Drugs based on engineered proteins represent a new frontier for pharmaceutical
makers. Even after they discover a protein that may form the basis of the next
wonder drug, however, they have to confront a long-standing problem: how to
produce large quantities of the protein in a highly pure state. Now, a multi-institutional
research team including a biochemist at the National
Institute of Standards and Technology (NIST) may have found* a new solution
in an enzymatic "food processor" they can activate at will.
The team has found an efficient method of harvesting purified protein molecules
by altering an enzyme that soil bacteria use to break down their food. In its
natural form, this enzyme would be of little use to drug developers, but the
team has modified it so that it can be activated at the desired moment. By creating
essentially an "on-switch" for the enzyme's activity, the team has
found a way to separate a single, desired protein from the mixture of thousands
generated by a living cell, which remains biotechnology's natural protein factory
Bacteria use the enzyme, called subtilisin, as a sort of food processor: After
producing it internally, they release the enzyme into the soil, where it uses
a minuscule "blade" to chop up proteins into digestible pieces. Because
it could damage the bacterium's interior, the blade has a protective sheath
that only comes off once the enzyme has exited the cell.
"The enzyme and sheath are strongly attracted to each other. The enzyme's
first act is to cut the sheath away," says NIST's Travis Gallagher. "The
method takes advantage of their attraction in order to isolate the protein we
The team first creates many "sheathless" copies of the enzyme, which
are modified to function only in the presence of a triggering molecule such
as fluoride. The modified enzymes are bound to the surface of a strainer. Then
the team uses engineered cells to generate mass quantities of a potentially
therapeutic protein, each copy of which has a subtilisin sheath attached to
it. After harvesting these proteins along with the thousands of others that
grow in the cellular interior, they filter the mixture through the strainer,
where the protein-sheath pairs are caught and stuck fast to the subtilisin while
the rest of the mixture drains away.
At this point, the team flicks their switch. They add a bit of fluoride and
the enzyme snips the bond between sheath and protein, releasing the desired
protein free of almost all impurities. "The technique can conceivably be
used to obtain any protein you like, and the process is repeatable, as the sheaths
can be removed for another round of purification," Gallagher says. "For
most proteins, the method can achieve greater than 95 percent purity at a single
The research team also includes members from Potomac Affinity Proteins, LLC
(PAP) and the University of Maryland Biotechnology Institute (UMBI). UMBI holds
the patent, which has been licensed to PAP. The research was supported by grants
from the National Institutes of Health and the Bill and Melinda Gates Foundation.
* T. Gallagher, B. Ruan, M. London, M. Bryan and P.N. Bryan. Structure of a
switchable subtilisin complexed with substrate and with the activator azide.
Biochemistry, DATE, pages, DOI.