Soluble protein expression: single-protein production system
The SPP (single protein production) system utilizes a modified version of Takara's cold-shock expression vectors along with expression of the endoribonuclease MazF to achieve remarkable protein purity and stable isotopic labeling (up to 90% of the newly synthesized protein) of target proteins.
Overview
Principle of the SPP system
The MazF (mRNA Interferase) protein is a sequence-specific endoribonuclease that specifically cleaves single-stranded RNAs at their ACA sequences. As a result, the expression of MazF virtually eliminates expression of most cellular mRNAs while allowing synthesis of the target protein for up to 72 hr post-induction. The SPP system for cold-shock expression consists of two co-expressed plasmids: an ACA-less pCold expression vector for expression of an ACA-less version of the target gene, and a vector carrying the MazF gene for inducible expression of the MazF protein. This system suppresses host protein expression more effectively than the pCold system alone, resulting in higher purity and labeling efficiency for a wider variety of desired proteins. This also enables soluble protein expression applications such as structural analysis by NMR (nuclear magnetic resonance) or other technologies.
The SPP system was developed by Takara Bio in collaboration with Dr. Masayori Inouye at the University of Medicine and Dentistry of New Jersey, USA.
More Information
Applications
- Purified protein expression
- Radioisotope labeling (up to 90% of newly expressed cellular protein is labeled target protein)
- NMR analysis
Components
| SPP System II | Cat. # 3368 |
| pCold II (SP-4) DNA | 20 µg (0.5 µg/µl) |
| MazF (mRNA Interferase) Expression Plasmid pMazF DNA | 0.5 µg (20 ng/µl) |
| Positive Control pCold I (SP-4) envZB DNA | 0.2 µg (20 ng/µl) |
| SPP System III | Cat. # 3369 |
| pCold III (SP-4) DNA | 20 µg (0.5 µg/µl) |
| MazF (mRNA Interferase) Expression Plasmid pMazF DNA | 0.5 µg (20 ng/µl) |
| Positive Control pCold I (SP-4) envZB DNA | 0.2 µg (20 ng/µl) |
| SPP System IV | Cat. # 3370 |
| pCold IV (SP-4) DNA | 20 µg (0.5 µg/µl) |
| MazF (mRNA Interferase) Expression Plasmid pMazF DNA | 0.5 µg (20 ng/µl) |
| Positive Control pCold I (SP-4) envZB DNA | 0.2 µg (20 ng/µl) |
The expression plasmid has been prepared by inserting the ORF of E. coli-derived protein envZB without ACA sequence into the pCold I (SP-4) DNA. Estimated molecular weight of expressed protein is 19.6 kDa.
References
Qing, G. et al. Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol 22, 877–882 (2004).
Suzuki, M., Zhang, J., Liu, M., Woychik, N. A. & Inouye, M. Single protein production in living cells facilitated by an mRNA interferase. Mol. Cell 18, 253–261 (2005).
Zhang, Y. et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli. Mol. Cell 12, 913–923 (2003).
Zhang, Y., Zhang, J., Hara, H., Kato, I. & Inouye, M. Insights into the mRNA cleavage mechanism by MazF, an mRNA interferase. J. Biol. Chem. 280, 3143–3150 (2005).
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