GeneArt® Chlamydomonas Protein Expression Kit

Catalog number: A24244

Invitrogen™  Related applications: Protein Expression

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Description

The GeneArt® Chlamydomonas Protein Expression Kit is our second-generation Chlamydomonas cloning and expression system. Like the first-generation GeneArt® Chlamydomonas engineering kits, this new system offers transgene expression from the nuclear genome of eukaryotic green alga Chlamydomonas reinhardtii 137c, 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. The kit includes frozen Chlamydomonas reinhardtii 137c cells, MAX Efficiency® Transformation Reagent for Algae, expression vector, One Shot® TOP10 competent E.coli cells, and easy-to-follow protocols. Our Gibco® TAP Media, offered separately, is optimized for the growth and maintenance of Chlamydomonas.

• Express up to 1% total soluble protein of 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
• Compatible with seamless assembly for creation of your constructs
• Enables you to receive reliable results with exceptional strain viability and purity

New and Improved Chlamydomonas pChlamy_4 Vector
Transgene expression from the Chlamydomonas nuclear genome offers several advantages over chloroplast expression, such as post-translational modifications and protein-targeting and/or secretion. However, expression from the nuclear genome has typically been less than robust and the molecular mechanism(s) of poor expression are not completely understood. Possible reasons include poor promoters, genome integration position effects, and transgene silencing. The GeneArt® Chlamydomonas Protein Expression Kit provides new solutions for high levels of expressed protein and selection against silencing. Our pChlamy_4 vector is also compatible with seamless cloning, an optional cloning method that results in no extra sequences in your final construct.

Features of the vector include:

• Endogenous and constitutive promoter, RbcS2, is combined with activator, Hsp70A, resulting in increased expression of your gene of interest
• Fusion of your gene of interest to 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
• 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 no tag at all

Selection Against Silencing
In order to circumvent the transgene silencing that often occurs in C. reinhardtii, our new 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.

MAX Efficiency® Transformation Reagent for Algae
One of the biggest hurdles in research and development with Chlamydomonas has been the introduction of exogenous DNA into Chlamydomonas strains due to rigid cell walls. Methods such as glass bead agitation, electroporation, and microparticle bombardment are available but often result in low transformation efficiency. MAX Efficiency® Transformation Reagent for Algae, when used to pretreat the cells prior to electroporation, enhances transformation efficiency for multiple strains of Chlamydomonas species. It increases the permeability of the Chlamydomonas cell wall and facilitates increased delivery of DNA into the cell nucleus by electroporation. To date, we have seen >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.

Versatile and Proven Algal Model Organism
Chlamydomonas reinhardtii is a freshwater, green microalga that has been a popular model organism for physiological, molecular, biochemical, and genetic studies and has recently gained attention as a platform for the production of therapeutic proteins and vaccines. It has served as a genetic workhorse and model organism for understanding everything from mechanisms of photosynthesis and nutrient regulated gene expression, to the assembly and function of flagella. Green algae are used as a platform for the production of biofuel and bio-products, due mainly to their rapid growth and ability to convert sunlight and C02 to energy. As C. reinhardtii propagates by vegetative division, the time from initial transformation to product production is significantly reduced relative to plants, requiring as little as six weeks to evaluate production at flask scale, with the potential to scale up to 64,000 liters in four to six weeks.

Gibco® TAP Media— Optimized for Chlamydomonas
Gibco® TAP Media, available separately, is optimized for the growth and maintenance of Chlamydomonas. The 1X formulation lets you avoid laborious media preparation steps. Award-winning bottle design is easier to use in the biosafety cabinet, 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 results in better overall efficiency and more robust data.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Promoter: Hsp70A-RbcS2
Cell Line: Chlamydomonas
Expression: Algae
Product Size: 10 reactions
Detection Method: Gel Electrophoresis
Number of Reactions: 10 Reactions
Protein Tag or Fusion: His Tag (6x)
Selection Agent (Eukaryotic): Hygromycin
High Throughput Compatibility: Not High Throughput-Compatible (Manual)

Documents

Manuals & protocols

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