Bioseparations & Protein Purification using Controllable Self-Cleaving Inteins

This technology provides a novel accelerated autocatalytic cleaving element that can be used to make ligand binding domains self-cleaving in affinity fusion mediated protein purification. 

Protein splicing has the potential to regulate activity of the host protein, because protein function is sequence linked. Conditional or controllable inteins whose activity is triggered by application of small molecules or temperature changes is more appealing because inducible inteins can be dropped into target proteins with standard molecular biological techniques. Current affinity-fusion protein purification provides a rapid means of purifying arbitrary proteins in a single step through genetic fusion of the desired protein to a ligand-binding domain. A major drawback has been the time required for completion of the cleavage reaction, currently in excess of 3 days at 4°C.

Our technology is based on smaller inteins (mini-inteins ΔI), an intein with improved splicing activity (splicing mutant, SM) and an intein with pH-controllable cleavage activity (cleavage mutant, CM) for fusion-based protein purification. Our enhanced SM functions in undesirable environmental conditions using a phage display system, because the phage particle is variable in different environments. Our invention provides a pH-sensitive cleaving mutant with greatly accelerated cleavage, decreasing the time for C-terminal cleavage from several days to hours at 4°C, or to minutes at higher temperatures. Furthermore, this intein mutant does not require addition of any exogenous factors to the product stream; the cleavage reaction is induced purely through a mild pH shift in the running buffer. Not only have individual superior inteins been identified, but also key cleavage residues and a method to generate inteins that are not subject to the limitations of commercially available intein cleavage systems.

Applications

  • Optimal intein fusion to a desired target protein for purification or study of the target protein
  • Rapid quantitation and purification of recombinant proteins
  • Developing molecular tools for clinical diagnostics
  • Use in large scale pharmaceutical production

Advantages

  • Controllable self-cleaving inteins can be dropped into target proteins with standard molecular biological techniques versus splicing inteins which require protease addition
  • Purification of intact precursor through accelerated C-terminal cleavage reaction without N-terminal cleavage.
  • Accelerated intein cleavage reactions making this technique more attractive for scaling up intein-based protein purification
  • One-step purification method synthesized on the column
  • Mutant mini-inteins with accurate but reduced splicing activity can be utilized due to deletion of endonuclease activity
  • Regulate activity of the host protein
  • New insight into structural and functional roles of conserved residues in protein splicing

State of Development

Many inteins are available that have various pH and temperature sensitive cleavage characteristics. Custom inteins can be created by request.

Patents

USPTO no. 6,933,362

Licensing Potential

Available for licensing.

Inventor

belfordMarlene Belfort, Ph.D.
Dr. Marlene Belfort has been actively engaged as a collaborator, mentor and reviewer in the field of inteins for over 15 years. She has been fascinated by their evolution and convinced of their utility and states that, “understanding their mechanism is the key to understanding the role of exteins in cellular evolution for biotechnological advancement”. Using a combination of genetics, biochemistry, structural biology and quantum mechanical modeling, her group has key residues that have aided in identifying acid and base catalysts that coordinate activities at splice sites which have shown the importance of allosteric effects for splicing. Her work has demonstrated the dramatic effects of the flanking extein sequences in splicing sequences in splicing and we designed an extein-based redox trap that no only promises further mechanistic insight, but also have implications for the persistence of inteins within cells. Her theory is that some inteins not only adapt to their extein, but regulate splicing in a manner consistent with cellular environment providing benefit to the cell. This challenges the “selfish intein” hypothesis.

Marlene holds two patents for intein technology and recently applied for a third. Sheco-developed an intein-based protein purification system for research, industrial-scale and microfluidic separation (Wood et al., 1999; Wood et al., 2000; Wu et al., 2002; Miao et al., 2005), several functional assays, and aprotease assay (Callahan et al., In press) that forms the basis of anintein-based botulinum sensor. Additionally, she co-discovered an anti-mycobacterial lead compound, the antineoplastic agent cisplatin.

