[Federal Register Volume 76, Number 37 (Thursday, February 24, 2011)]
[Notices]
[Pages 10378-10379]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 2011-4168]
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DEPARTMENT OF HEALTH AND HUMAN SERVICES
National Institutes of Health
Government-Owned Inventions; Availability for Licensing
AGENCY: National Institutes of Health, Public Health Service, HHS.
ACTION: Notice.
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SUMMARY: The inventions listed below are owned by an agency of the U.S.
Government and are available for licensing in the U.S. in accordance
with 35 U.S.C. 207 to achieve expeditious commercialization of results
of Federally-funded research and development. Foreign patent
applications are filed on selected inventions to extend market coverage
for companies and may also be available for licensing.
ADDRESSES: Licensing information and copies of the U.S. patent
applications listed below may be obtained by writing to the indicated
licensing contact at the Office of Technology Transfer, National
Institutes of Health, 6011 Executive Boulevard, Suite 325, Rockville,
Maryland 20852-3804; telephone: 301/496-7057; fax: 301/402-0220. A
signed Confidential Disclosure Agreement will be required to receive
copies of the patent applications.
Recombinant BoCPB: An Enzymatic Reagent for Removing Disordered,
Positively Charged C-terminal Residues From Recombinant Proteins
Description of Technology: Affinity tags are commonly used to
facilitate the purification of recombinant proteins, but concerns about
the potential impact of the tags on the biological activity of the
target proteins makes it necessary to remove them in most cases.
Proteases with high sequence specificity, such as tobacco etch virus
(TEV) protease, are typically used for this purpose. Affinity tags on
the amino-terminus (N-terminal tag) can be cleaved by TEV protease to
yield a recombinant protein product with only one nonnative residues on
its C-terminus (usually G or S). In contrast, removal by TEV protease
of tags added to the carboxy-terminus (C-terminal tag) of proteins has
proven to be somewhat problematic, yielding a recombinant protein
product with six nonnative residues on its C-terminus (ENLYFQ). Since
C-terminal affinity tags are potentially very useful, particularly when
used in combination with N-terminal tags in an ``affinity sandwich''
format, it would be very desirable to have a reagent to remove the C-
terminal affinity tags without leaving extra nonnative residues behind.
Previously, the NIH inventors created a tagged version of a fungal
carboxypeptidase from Metarhizium anisopliae (MeCPA) that is capable of
removing histidine residues and many other types of amino acids from
the C-termini of recombinant proteins. The only limitation of the MeCPA
enzyme is that it does not remove positively charged residues (arginine
and lysine). To overcome this drawback of MeCPA, the NIH inventors have
now cloned, expressed and purified bovine carboxypeptidase B (BoCPB),
which is specific for the removal of these positively charged residues.
Like the genetically engineered MeCPA, the recombinant BoCPB has a C-
terminal polyhistidine tag. This feature facilitates the purification
of the enzyme, and, because this His-tag as been engineered to be
immune to the action of MeCPA and BoCPB, it can be used to separate the
enzymes from the products of a carboxypeptidase digest. By using a
mixture of MeCPA and BoCPB, it should be possible to remove any short
affinity tag along with disordered C-terminal residues of a recombinant
protein with the exception of proline, which can be used as a ``stop
sign'' to facilitate the
[[Page 10379]]
production of a digestion product with a homogeneous C-terminus.
Applications
Removal short C-terminal affinity tags from recombinant
proteins without leaving any nonnative residues behind when used in
combination with MeCPA.
Identification and removal of disordered residues from the
C-termini of native (untagged) proteins, thereby increasing their
propensity to crystallize.
Inventors: David Waugh et al. (NCI)
Related Publications: None.
Patent Status: HHS Reference No. E-027-2011/0--Research Tool.
Patent protection is not being pursued for this technology.
Licensing Status: Available for licensing.
Licensing Contact: Whitney Hastings; 301-451-7337;
[email protected].
Collaborative Research Opportunity: The National Cancer Institute,
Protein Engineering Section, is seeking statements of capability or
interest from parties interested in collaborative research to further
develop, evaluate, or commercialize recombinant BoCPB and/or similar
enzymes. Please contact John Hewes, PhD at 301-435-3121 or
[email protected] for more information.
A DsbC Expression Vector for the Production of Proteins With Disulfide
Bonds in the Cytosol of E. coli
Description of Technology: Many proteins of biomedical importance
contain disulfide bonds and such proteins are notoriously difficult to
produce in Escherichia coli. Current methods to address this problem
either export the protein to the periplasmic space, which is a more
favorable redox environment for disulfide bond formation, or utilize
genetically modified strains of E. coli to alter the redox potential of
the cytosol (such as ``Origami'' or ``Shuffle'' cells). Unfortunately,
these methods generally result in very low yields of the desired
product, thus emphasizing the need for a novel method.
The NIH inventors have designed a DsbC expression vector that can
be used to improve the yield of correctly oxidized recombinant proteins
in the cytosol of E. coli. By overproducing DsbC on a separate plasmid
and coexpressing it with carboxypeptidases in the cytosol of E. coli,
the inventors were able to increase the amount of properly oxidized,
active carboxypeptidases that could be recovered from the cytosol by at
least 4-fold. Further, they believe that co-expression of DsbC from a
multicopy plasmid vector will also improve the yield of other disulfide
bond-containing proteins in E. coli.
Applications: Improving the yield of correctly oxidized recombinant
proteins in the cytosol of E. coli.
Advantages: Substantial increase in the amount of active
carboxypeptidases recovered from the cytosol and improved yield of
disulfide bond-containing proteins in E. coli.
Inventors: David Waugh et al. (NCI)
Related Publications
1. Prinz WA, Aslund F, Holmgren A, Beckwith J. The role of the
thioredoxin and glutaredoxin pathways in reducing protein disulfide
bonds in the Escherichia coli cytoplasm. J Biol Chem. 1997 Jun
20;272(25):15661-15667. [PubMed: 9188456]
2. Levy R, Weiss R, Chen G, Iverson BL, Georgiou G. Production of
correctly folded Fab antibody fragment in the cytoplasm of Escherichia
coli trxB gor mutants via the coexpression of molecular chaperones.
Protein Expr Purif. 2001 Nov;23(2):338-347. [PubMed: 11676610]
Patent Status: HHS Reference No. E-028-2011/0--Research Tool.
Patent protection is not being pursued for this technology.
Licensing Status: Available for licensing.
Licensing Contact: Whitney Hastings; 301-451-7337;
[email protected].
Dated: February 16, 2011.
Richard U. Rodriguez,
Director, Division of Technology Development and Transfer, Office of
Technology Transfer, National Institutes of Health.
[FR Doc. 2011-4168 Filed 2-23-11; 8:45 am]
BILLING CODE 4140-01-P