טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAiney Carmit
SubjectOverexpression and Purification of SLP-76 for the
Purpose of Structure-Function Analysis
DepartmentDepartment of Biotechnology
Supervisor Dr. Deborah Yablonski


Abstract

The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) is a 76-kDa evolutionarily conserved adaptor protein, which plays a role in the signal transduction pathways of receptors containing ITAM motifs. The essential role of SLP-76, in a subset of hematopoietic cell types, suggests that SLP-76 is a potentially potent and cell-type-specific therapeutic target. Our long-term goal is to use x-ray crystallography to determine the three dimensional structure of SLP-76. Thus, the main objectives of this study were to express SLP-76 and purify the protein. Various conditions were tested in order to set up optimal expression and purification systems for SLP-76. Overexpression of the SLP-76 gene was achieved using the T7 polymerase expression system. Optimization of the system included choice of host strains, and adjustment of temperature of induction, IPTG concentration, time point of induction, length of induction period and culture media. With this system the amount of SLP-76 protein achieved was about 30 mg per liter culture. SLP-76 was then purified by several chromatographic procedures. The first step was carried out by Metal Affinity Chromatography using Ni-NTA agarose resin. The second step was achieved by HiTrap Q HP anion exchanger. The final step was carried out by Gel Filtration Chromatography using Superdex200 analytical column. Following these purification steps, the protein was highly pure as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and FPLC gel filtration. However, minor contamination by truncated protein products could be observed, and if these products will interfere with subsequent crystallization studies, they will have to be removed in future work.