Awards & Honors: Distinguished Scientist, Wadsworth Center, Molecular Genetics

  • Fellow, American Association for Advancement of Science
  • Doctor Philosophiae Honoris Causa, Hebrew University
  • Distinguished Professor, School of Public Health, Biomedical Sciences
  • Alice Evans Award, American Society for Microbiology
  • MERIT Award NIH
  • Fellow, American Academy of Microbiology
  • Member, National Academy of Sciences (NAS)
  • Fellow, American Academy of Arts and Sciences

Education

  • Ph.D., University of California at Irvine (1972)
  • Postdoctoral training, Hebrew University, Jerusalem

Publications

Peer-reviewed Publications (of ~170)

Most relevant to current invention

Derbyshire, V., Wood, D.W., Wu, W., Dansereau, J.T., Dalgaard, J.Z. and Belfort,M. Genetic definition of a protein-splicing domain: functional mini-inteins support structure predictions and a model for intein evolution. Proc. Natl. Acad. Sci. USA (1997) 94, 11466-11471.

Derbyshire, V. and Belfort, M. Lightning strikes twice: intron-intein coincidence. Proc. Natl. Acad. Sci. USA (1998) 95,1356-1357.

Wood, D., Wu, W., Belfort, G., Derbyshire, V. and Belfort, M. A Genetic System Yields Self-Cleaving Inteins for Bioseparations. Nature Biotechnol. (1999) 17, 889-892.

Wood, D.W., Derbyshire, V.,Wu, W., Cartrain, M., Belfort, M., and Belfort, G. Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor. Biotechnol. Prog. (2000) 16,1055-1063.

Wu, W., Wood, D.W., Belfort, G., Derbyshire, V. and Belfort, M. Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable splicing. Nucleic AcidsRes. (2002) 30, 4864-4871.

Miao, J., Wu, W., Spielmann, T., Belfort, M., Derbyshire, V., and Belfort, G. Single-step affinity purification of toxic and non-toxic proteins on a fluidics platform. Lab on a Chip (2005) 5,248-253.

Hiraga, K., Derbyshire, V., Dansereau, J. T., VanRoey, P. and Belfort, M. Minimization and stabilization of the Mycobacterium tuberculosis recAintein. J. Mol. Biol. (2005) 354, 916-926.

Van Roey, P., Pereira, B., Li, Zh., Hiraga, K., Belfort, M., and Derbyshire, V. Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue. J.Mol. Biol. (2007) 367,162-173.

Amitai, G., Callahan, B.P., Stanger, M.J., Belfort, G. and Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. USA (2009) 106, 11005-11010.

Du, Z., Shemella, P.T., Liu, Y., McCallum, S.A., Pereira, B., Nayak, S.K., Belfort, G., Belfort, M. and Wang, C. Highly conserved histidineplays a dual catalytic role in protein splicing: a pKa shift mechanism. J. Am. Chem. Soc. (2009) 131, 11581-11589.

Hiraga, K., Soga, I., Dansereau, J.T., Pereira, B., Derbyshire, V., Du, Z., Wang, C., Van Roey, P., Belfort, G. and Belfort, M. Selection and structure of hyperactive inteins: peripheral changes relayed to the catalytic center. J. Mol. Biol. (2009) 393, 1106-1117.

Du, Z., Liu, Y., Ban, D.,Lopez, M.M., Belfort, M. and Wang, C. Backbone dynamics and global effects of an activating mutation in minimized Mtu RecA inteins. J. Mol. Biol. (2010) 400, 755-767.

Callahan, B.P., Stanger, M.J. and Belfort, M. Cut and Glow: Protease activation of split green fluorescent protein Chem Biochem (2010). In press.

Additional Recent Publications

Coros, C.J., Piazza, C. L., Chalamcharla, V.R., Smith, D. and Belfort, M. Global regulators orchestrate group II intron retromobility. Mol. Cell(2009) 34, 250-256.

DiChiara, J.M., Contreras-Martinez, L.M., Livny,  J., Smith, D., McDonough, K.A. and Belfort, M.  Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in M. tuberculosis and M. smegmatis. Nucleic Acids Res. (2010) 38, 4067-4078.

Chalamcharla, V.R., Curcio, M.J. and Belfort, M. Nuclear expression of a group II intron is consistent with spliceosomal intron ancestry. Genes Dev. (2010) 24, 827-836.

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:  diane.borghoff@healthresearch.org  Website:  www.healthresearch.org