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A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii

Paper ID Volume ID Publish Year Pages File Format Full-Text
22934 43401 2015 7 PDF Available
Title
A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii
Abstract

•A special vector for chloroplast transformation of C. reinhardtii is described.•The transformation vector allows modular cloning by Golden Gate shuffling.•Selection of transplastomic algae strains does not involve antibiotics.•The system has been tested using various regulatory sequences attached to a foreign gene.

In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts.

Keywords
Algal biotechnology; Chlamydomonas reinhardtii; Fud7; Transgenic chloroplast; Rekombinant protein expression
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A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 195, 10 February 2015, Pages 60–66
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us