Molecular Mechanisms of Bacterial Superantigen Function
Abstract
Plan Objectives: 1. clone and express a human T cell receptor (TCR) gene in bacteria. 2. clone and express a human T cell receptor gene in insect cells. 3. determine the binding kinetics of the soluble T cell receptor to complexes of HLA-DRl/HA and Ses in real time using an optical biosensor. Approach: We cloned individual c DNA of a and b chains of a human TCR, clone 1, in bacteria. Acidic and basic sequences of Leucine zipper were added to either gene to facilitate proper protein folding. Each gene was expressed in bacterial expression system. Accomplishments (by objective): 1. Beta chain of TCR was produced in inclusion bodies of the expressing bacteria to over 90% purity. 2. Alpha chain of TCR was produced in inclusion bodies but mixed with many other proteins. 3. At present, we can properly fold TCR from bacteria, as confirmed by ELISA, but with a low yield. We attempted to optimize refolding of TCR by; a) mixing a larger proportion of chain protein with the beta chain, b) try several protein folding methods, and c) use other strains of bacteria expressing the gene to increase compatibility with the a chain. Adding 5% glycerol helped refolding.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 2001
- Accession Number
- ADA396810
Entities
People
- Scheherazade Sadegh-nasseri
Organizations
- Johns Hopkins University