the Spacer Cleavage Sites

To date, all of the constructs representing modifications of the expected cleavage sites at the amino and carboxyl ends of the spacer have been analyzed with regard to protein expression and enzyme activity. The results have been summarized in Table 1. Site directed mutations in or at the cleavage sites (Class I) do not appear to disrupt the processing of the large subunit from the small subunit based on:

  1. the presence of enzyme activity;
  2. and analysis of protein size via sodium dodecyl sulphate-polyacrylamide gel electrophoresis separation and western detection.

Comparison of the large subunit of pCA1 in the constructs indicates that the small subunit is not present. Nonprocessed pCA1 would be expected to have an increase of approximately 4kDa if the small subunit peptide remained attached. A construct (SDM-IRS) that inserts additional amino acid residues (V-N and T-S at the amino and carboxyl termini, respectively) at the sites of cleavage does not appear to interrupt processing. Analysis of protein expression levels and distribution also provides supporting evidence for the proper processing of the spacer in the Class I constructs. All Class I constructs demonstrate significant levels of protein expression and expression can be detected in 65 percent or more of the plants assayed, indicating protein stability.

Table 1. Protein Expression and Enzyme Activity of Constructs
Class Construct Expressed Protein Enzyme Activity Distribution
(+) pCA1 Total Total
  Atl-Cah1 + + 17 21
I SDM12-3 + + 17 21
I SDM13-2 + + 13 13
I SDM14-4 + + 13 13
I SDM15-1 + + 20 20
I SDM22-2 + + 6 6
I SDM23-1 + + 2 3
I SDM-IRS1 + + 19 29
II SDM8-7-2 +* - 2 15
II SDM17-1 +* - 2 39
II SDM-LS + NA 12 ~50
III SDM-HIS1 + + 4 6
III SDM-RS + + 10 14

Disruption of Large/Small Subunit Association

The Class II mutations represent disruptions of large/small subunit interactions through the disruption of cysteine residues involved in disulfide bond formation (SDM8*; SDM17) and expression of the large subunit alone (SDM-LS). As previously reported, few plants have been identified expressing detectable levels of protein, suggesting that the inability to form the heterotetramer (2X[large subunit-small subunit]) results in protein instability. Screening of additional plants has yielded one additional SDM17 plant. SDM8* screens are in progress. The first round of SDM8* screens yielded two plants with low levels of detectable protein (LS), and seeds from these plants were harvested and grown to produce bulk flats of Arabidopsis in an effort to purify the protein. The vegetative parts of six plants were harvested and the total soluble protein was extracted and loaded on an affinity column. Analysis of the elution fractions resulted in the detection of purified pCA1. Although the protein was concentrated and purified from the extract, the concentration remained too low to detect the small subunit. Further analysis and optimization of the affinity purification will provide information about the processing of this mutant. Purification on the SDM8* protein will allow us to determine if the spacer peptide was processed properly in the absence of the large/small subunit interactions, thus providing insight about whether carbonic anhydrase is cleaved and then assembled or if it is assembled and then cleaved.

Large Scale Modification of the Spacer Peptide

The Class III mutations represent large-scale changes that are designed to disrupt or change entirely the physical nature of the spacer. These mutations include deletions of predicted critical regions and the design of artificial spacers based on our understanding of the spacer. The SDM-HIS1 construct completely replaces the 5-Histidine repeat at the amino terminus of the spacer with a Serine residue and has been shown to have no apparent effect on the processing of the protein based on the large subunit size (MW) and enzyme activity. Plants expressing the first artificial spacer designed (SDM-RS1) have been analyzed for protein expression and activity and demonstrate both. Plants appear to have slightly lower levels of expressed protein compared to the SDM11-2 plants, but this may be caused by the number of gene insertions present. Interestingly, comparison via western analysis of the large subunit peptide from SDM-RS1, SDM-HIS1, and SDM11-2 indicates no significant difference in molecular weight, suggesting that processing is similar for each.

In the interest of characterizing the spacer properties responsible for peptide cleavage, a second artificial spacer has been designed to investigate the role of overall peptide charge in processing. The apparent processing of SDM-RS1 and the processing of all Class I mutants support the hypothesis that a conserved cleavage site is not present or required. Instead, the overall peptide charge may be contributing directly to processing. To investigate this hypothesis, a second artificial spacer has been designed, SDM-ASP1 (Acidic Spacer) in which all basic residues have been substituted with acidic residues in the spacer peptide. This construct expands on the design of SDM-HIS1, in which the 5-His repeat has been deleted. SDM-ASP1 does not contain the 5-His repeat (not substituted) and all subsequent basic residues are converted to acidic residues. The artificial spacer has been synthesized and currently is being inserted in place of the endogenous spacer of the SDM-IRS1 construct. Analysis of the expression and processing of SDM-ASP1 will provide information regarding the essential properties of the spacer and allow insight into the mechanism involved in the proteolytic processing.

