Principal Technology

The Pt-Fold Protein Refolding Technology

ProteomTech’s core technology, Pt-Fold, was initially developed over a fifteen-year period by Dr. Xinli Lin, ProteomTech’s cofounder and CSO. The Pt-Fold technology has been further refined for over 5 years in ProteomTech. At present, Pt-Fold has matured into one of the most advanced protein refolding technology, which includes a unique set of procedures for the refolding or re-naturing of denatured proteins, especially proteins expressed in E. coli as inclusion bodies.

ProteomTech has exclusive license for the patent for high-throughput purification and refolding of inclusion bodies titled, "Universal procedure for refolding recombinant proteins". Its sole inventor, Dr. Xinli (Henry) Lin, ProteomTech’s Chief Scientific Officer, has developed the Pt-Fold technology over a fifteen-year period.

Using its proprietary protein refolding technology, PTI is capable of producing proven and promising therapeutic proteins that are difficult to produce by other methods, thus expanding the candidate pool of protein drugs.

By producing recombinant proteins in E. coli using Pt-Fold, PTI is able to produce high quality protein drugs in much shorter development time and at much lower cost compared to mammalian cell culture or other recombinant protein expression systems.

The structure of the streptokinase/ plasminogen complex was solved and published in 1998 in Science using the proteins Dr. Lin had successfully refolded and purified. Dr. Lin has also refolded and purified beta-secretase (BACE), implicated in Alzheimer’s disease, in quantities sufficient for structural studies. BACE is believed to be critical for developing drugs for the prevention and treatment of Alzheimer’s disease. These results have been published in PNAS and Science. In addition, the human proteins pro-insulin, pro-urokinase, POMC, pepsinogen, VEGI (vascular endothelial growth inhibitor, an anti-angiogenesis protein with potential anti-cancer properties), as well as more than 200 other proteins, have been refolded and purified. Other techniques, secrets and know-how have been developed since the inception of the company.

The PT-Fold Method

This unique method consists of the following steps:

1. Synthesis of a recombinant protein in E. coli as inclusion bodies,
2. Isolation of the inclusion bodies to high purity.
3. Solubilization of the inclusion bodies in a proprietary buffer solution.
4. Protein refolding using procedures designed for the specific class of protein.
   1. Unique protein refolding technology “Pt-Fold Technology.
   2. Optimization of refolding conditions to maximize yield, functionality and stability over time.
5. Reconcentration by ultrafiltration.
6. Purification of the refolded protein using various column chromatographic procedures.

Advantages of Inclusion Body Expression

1. E. coli expression is more efficient and less expensive compared to mammalian, insect, and yeast cultures.
2. Some proteins are harmful to the host when expressed in their native forms; thus, insoluble expression is the preferred way to obtain large quantities of the recombinant proteins. In fact, most important proteins are highly regulated in mammalian cells, partially because over-expression of these proteins in their native form is harmful to the cell.
3. Recombinant proteins are more stable in their inclusion body form; thus, in most cases, the common problems of degradation and instability related to recombinant protein production can be avoided.
4. High levels of expression can be achieved for most proteins (200 to 400 mg of inclusion bodies per liter of bacterial culture is routinely obtained).
5. The inclusion bodies are easily purified to greater than 90% purity with a simple freeze/thaw and detergent washing procedure, making parallel production of many proteins possible.
6. Inclusion bodies processes are scalable. Several therapeutic proteins on the market are manufactured by inclusion body processes, including human growth hormone (HGH), human insulin and alpha-inteferon.

Uniqueness of the method

Dr. Xinli Lin is the sole inventor of the pH Shift technology, patented by the Oklahoma Medical Research Foundation, with an exclusive license granted by OMRF to ProteomTech.

No similar procedure has been published or patented. Other techniques, secrets and know-how have been developed since the inception of the company and are now part of the Pt-Fold technology platform.

Proteins Produced

Following is a list of the types of proteins produced utilizing Pt-Fold technologies. To date, ProteomTech has successfully refolded more than 200 proteins.

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

©2009 ProteomTech, Inc.