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- What does ProteomTech
do?
ProteomTech, Inc., a biotech company located
in the San Francisco Bay Area, has developed
Pt-Fold, a protfolio of unique, proprietary
technologies for correctly refolding
proteins expressed in E. coli as inclusion
bodies.
- What is Pt-Fold?
Pt-Fold refers to the technology
platform consisting of multiple technologies
and methods invented by ProteomTech scientists.
These high-throughput refolding procedures
can be used for the production of research
quantities of large numbers of recombinant
human proteins and for large-scale production
of proteins of therapeutic interest. Proteins
produced using Pt-Fold have native activity
and have been crystallized for structural
determination. A general description
of Pt-Fold can be found on our
website.
- What is unique
about your Pt-Fold Technology?
It is a highly controlled process which
guides the protein through stepwise procedures,
gradually rebuilding its native structure.
Other labs may use similar procedures,
but we fully developed the technology
and patented it. The Pt-Fold technology
platform also includes other formulations
and know-how that are unique to our company,
which have not been disclosed. Since we
specialize in refolding, we believe we
have more experience than any one in the
refolding area. Since the inception of
the company, we have successfully refolded
more than 250 different proteins.
- What starting
material do you accept?
cDNA, or expression plasmid in E. coli,
preferred;
cell paste or freshly isolated inclusion
bodies accepted (if properly stored and
shipped);
purified proteins in denaturing solution
(as long as the constituents of the solution
are revealed and we accept it as suitable
for our refolding procedures)
- What information
do you need about my protein?
Protein molecular weight: Size (KD), length
of protein (# of amino acids); pI; # of
cysteines or internal disulfide linkages;
protein family (i.e., MAP kinase, serine
protease, etc.) ; domain or whole protein?
If functionality is an acceptance criterion,
the activity assay buffer conditions.
If crystallization is a goal, the conditions
for crystal trials.
- What is the
success rate of your Pt-Fold Technology?
We have increased our success rate from
40% with our original, single procedure,
to >80% using more than forty new variations.
- Can you refold
proteins with many internal disulfide
bonds?
Yes, we have successfully refolded proteins
with as many as 13 disulfide bonds (urokinase),
with demonstrated enzymatic activity.
- What types
of proteins have you successfully refolded?
| Cell Receptors |
|
Splicing Factors |
| Proteinases |
|
Adaptor Proteins |
| Proteinase Inhibitors |
|
Angiogenesis related |
| Signal Transduction Related |
|
G Protein Regulational Proteins |
| Kinases |
|
G Protein Family |
| Phosphatases |
|
Cellular Enzymes |
| Extracellular Matrix |
|
Calcium Binding Proteins |
| Transcription Factors |
|
Mitochondrial Proteins |
| Oncogenes |
|
Hypothetical Proteins |
| Cytokines |
|
CD protein Extracellular Domain |
- What quality
control steps do you take?
- OD measurement for protein quantity
and yield
- single, sharp gel filtration peak
- non-reduced SDS-PAGE for identity
and purity
- protein assay for more accurate
measuring of protein quantity
- activity measurement if available
- Other QC tests, including CD and
light scattering can be specified
by contract.
- What 3rd party
quality validation do you have?
Besides publications in journals such
as Science and PNAS, our collaborators
and customers perform a variety of tests,
including functional assays and crystallization
studies. Thus they are the independent
"judge" of our refolding process.
For internal protein drug development
activities, besides performing our own
functional tests and QA, we send our refolded/purified
proteins to other reputable labs for independent
testing. For example, we sent our Pt-Fold
produced pro-urokinase to two outside,
experienced labs. Both labs proved by
direct comparison that our pro-urokinase
is functionally the same as mammalian
expressed enzyme. For VEGI studies, we
also validated the function of the protein
by collaboration with outside, reputable
academic labs.
- What is the
protein size range you have successfully
worked with?
From 6 KD to 135 KD. We can also co-refold
individual domains of much larger proteins
(streptokinase-plasminogen complex is
an example).
- What success
has ProteomTech had with refolding isolated
domains, e.g. of transmembrane proteins?
Two examples of refolding proteins which
carry transmembrane domains are BACE1
(beta-secretase), implicated in Alzheimer’s
disease, and VEGI (vascular endothelial
growth inhibitor), a potential cancer
therapeutic.
The structure of BACE1 was solved using
protein refolded by our CSO, Xinli Lin
(published in Science). This had the transmembrane
domain removed.
For VEGI we have refolded several isoforms,
including some with transmembrane domains
removed. These materials have been used
for preliminary animal studies.
- What quantity
of a given protein can you produce?
We usually deliver 5-10 mg during feasibility.
We are capable of producing gram quantities
for subsequent delivery.
- What about
post-translational modifications (PTM)?
It is recognized in the field that for
drug discovery and development, PTM are
becoming more and more important. Since
Pt-Fold applies to E. coli systems,
we are frequently asked if it is applicable
to human (or mammalian) expression systems.
E. coli expressed proteins can have proper
PTM. For example, we have expressed in
E. coli and refolded/purified kinases
for two customers, who have tested and
shown that the kinases are active, and
properly phosphorylated. On the other
hand, E. coli does not have a proper glycosylation
system, thus E. coli expressed human proteins
are not glycosylated. However, it is widely
believed that in mammalian cells, glycosylation
is not usually required for activity/function
of the protein. It is accepted that cells
use glycosylation to label a protein for
targeting to a particular cellular compartment,
or just to make a protein more soluble
in a particular cellular environment.
Thus the E. coli expressed/refolded, non-glycosylated
human protein can be functionally the
same as the "native" protein
expressed from mammalian cells. For example,
publications document that Alzheimer's
beta-secretase refolded from E. coli is
functionally the same as the mammalian
expressed enzyme. Furthermore, because
of the lack of glycosylation, E. coli
expressed/refolded enzymes are better
for structural studies (crystallization)
and drug development, as shown in the
publication in Science for the structure
of beta-secretase. Finally, our data,
our customers and other publications have
proven that the E. coli refolded urokinases
(non-glycosylated) have the same activity
as that of mammalian expressed (glycosylated).
- What is the
turnaround time for a given protein? How
long will it take to refold a protein?
Eight to twelve weeks depending on the
difficulty of the protein.
- Will the final
protein be shipped in physiological buffer
or buffer with detergent?
There are multiple options, including
adapting the final folding buffer to a
particular assay you may want to perform,
or removing detergents for crystallization.
Protein samples are shipped at
4 degrees C.
- What about
technology licensing for large-scale manufacturing?
ProteomTech is interested in negotiating
technology transfer agreements for Pt-Fold,
for process scale-up and manufacturing
for a specific protein.
- Can I utilize
Pt-Fold for high throughput refolding
in my own laboratories?
For internal production of research quantities
of proteins from inclusion bodies, ProteomTech
plans to commercialize automated instrumentation
and reagents developed internally for
screening protein refolding conditions.
Please contact us to discuss “early
access”.
- Do you use
tags?
Tags are not required for our purification
protocols. No fusion tags are required
if the protein is expressed well in E.
coli, but His-tags or others may enhance
the expression level for higher yield.
- Who are your
customers?
Big pharma, biotech, academic/government
labs, including Abbott Laboratories, Aventis,
Celera, Dyax, Exelixis, Johnson &
Johnson, Structural Genomix, Takeda Pharmaceuticals,
Tularik, UCSF, USDA, Xerion Pharma
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