Slides3 11.45.23 PM - MIT OpenCourseWare http:/ocw.mit.edu...

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MIT OpenCourseWare http://ocw.mit.edu 5.36 Biochemistry Laboratory Spring 2009 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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His-tag Affinity Tags for Protein Purification I. Overview of Protein Expression and General Strategies for Protein Purification II. Affinity tags for Protein Purification A. Characteristics of tags B. Common affinity tags GST-tag FLAG-tag C. Cleavage of affinity tags III. SDS gel analysis of purified proteins His-tag
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Our progress so far for H396P Abl(229-511) expression and isolation: BL21(DE3) cell Prior to Session 2, BL21(DE3) expression cells co-transformed with 1) an H396P Abl-encoding _____-resistant vector and kan 2) a YopH Tyr phosphatase -encoding ______-resistant vector strep were spread onto a LB-agar plate with antibiotics for colony selection. (Session 5) grow up spin down and Pick a single overnight freeze cell pellet colony kan/strep plate Lyse cells and Isolate the starter culture 500-mL culture H396P Abl protien Induce protein over-expression
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Successful Abl kinase domain expression in bacteria requires co- expression with a phosphatase! High yield bacterial expression of active c-Abl and c-Src tyrosine kinases Markus A. Seeliger 2 1, Matthew Young 2 1, M. Nidanie Henderson 2 1, Patricia Pellicena 2 1, David S. King 1, Arnold M. Falick 1, John Kuriyan 3 2 1 * Received: 3 August 2005; Revised: 2 September 2005; Accepted: 4 September 2005 Seeliger, M.A. et al. Protein Sci . 14 , 3135-3139 (2005)
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Successful Abl kinase domain expression in bacteria requires co- expression with a phosphatase! High yield bacterial expression of active c-Abl and c-Src tyrosine kinases Markus A. Seeliger 2 1, Matthew Young 2 1, M. Nidanie Henderson 2 1, Patricia Pellicena 2 1, David S. King 1, Arnold M. Falick 1, John Kuriyan 3 2 1 * 4 September 2005 Prior to 2005, expression of active Abl kinase domain was carried out • in insect cells. Insect cells yield milli gram quantities of protein, but are time consuming and expensive to maintain. • in bacteria with very low yields ( micro grams). While biochemical studies can be carried out with tiny amounts of protein, milligram quantities are required for biophysical and structural studies, such a crystallography and NMR.
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Successful Abl kinase domain expression in bacteria requires co- expression with a phosphatase! High yield bacterial expression of active c-Abl and c-Src tyrosine kinases Markus A. Seeliger 2 1, Matthew Young 2 1, M. Nidanie Henderson 2 1, Patricia Pellicena 2 1, David S. King 1, Arnold M. Falick 1, John Kuriyan 3 2 1 * 4 September 2005 Seeliger and co-workers hypothesized that the low yields of soluble Abl in E. coli were due to the toxicity of Tyr kinase activity in bacteria. • Phosphatase co-expression prevents high levels of toxic kinase activity.
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This note was uploaded on 12/16/2011 for the course BIOLOGY 101 taught by Professor Mr.wallace during the Fall '11 term at Montgomery College.

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Slides3 11.45.23 PM - MIT OpenCourseWare http:/ocw.mit.edu...

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