A Novel Negative Regulator of Angiogenesis
Abstract
The aim of this proposal is to purify a sufficient amount of VEGI, a novel inhibitor of angiogenesis we discovered recently, in order to determine its anticancer activity in animal models. Initially we planned to express the protein in insect cells; however, we found out that we can express the protein in E. coli with good yield and very high activity. We have developed two bench-top procedures to purify VEGI protein. When expressed in E. coli, VEGI protein is basically found in the inclusion bodies. Upon refolding, the protein, which is tagged with 10-histidine residues at the N-terminus, is purified by a one-step affinity chromatography using Ni-sepharose resin. The second purification procedure involves dissolving the inclusion bodies in SDS and purify the denatured VEGI protein by using a gel filtration column; the purified VEGI protein is then refolded by dilution (Scheme 2). The yield from either procedure is 2-5 mg/L, which is reasonable for bench-top production. The specific activity of the protein preparation, as determined by the IC50 value for the inhibition of endothelial cell growth, is 6 ng/ml, representing nearly 500-fold improvement as compared to our earlier preparations. These procedures are simple and ready for scale-up studies.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 01, 1999
- Accession Number
- ADA385777
Entities
People
- Luyuan Li
Organizations
- Georgetown University