Mechanisms of ErbB2 Tyrosine Kinase Regulation in Breast Cancer
Abstract
The c-erbB-2 proto-oncogene (also called HER2 or neu) encodes the ErbB2 protein, a transmembrane receptor tyrosine kinase (TK) whose overexpression is an important determinant in the initiation and progression of human breast cancer. Phosphorylation of downstream targets by the ErbB2 TK is the key event leading to activation of the signaling pathways associated with development and progression of aggressive breast cancer. This project focuses on the elucidation of the three-dimensional structure of human ErbB2 TK in the apo form and in complex with non-hydrolyzable ATP analogs and EGFR/ErbB2 family-specific inhibitors, using X-ray crystallography. To crystallize the ErbB2 protein, the DNA fragments encoding ErbB2(704-990) and ErbB2(704-1255) were cloned into the eukaryotic expression vector pGAPZA for production in P. pastoris cells. Each construct was introduced into P. pastoris by electroporation and the clones, which can express recombinant ErbB2(704-990) and ErbB2(704-1255) proteins, were induced for large scale protein preparation. Unsuccessful attempts to purify the proteins lead to a new strategy for producing these recombinant proteins in baculovirus expression system. The expression vector pFastBac1 was chosen for production of ErbB2(704-990) and ErbB2(704-1255) proteins in Sf9 and Sf21 insect cells. So far, Bacmids containing the DNA fragments were made and verified by PCR. It is still in progress to transfect the insect cells with the recombinant Bacmids for the production of ErbB2 proteins.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2005
- Accession Number
- ADA446952
Entities
People
- Gabriel Birrane
- John A. Ladias
- Lina Gu
Organizations
- Beth Israel Deaconess Medical Center