To date, engineered
E.coli has been used extensively as the cellular host for foreign protein expression due to its rapid growth rate, capacity for continuous fermentation, and relatively low cost. Many commercial expression systems designed for various applications and compatibilities are available. Over the years, some
E.coli strains have been employed in the production of heterologous proteins due to the availability of several well-established expression systems.
Codon bias problems become highly prevalent in recombinant expression systems. By increasing the copy number of the limiting tRNA species, E.coli can be controlled to match the codon usage frequency in heterologous genes. Rosetta-gami™ B Host Strains have been developed by Novagen for researchers to express the eukaryotic proteins that contain codons rarely used in E.coli.
Rosetta-gami™ B Host Strains are BL21 derivatives, and are designed to enhance the expression of eukaryotic proteins that contain rare codons. These strains supply tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid named pRARE. Thus, the Rosetta strains provide for "universal" translation where translation would otherwise be limited by the codon usage of E.coli.
Furthermore, the highly concentrated proteases of the intracellular environment may also alter some characteristics of the final product, prohibiting the expression of some bioactive proteins in E.coli. Rosetta-gami™ B Host Strains do not contain the lon protease and another outer membrane protease, OmpT. The lack of these two key proteases reduces degradation of heterologous proteins expressed in the strains. The trxB/gor mutant facilitates cytoplasmic disulfide bond formation, which is helpful for the folding of proteins in vivo.
The process from gene to expressed protein is detailed and summarized as follows: The gene of interest is analyzed to confirm the number and frequency of rare codons in the sequences. The gene is cloned into an expression plasmid. It is important to make sure that this candidate expression plasmid can co-exist with the plasmid pRARE, which is already in the Rosetta strain. (pRARE contains a p15a replicon and is compatible with most cloning vectors.) Following cloning, the recombinant plasmid is checked by restriction analysis, PCR or sequencing. The confirmed plasmid is then transformed into Rosetta cells. The expression of the protein of interest is precisely controlled with IPTG concentration due to deletion of lacZY. The expression product is analyzed by SDS-PAGE and can be further analyzed via Western blot.
In our laboratory, we have expressed antigenic proteins of many pathogens, including viruses, using Rosetta-gami™ B host strains; some of these proteins were used to immunize experimental animals to get neutralizing antibodies. At the same time, the prokaryotic expressed proteins were used as diagnostic reagents for detecting antibody changes.
This product is an efficient expression host strain for many genes including eukaryotic genes. However, like other common prokaryotic strains, Rosetta-gami™ B host strains also lack post-translational modifications; therefore, whether it is suitable for producing particular proteins that require post-translational modification needs further investigation. Moreover, in order to prevent the loss of pRARE, the addition of chloramphenicol is helpful in the preparation of competent cells or in the process of inducing expression.
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Xiaofeng Ren
Research Scholar
College of Veterinary Medicine
Northeast Agricultural University
China
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