USE OF GREEN ALGAE FOR HETEROLOGOUS PROTEIN EXPRESSION AND PRODUCTION.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0209124
• Project Start Date: Oct 1, 2006
• Project End Date: Sep 30, 2008
• Program Code: [(N/A)]- (N/A)

Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007

Performing Department
PLANT SCIENCE

Non Technical Summary
Expression of recombinant proteins is not trivial: different systems for expression of recombinant proteins have been designed but no system yet is available that can combine high protein yield, ease of manipulation and low cost of operation. We propose to develop a unicellular green algae transformable system that will allow the easy nuclear or chloroplastic expression of proteins of interest at very high level and low cost.

Animal Health Component

40%

Research Effort Categories

Basic

30%

Applied

40%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
204	1899	1040	50%
204	7010	1040	50%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest);

Subject Of Investigation
7010 - Biological Cell Systems; 1899 - Oilseed and oil crops, general/other;

Field Of Science
1040 - Molecular biology;

Keywords

algae

transformation

recombinant protein

gfp

Goals / Objectives
Expression of functional proteins especially enzymes, pharmaceutical proteins and membrane proteins is not trivial. Different systems for expression of recombinant proteins have been designed. Each system offers distinct advantages in terms of protein yield and ease of manipulation and cost of operation. Following are examples: E. coli, yeast: either of these system is very useful for simple proteins, however, complex proteins may not be properly folded or functional. Baculoviral: insect cells can produce large quantities of foreign protein; however the results are not reproducible and it is very costly. Transgenic mammalian cells: this is a very useful system for complex proteins but very expensive. Plants are now used as bioreactors (Abranches et al., 2005): foreign proteins can be expressed in leaf or seed tissue. However, the level of expression of foreign proteins can differ from plant to plant. Chloroplast transformation helps achieve very high levels of protein expression. However, some post-translational modifications needed for proper functioning of the foreign DNA may not occur in the chloroplast. Additionally, the length of time required from the initial transformation event to having usable (mg to g) quantities of recombinant protein takes years. Finally, when working with plants that are to be introduced into the fields additional strategies are needed to significantly reduce the potential for gene flow to surrounding crops. Green algae: this is an excellent expression platform for complex recombinant proteins. Indeed, in selected green algae species, recombinant DNA can be introduced into the genomes of the nucleus, the chloroplast, and even the mitochondria, and thus the system offers both prokaryotic (chloroplast, mitochondria) and eukaryotic translation systems for a tailored expression of virtually any protein at low cost and in a perfectly controlled and safe environment. We propose to develop a unicellular green algae transformable system that will allow for the nuclear or chloroplastic expression of proteins from various sources and for different projects. We hypothesize that: Unicellular green algae transformed with a GFP recombinant DNA fused to a signal peptide nucleotide sequence will be post-translationally modified appropriately and will be secreted into the culture medium from which it will be easily purified. Algae transformed with a GFP recombinant DNA targeted to the chloroplast will produce the highest levels of GFP expression. To test these hypotheses the following specific aims are proposed. 1: Determination of optimal culture conditions for unicellular green algae protein expression. 2: Generate the GFP sequence with the right codon usage and amino acid changes to enhance fluorescence of the GFP . 3: Construct vectors for nuclear and chloroplast transformation of unicellular green algae. 4: Perform nuclear and chloroplast transformation of the algae with the GFP recombinant DNA.

