Description:
Inteins are self-splicing proteins that have the ability to synthesize peptide bonds in a precise manner at virtually any site. A major barrier to applied uses, since inteins splice autocatalytically, is controlling their activity in a way that is widely usable and flexible. Our improved method to control protein splicing uses a single engineered disulfide bond that forms reversibly according to redox changes. Oxidative conditions in bacteria and in vitro favor disulfide bond formation. This interaction, which has been validated by x-ray crystallography, "traps" a catalytic intein residue to block splicing. By adding a reducing agent, the disulfide bond is rapidly broken and splicing ensues. For the first time, intein self-splicing can be controlled utilizing redox changes in vitro, in bacteria and in crystallo.
REDOX-TRAPPED INTEIN
Background:
Inteins are protein domains that self-excise from internal positions of selected substrate proteins, called exteins. The excision and rejoining event, referred to as protein splicing, is traceless and is auto-catalyzed by the intein. Since the discovery of protein splicing > 600 inteins have been identified as intervening sequences in diverse proteins from all domains of unicellular life. On the basis of their unique catalytic activity, a number of these inteins have been developed into commercial reagents of broad use to molecular biologists, protein chemists, and chemical biologists. The three most widely used intein-based techniques are the in vitro practices of bioseparations and expressed protein ligation along with in vivo methods to control the synthesis of a reporter protein.
Currently, the main bottleneck to maximizing the efficiency of intein-based applications is the constitutive autocatalytic nature of intein activity. Thus, the unique capacity of inteins to make and break specific peptide bonds is not regulated endogenously, rather catalysis appears to ensue once the intein is folded. That lack of control as the intein-fusion protein is expressed causes assay background in vivo or reduces precursor yield for the in vitro techniques.
Previous efforts to regulate inteins focused on mutating residues at the intein active site or interrupting the intein domain with an allosteric protein element. These strategies have the potential to alter intein folding and thereby impinge on intein solubility and catalytic efficiency. Furthermore, some of these approaches require introduction of synthetic amino acid analogues through manipulations in vitro, thus making the procedure cumbersome for in vivo applications.
We have developed an alternative method to solve the intein controllability problem that preserves intein structure, is implemented through standard mutagenesis, and makes use of a physiological trigger. This "redox trapping" strategy imparts control over intein activity through an engineered disulfide bond between the intein's catalytic cysteine and a second cysteine in the flanking N-extein sequence. As in other engineered redox traps, the intein is catalytically competent in the disulfide form, but incapable of functioning normally until activated by a reducing agent. Because our redox-trapping strategy requires mutations in the flanking N-extein, intein structure, function, and solubility are maintained. We have used this strategy to impart controllability to divergent inteins in vitro and in living bacteria.
Applications
- Bioseparation practices in vitro
- Methods to control the synthesis of a reporter protein in vivo
Advantages
- Controlled by a natural trigger: redox changes
- Implemented by standard mutagenesis
- Does not compromise intein structure or function
- Faster response compared to inducible genes
State of Development
Early research stage
Patents
USPTO # 61/433,730
Licensing Potential
Available for license
Inventors
Brian Callahan, Ph.D.
Marlene Belfort, Ph.D.
Natalya Topilina, Ph.D.
Patrick Van Roey, Ph.D.
Matthew Stanger, B.S.
Publications
Callahan, Brian P., Topilina, Natalya I., Stanger, Matthew J., Van Roey, Patrick and Belfort, Marlene. Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications. Nature Structural and Molecular Biology received 4 October 2010; accepted 21 January 2011; published online 3 April 2011; doi:10.1038/nsmb.2041.
Summary
Marketing Summary - A Redox Trap to Control Intein Activity
Contact
Diane L. Borghoff, B.S., M.S.
Marketing & Licensing Associate – Intellectual Property
Health Research, Inc.
150 Broadway – Suite 560, Menands, New York 12204-2719 U.S.A.
Phone 518-431-1213 Fax 518-431-1234
E-mail: DLB22@healthresearch.org Website: www.healthresearch.org
