FOR SALE: Difficult and
Medically Important Proteins.
FAQ
What does ProteomTech do?
What is Pt-Fold?
What is unique about your Pt-Fold Technology?
What starting material do you accept?
What information do you need about my protein?
What is the success rate of your Pt-Fold Technology?
Can you refold proteins with many internal disulfide bonds?
What types of proteins have you successfully refolded?
What quality control steps do you take?
What 3rd party quality validation do you have?
What is the protein size range you have successfully worked with?
What success has ProteomTech had with refolding isolated domains, e.g. of transmembrane proteins?
What quantity of a given protein can you produce?
What about post-translational modifications (PTM)?
What is the turnaround time for a given protein? How long will it take to refold a protein?
Will the final protein be shipped in physiological buffer or buffer with detergent?
What about technology licensing for large-scale manufacturing?
Can I utilize Pt-Fold for high throughput refolding in my own laboratories?
Do you use tags?
Who are your customers?
- What does ProteomTech do?
ProteomTech, Inc., a biotech company located in the Orange County Area, has developed Pt-Fold, a portfolio 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.
BREAKING NEWS:
ProteomTech, Inc. and
Collaborators Make Major Breakthrough
in Stem Cell Research