Project Methods
Determination of optimal culturing conditions for unicellular green algae protein expression. Algae will be cultured in TAP (tris acetate phosphate) culture medium under continuous illumination and agitation. We will test different light, temperature, shaking speed conditions to obtain optimal algal growth and protein expression. Protein content will be measured by standard Biorad methods performed in our lab. Generate the GFP sequence with the right codon usage and amino acid changes to enhance fluorescence of the GFP. The use of codon bias for gene expression into the algae is key for the accumulation of the recombinant protein (Mayfield et al., 2003; Mayfield and Franklin, 2005). The C. reinhardtii codon usage will be taken from: http://www.bio.net/bionet/mm/chlamy/1997-March/000842.html. Changes will be made to the wild type GFP sequence to enhance the fluorescence(Heim and Tsien, 1996). We will take into consideration codon usage to generate the recombinant DNA for expression in nucleus and chloroplast genomes. Both GFP sequences will be generated using the method developed by Stemmer et al. (1995) which allows the generation of a complete gene from a pool of primers, each 40 nucleotides in length. The terminal primers will contain restriction sites for insertion in the expression vectors. Construct vectors for transformation of unicellular green algae. Depending on the protein that we will want to express in algae, transformation of the nucleus or of the chloroplast will be recommended. So we will construct two types of vectors: one for expression in the nucleus (in this case, the protein will be targeted for secretion in the medium for easy purification), and one for expression in the chloroplast. These vectors will be ready to use expression systems that will only require the insertion of the recombinant DNA before transformation of the algae. We will insert control sequences in the expression vectors. Transform the algae with the codon optimized GFP. Transformation techniques: Different methods for nuclear and chloroplast transformation are present in the literature (Kindle, 1990; Shimogawara et al., 1998; Qin et al., 2005). We have available in our lab the equipment necessary to perform electroporation transformation of algae (Qin et al., 2005). Check GFP expression in the algae: We will assay GFP accumulation in the media (if nuclear expression) or in the soluble protein extract (if chloroplast expression). Franklin et al. (2002) developed a rapid assay to quantify GFP accumulation in soluble protein extract that we should be able to easily adapt to our excreted proteins. Integration of the GFP modified sequence in the nucleus or chloroplast genomes will be determined by PCR and southerns. Expression level of the protein and whether the quantity of protein present is in direct correlation with the amount of mRNA produced for the protein will be assayed by northern and real-time PCR. Statistical methods: Statistics will be used (SAS, ANOVA) to assess the differences in fluorescence between the different conditions tested and determine which growth conditions lead to the maximum expression level of the GFP protein.

Progress 10/01/06 to 09/30/08

Outputs
OUTPUTS: Activities: 1.Both the carborundum mediated transformation technique and biolistics were used to introduce a plasmid bearing the streptomycin gene into algal cells. 2. Confirmation of the effects of the stable isotopes: In preparation to examine proteins produced by the transformed cells, we examined the effect of growth of algae in medium containing stable isotopes. Using real-time RT-PCR we measured the expression of stress related genes. It was especially important for the algae grown in 15N: since the isotope appeared to have no visible physiological or morphological effects. The expression of the heat shock protein 70 (Hsp70) was assayed in the algae grown in regular media, in media containing 100% 15N and in media containing 60% D2O. The 18S gene was used as the normalizer and the regular media as the calibrator. PARTICIPANTS: Undergraduates Geraets and Kollars had the opportunty to learn basic molecular biology tools and to present their research at the SD Academy of sciences. They both published their research in the SDSU undergraduate journal. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
1. We have successfully conferred streptomycin resistance to algae by transfer of a plasmid bearing the gene that confers resistance. The transformed cells will be grown, DNA isolated and PCR performed to verify the success of both nuclear and chloroplast transformation. 2. The expression of Hsp70 was found to be down-regulated in 15N-containing media, and very highly up-regulated in media containing 60% D2O (3.37 fold). Thus we have concluded that 15N will serve as a valuable tool to follow protein turnover in transgenic algal cells.

