Isotopic Enrichment of Tryptic Fragments
Rutgers scientists have developed two Single Protein Production (SPP)-based systems to obtain high levels of specific isotopically enriched proteins: an independently inducible system (cSPP(tet)) and an amino acid auxotroph-based dual inducible system.
The proprietary SPP system, developed at Rutgers and currently licensed to Takara Bio Inc., essentially turns living E. coli cells into a bioreactor for producing a single protein of interest at high levels. The system utilizes an E. coli endoribonuclease, MazF, to selectively cleave cellular mRNAs at the ACA sequences and thus completely inhibit protein synthesis. However, MazF-induced E. Coli cells are in semiquiescent state and are still capable of synthesizing the protein of interest, of which the mRNA is engineered to contain no ACA base triplet. The SPP system can be further condensed 10-40 fold, without any reduction in protein production (condensed SPP, or cSPP). This greatly facilitates the production of recombinant proteins enriched with deuterium or other isotopes and amino acids analogues at a fraction of the cost of conventional methods.
In a previous cSPP system to produce isotope enriched proteins for NMR studies, both MazF and the target protein are induced at the same time, resulting in 10-20% of target protein produced without isotopic labeling.
The novel independently inducible cSPP system (cSPP(tet)) temporally separates the induction of MazF (by IPTG), required to convert cells into a semiquiescent state prior to condensation, from the expression of the target protein in a tetracycline-inducible vector. This unique independent inducible induction of MazF and target protein avoids production of the unlabeled protein fraction and improves the isotope enrichment to >95%. This is particularly important for producing perdeuterated, 15N,13C-enriched proteins.
The amino acid auxotroph–based dual inducible cSPP system can achieve very tight induction without the need for the tetracycline-inducible component. It is unique in a way that the production of target protein is induced by the addition of amino acid histidine or tryptophan in the medium a few hours after His-less or Trp-less MazF induction. This system can achieve almost complete suppression of the production of the unlabeled target protein, and highly improve the isotopic enrichment efficiency to >98%.
- Production of isotope labeled proteins for NMR analysis, particularly perdeuterated proteins
- Production of proteins with specific amino acids being replaced with their analogues
- High-level production of a single target protein
- Utility for producing otherwise toxic proteins
- Utility for producing membrane proteins
- High purity of isotope labeled protein - no need for further purification
- The cell culture can be condensed 10-40 times, leading to a saving of up to 97.5% in the costs of expensive reagents. This is particularly important for producing perdeuterated protein samples.
- >95 - 98% isotope enrichment efficiency
Intellectual Property & Development Status:
Patents issued and pending. The background technology SPP system is a proprietary technology owned by Rutgers and currently exclusively licensed to Takara Bio Inc.. Future licensee to the independently inducible and/or dual inducible cSPP technologies will need to acquire a sublicense from Takara.