[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

    <bullet> Removal short C-terminal affinity tags from recombinant 
proteins without leaving any nonnative residues behind when used in 
combination with MeCPA.
    <bullet> 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; 
<a href="/cdn-cgi/l/email-protection#86eee7f5f2efe8e1f1c6ebe7efeaa8e8efeea8e1e9f0"><span class="__cf_email__" data-cfemail="acc4cddfd8c5c2cbdbecc1cdc5c082c2c5c482cbc3da">[email protected]</span></a>.
    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 
<a href="/cdn-cgi/l/email-protection#deb6bba9bbadb49eb3bfb7b2f0b0b7b6f0b9b1a8"><span class="__cf_email__" data-cfemail="a5cdc0d2c0d6cfe5c8c4ccc98bcbcccd8bc2cad3">[email protected]</span></a> 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; 
<a href="/cdn-cgi/l/email-protection#147c7567607d7a73635479757d783a7a7d7c3a737b62"><span class="__cf_email__" data-cfemail="5f373e2c2b363138281f323e36337131363771383029">[email protected]</span></a>.

    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


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