Endogenous Spacer Peptide (SDM-IRS1)


Acidic Artificial Spacer (SDM-ASP1)


Presently, a concentrated effort is under way to purify the large and small subunit peptides for sequence analysis to determine the exact site of cleavage in Arabidopsis. This will be compared to the sequence data previously obtained from tobacco and Chlamydomonas to determine the level of conservation for processing between organisms. Vegetative material currently is being generated and bulked for the Class I and II mutants, and Class III mutants will follow after generation of significant seed.

Class IV Mutants: Disruption of Putative Internal Cleavage Sites in the Spacer

This is a new class of SDM mutants designed to investigate the hypothesis that spacer cleavage is the result of proteolysis at specific sites within the peptide, followed by “trimming” of the peptides via carboxyproteases or other endoproteolytic activity. This scenario would be the result of a multiple step mechanism, in which the peptide is cleaved at a putative internal site and then processed to the recognized amino and carboxyl terminal residues of the small and large subunits, respectively. Endoproteolytic cleavage is known to occur at basic residue sites, specifically -R-, -R-R-, and -K/R-R- sites. The spacer peptide contains two such -R-R- sites. Three SDM constructs currently are under development, in which the -R-R- sites are eliminated individually (SDM30, SDM31) and simultaneously (SDM32). The constructs have been inserted into cloning vectors for sequence confirmation and presently are being inserted into the binary vector.

Modifications of the ER-Leader Peptide

The Cah1 gene with the endogenous Chlamydomonas leader sequence has been inserted into the binary vector and transgenic plants have been developed. This construct (+)CL-Cah1 will determine if the Chlamydomonas leader sequence can be recognized by higher plants. Of 32 plants selected via herbicide treatment, only two were found to contain very low levels of pCA1. This suggests that the endogenous Chlamydomonas leader is not efficiently recognized by Arabidopsis.

A second construct lacking any ER leader sequence [(-)CL-Cah1] also has been developed and transgenic Arabidopsis have been analyzed. This construct will help determine if the expressed protein can exist as a stable protein in the cytosol and if processing can occur without movement through the endomembrane system. Of 72 plants assayed for protein expression, 0 plants were found, indicating that the protein is not stable in the cytosol, but must be transported to the endomembrane system.

Foreign Gene Expression of Antibody Heavy and Light Chains

The third project consists of the simultaneous expression of the heavy and light chains of an antibody in Arabidopsis using the spacer technology from Cah1 in Chlamydomonas. This project is designed to exploit the unique processing of Cah1 for the expression of multiple foreign genes simultaneously.

Following the recommendation of Dow, I have requested the transfer of the two public domain antibody genes, lchainMGR48m (AY311598) and hchainMGR48m (AY311599), from Plant Research International B.V. (PRIBV) (through Iowa State University). I am in contact with representatives from PRIBV and Iowa State University, and am still awaiting approval of the material transfer.

Simultaneous Expression of Unrelated Genes: GFP and Large Subunit

To advance the foreign gene expression project, an alternative approach was devised. A chimeric construct comprised of the large subunit of Cah1 and the green fluorescent protein (GFP) has been developed. This construct will allow investigation of the processing through combined analysis using fluorescence from the reporter gene (GFP) and western analysis to detect the properly processed Cah1 large subunit.

Currently, we have identified 12 of approximately 50 transgenic Arabidopsis plants that express detectable levels of the large subunit truncated protein. The low level may indicate some instability, however, the presence of detectable levels of Cah1 large subunit indicates that it is an acceptable reporter candidate. Plants containing the AtL-GFP and AtL-GFP-Spacer-Large Subunit constructs have been developed and analyzed via western blots. Primary antibodies specific for the large subunit region and the GFP protein were used to determine the level of expression and the presence of processing. Seven GFP-only plants were identified through herbicide selection, and all seven contained detectable levels of a truncated GFP protein (~5kDa smaller). This is similar to results previously obtained from tobacco, indicating that ER-processed GFP may undergo additional cleavage. Seventeen herbicide-selected plants containing the AtL-GFP-Spacer-Large Subunit construct were identified and all 17 expressed detectable levels of the truncated GFP. Three of the plants also expressed detectable levels of large subunit at approximately 35kDa, indicating that the large subunit peptide was effectively cleaved from the GFP protein. These results indicate that the peptide can be processed in a non-native configuration with nonrelated gene products. These results strongly support the applied potential for simultaneous expression of foreign genes such as heavy and light antibody chains, as well as other potential gene products of interest.


Our conclusions are that the system appears to function as predicted and should be useful for the intended applications.

Journal Articles:

No journal articles submitted with this report: View all 2 publications for this subproject

Supplemental Keywords:

sustainable industry, sustainable business, waste, agricultural engineering, bioremediation, environmental engineering, geochemistry, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology, sustainable industry/business, environmental chemistry,, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Genetics, Technology, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, agrobacterium, bioengineering, anti-pest proteins, transgenic plants, biomass crop plants, plant genes, biotechnology, remediation, plant biotechnology, cloning

Relevant Websites: Exit

Progress and Final Reports:

Original Abstract
  • 2003

  • Main Center Abstract and Reports:

    R829479    The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program

    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