Publications

• PUBLICATIONS, PRESENTATIONS, TALKS: Kollars, B., R. Geraets, M-L. Sauer, J. Cohen, F. Sutton. 2008. Deuterium Effects on Chlamydomonas reinhardtii. SDSU Journal for Undergraduate Research.
• Geraets, R., B. Kollars, M-L. Sauer, J. Cohen, F. Sutton. 2008. 15N Effects on Chlamydomonas reinhardtii. SDSU Journal of Undergraduate Research.
• Sauer M-L., W-P. Chen, B. Kollars, R. Geraets, J. Cohen, F. Sutton(2008) Stable Isotope Effects on Chlamydomonas reinhardtii stress-related gene transcription. Oral presentation at the South Dakota Academy of Sciences, Chamberlain, SD, April 4, 2008.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Development of algal system. We started the growth of 8 different strains of unicellular green algae: Seven Chlamydomonas reinhardtii strains and one Dunaliella tertiolecta strain. Among the seven strains of C. reinhardtii, five strains (CC-124, CC-125, CC-620, CC-621 and CC- 1266) are wild type strains of different mating types and two strains (CC-400 and CC-3491) are cell wall deficient mutants. The algae are grown mixotrophically both on TAP (tris acetate phosphate) agar plates and in TAP liquid cultures. When grown in liquid culture, the algae is inoculated in culture tubes containing the media and the tubes laid horizontally and shaken constantly under a 16 hour light/8 hour dark photoperiod. Nuclear transformation of the unicellular green algae Chlamydomonas reinhardtii. Different transformation methods were tested and optimized: 1- Transformation by electroporation: cell wall deficient mutants were used or wild type strains whose cell walls were removed with autolysin (enzyme produced by the algae during mating). Algae and DNA were placed in electroporation cuvettes and a strong electric field applied to transfer DNA into the nucleus. The algae were then removed from the cuvettes and plated on media containing a selection agent to select only transformed cells. 2- Transformation with glass beads on cell wall deficient strains. Cell wall deficient strains, DNA and galss beads were vortexed together. After vortexing, the algae were plated on selective media to select only the cells that incorporated the DNA of interest. 3- Transformation with carborundum on cell wall deficient strains and wild type strains. Cell wall deficient strains and wild type strains (the cell wall was not remove with this method) were used in this assay. After mixing in a tube the algae, carborundum and the DNA, the tubes were placed for several minutes in the FastPrep-24 (MP Biosciences). This machine is a high speed vortexer and is used generally as a tissue homogenizer. The samples were then plated on selective media which allowed the selection of the algal cells that had been transformed with the recombinant plasmid DNA. Colonies that consistently grew on the selective media were assayed for incorporation of the transgene by PCR with transgene specific primers. Chloroplast transformation of the unicellular green algae Chlamydomonas reinhardtii. We started the process of developing our chloroplast transformation protocol. Tutoring: 3 undergraduate students were trained to grow and maintain the algal cultures and participated in the experiments involving the algae. They prepared media, transferred the strains monthly to new media, prepared liquid cultures for different experiments. Networks and/or collaborations fostered by the project or activity. We started a network with the researchers at the University of Minnesota that also work with the algae. PARTICIPANTS: PI: Dr. Marie-Laure Sauer. Dr. Sauer developed and conducted the research. Co-PI: Dr Fedora Sutton: Dr. Sutton helped in the development of the research. Undergraduate students: Ryan Geraets and Brett Kollars. The two undergraduate students were trained in sterile culture techniques, they made media to grow the algae and helped maintain the different strains. Collaborators at the University of Minnessota, in particular Dr. Carolyn Silflow. Dr. Silflow provided some of her protocols for the growth, DNA, RNA and protein extraction of algae.

Impacts
We are now able to grow different strains of algae both in liquid and on solid media. We have refined the culture of the cell wall deficient mutants. Where the wild type strains are grown in TAP media in liquid and TAP media with 1.5% agar on solid media, the cell wall deficient mutants are grow in TAP media with 1% sorbitol in liquid. Also, when grown on plates, they are cultured on TAP with only 0.8% agar. The nuclear transformation using electroporation was not successful. This transformation method is time consuming and because of many steps the potential for contamination of our samples was increased. After several attempts to improve this method, we decided to focus on faster and more efficient methods. Both the transformation methods with glass beads and carborundum allowed us to obtain transformed algae. The glass bead method worked very well with cell wall deficient mutants but did not work on wild type strains unless we removed the cell wall first. For this reason we decided to develop a method that would allow the transformation of wild type strains of algae that had their cell wall intact. We were successful and developed the carborundum method of algal transformation. The carborundum method represents a major improvement over other methods because it allows the transformation of both cell wall deficient strains and wild type strains with an intact cell wall. The method is very easy, fast and efficient. We will publish this new technique as it would benefit other researchers.

Publications

• No publications reported this period