Crystallization trials of Refolded Breast Tumor Kinase (BRK) and Peroxisome Proteins

Abstract

Protein expression and purification are essential steps and initial conditions for crystallization works and crystal structure studies. Here, we are presenting two different parts that is much relative to the protein expression and purification experiments. The first part, also a major project, is focusing on the preliminary crystallization trials of refolded Breast tumor kinase (Brk). Breast tumor kinase (Brk), a non-receptor tyrosine kinase that is overexpressed in a high percentage of breast carcinomas, has been found to be constitutively expressed in a large proportion of cutaneous T-cell lymphomas and other transformed T- and B-cell populations. Brk also has conferred in vivo oncogenicity on the BaF3 cells. Furthermore siRNA-mediated inhibition of endogenous Brk in malignant T-cells has diminished their growth and survival capacity. However, the role of Brk in cell transformation remains poorly defined. To determine the actual mechanism that is responsible for the Brk-mediated control over basic cell functions, we propose to elucidate the three-dimensional structure of Brk using X-ray crystallography and correlate with its function. The structure of Brk (and its mutants) will eventually help to identify potential therapeutic targets for lymphomas and, possibly, other malignancies. Furthermore, studies are underway to identify the interacting partners of Brk in lymphoma. The recombinant human Brk protein was expressed in bacteria in the insoluble form and refolded. The refolded protein was characterized by several biophysical methods, like, intrinsic tryptophan fluorescence and circular dichroism spectroscopy. The second part is mainly concerning on expression and purification of peroxins (peroxisome factor 8 and peroxisome factor 20). Peroxins are proteins that are required for various aspects of peroxisome biogenesis including assembling peroxisome membrane, importing most of the peroxisomal matrix proteins, peroxisome proliferation and peroxisome inheritance. Two of such peroxins, Pex8 and Pex20 that are involved in Peroxisome targeting signal 2 (PTS2) pathway, are known to play key roles in transporting important peroxisomal matrix proteins into the matrix of the peroxisome. These proteins also interact with the Pex7 receptor to translocate proteins that have the PTS2 sequence into the peroxisome. Even though most of the activities and functions of these proteins have been predicted, full understanding of these proteins will be possible after theirs structure are known. In the present study, we have successfully over-expressed and purified GST-Pex8 and GST-Pex20.