Chlamydomonas Protein Expression

Green algae are used as the basis for platforms for the production of biofuels and bioproducts, due mainly to their rapid growth and photosynthetic ability. Because C. reinhardtii propagates by vegetative division, the time from initial transformation to product synthesis is significantly reduced relative to plants, requiring as little as 6 weeks to evaluate production at the flask scale, with the potential to scale up to 64,000 L in 4 to 6 weeks.

C. reinhardtii 137c is a eukaryote with a large genome size of 121 Mb, and a versatile and proven model organism. Our Invitrogen GeneArt Chlamydomonas Protein Expression Vector offers transgene expression from the nuclear genome but is optimized for high-level expression, provides selection against gene silencing, and offers dual protein tags for detection and/or purification of your gene of interest. Gibco TAP Growth Medium, offered separately, is optimized for the rapid growth and maintenance of Chlamydomonas.

• Express up to 1% total soluble protein from your gene of interest
• Select against gene silencing, even over multiple passages
• Detect and purify your gene of interest with 6His TEV and/or V5-His epitope tags
• Enable reliable results with exceptional strain viability and purity
• Use (optional) seamless assembly to create your constructs

Transgene expression from the Chlamydomonas nuclear genome via the pChlamy_4 vector offers several advantages over chloroplast expression, such as posttranslational modifications and protein targeting and/or secretion.

• The endogenous and constitutive promoter RbcS2 is combined with an activator (Hsp70A), resulting in increased expression of your gene of interest.
• Fusion of your gene of interest to the bleomycin/Zeocin resistance gene, sh-ble, circumvents silencing.
• Self-cleaving sequence for the 2A peptide from foot-and-mouth disease virus (FMDV) mediates proper cleavage between resistance markers and protein of interest to yield two discrete proteins.
• A 3′ UTR for proper transcript termination and possible additional benefits like increased translation efficiency, mRNA stability, and polyadenylation signals.
• Dual protein tags 6His TEV and V5-His epitopes can be fused to both or either ends of your gene of interest, or you can elect to have no tag at all.
• Use (optional) seamless assembly to create your constructs.

The gene for the hydrolytic enzyme xylanase (xyn1) from Trichoderma reesei was cloned into the vector pChlamy_4 and transformed into Chlamydomonas reinhardtii 137c. Xylanase activity was measured using the EnzChek Ultra Xylanase Assay Kit (Cat. No. E33650). Xylanase activity levels from the pChlamy_4 constructs ('Prot Exp') were 17-fold over previous observations ('Genetic Eng').
 

The gene for the hydrolytic enzyme xylanase (xyn1) from Trichoderma reesei was cloned into the vector pChlamy_4 and transformed into Chlamydomonas reinhardtii 137c. Xylanase expression was measured by western blot analysis, and the results showed that xylanase protein represented approximately 1% of the total soluble protein.
 

In order to circumvent the transgene silencing that often occurs in Chlamydomonas reinhardtii, our pChlamy_4 vector is designed so that proteins are expressed as transcriptional fusions with the bleomycin/Zeocin resistance gene sh-ble (Rasala et al., 2012). The self-cleaving sequence for the 2A peptide from the foot-and-mouth disease virus (FMDV) is placed between the antibiotic resistance gene and the gene of interest. It encodes a short ~20 amino acid sequence that mediates proper cleavage to yield two discrete proteins. With this system we have seen positive transformants maintain high expression levels for much longer than with other systems, even after many passages with or without selection pressure (see figure below).

The gene for the hydrolytic enzyme xylanase (xyn1) from Trichoderma reesei was cloned into vector pChlamy_4 and transformed into Chlamydomonas reinhardtii 137c. Xylanase activity was measured daily for 2 weeks using the EnzChek Ultra Xylanase Assay Kit (Cat. No. E33650) and was compared to expression levels when the gene was expressed in other systems. Fusion of the xylanase gene bleomycin/zeocin resistance gene, sh-ble, in pChlamy_4 vector, circumvents silencing so proteins are expressed through many cell passages with or without selection pressure.
 

One of the biggest hurdles in research and development with Chlamydomonas has been the introduction of exogenous DNA into Chlamydomonas strains. Methods such as glass bead agitation, electroporation, and microparticle bombardment are available but often result in low transformation efficiency. Invitrogen MAX Efficiency Transformation Reagent for Algae, when used to pretreat cells prior to electroporation, enhances transformation efficiency for multiple strains of Chlamydomonas. It increases permeability of the Chlamydomonas cell wall and facilitates increased delivery of DNA into the nucleus by electroporation. To date, we have seen a >200-fold increase in transformation efficiency over previously recommended transformation conditions, in 10 different Chlamydomonas strains, including wild type and mutants, using circular or linear DNA as well as PCR fragments.

Gibco TAP Growth Medium, offered separately, is optimized for the growth and maintenance of Chlamydomonas. The 1X formulation lets you avoid laborious media preparation steps. The award-winning bottle design makes manipulation in the biosafety cabinet easier, minimizes the risk of contamination, and helps you perform cell culture more consistently. Superior packaging and quality, greater reliability, and improved consistency in Chlamydomonas culture result in better overall efficiency and more robust data.