Catalytic Domains of [beta](1,4)-galactosyltransferase I Having Altered Donor and Acceptor Specificities, Domains That Promote In Vitro Protein Folding, and Methods for Their Use
Description of Invention:
[beta](1,4)-galactosyltransferase I catalyzes the transfer of galactose from the donor, UDP-galactose, to an acceptor, N-acetylglucosamine, to form a galactose-[beta](1,4)-N-acetylglucosamine bond. This reaction allows galactose to be linked to an N-acetylglucosamine that may itself be linked to a variety of other molecules. The reaction can be used to make many types of molecules having great biological significance. For example, galactose-[beta](1,4)-N-acetylglucosamine linkages are very important for cellular recognition and binding events as well as cellular interactions with pathogens, such as viruses. Therefore, methods to synthesize these types of bonds have many applications in research and medicine to develop pharmaceutical agents and improved vaccines that can be used to treat disease.
The present invention is based on the surprising discovery that the enzymatic activity of [beta](1,4)-galactosyltransferase can be altered such that the enzyme can make chemical bonds that are very difficult to make by other methods. These alterations involve mutating the enzyme such that the mutated enzyme can transfer many different types of sugars from sugar nucleotide donors to many different types of acceptors. Therefore, the mutated [beta](1,4)-galactosyltransferases of the invention can be used to synthesize a variety of products that, until now, have been very difficult and expensive to produce.
The invention also provides amino acid segments that promote the proper folding of a galactosyltransferase catalytic domain and mutations in the catalytic domain that enhance folding efficiency and make the enzyme stable at room temperature. The amino acid segments may be used to properly fold the galactosyltransferase catalytic domains of the invention and thereby increase their activity. The amino acid segments may also be used to increase the activity of galactosyltransferases that are produced recombinantly. Accordingly, use of the amino acid segments according to the invention allows for production of [beta](1,4)-galactosyltransferases having increased enzymatic activity relative to [beta](1,4)-galactosyltransferases produced in the absence of the amino acid segments.
Applications:
Synthesis of polysaccharide antigens for conjugate vaccines, glycosylation of monoclonal antibodies, and as research tools.
Development Stage:
The enzymes have been synthesized and preclinical studies have been performed.
Inventors: Pradman K. Qasba (NCI) Boopathy Ramakrishnan (NCI) Elizabeth Boeggeman (NCI)
Patent Status:
DHHS Reference No. E-230-2002/2 --
U.S. Patent Application No. 11/178,230 filed 08 Jul 2005, allowed
Foreign rights also available
Licensing Status: Available for exclusive or non-exclusive licensing.
Collaborative Research Opportunity:
The National Cancer Institute's Nanobiology Program is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize the use of galactose and modified galactose to be linked to an N-acetylglucosamine that may itself be linked to a variety of other molecules. Please contact John D. Hewes, Ph.D. at 301-435-3121 or hewesj@mail.nih.gov for more information.
For Additional Information Please Contact: John Stansberry Ph.D.
NIH Office of Technology Transfer
6011 Executive Boulevard, Suite 325
Rockville, MD 20852-3804
Phone: (301)435-5236
Email: stansbej@mail.nih.gov
Fax: (301) 402-0220
