Engineering a Cytolytic Human Protein into a Novel Prostate Cancer Protoxin
Abstract
The principle investigator has made substantial progress in executing the statement of work outlined in the proposal. In addition he has fulfilled the requirements mandated by his training program to date. First, the PI obtained the human C5 cDNA and substituted a PSA substrate sequence in place of the wild type activation sequence. Modifications were implemented based on results from homology modeling. Next, PSA mediated cleavage was confirmed by mass spectrometry. Recombinant production of the confirmed PSA cleavable C5 mutant was scaled up by an adenovirus for in vitro assays. Purification of the recombinant protein was achieved using His-chromatography. PSA-mediated cleavage of the mutant was analyzed by western blot which resulted in curious results. It was then determined wtC5 and wtC3 are substrates of PSA. Ongoing studies include reverse engineering wild-type C5 to make PAC-2 selectively activateable.
Document Details
- Document Type
- Technical Report
- Publication Date
- Mar 01, 2011
- Accession Number
- ADA600512
Entities
People
- M. B. Manning
Organizations
- Johns Hopkins University