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536lecntwtbnk_3 11.45.23 PM - MIT OpenCourseWare...

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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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________________________________________________________________________________ ________________________________________________________________________________ 5.36 Lecture Summary #3 Thursday, February 12, 2009 CI-M workshop on Feb. 19 th : Bring two hard copies of your mini-review outline and an annotated bibliography w/ at least 3 primary research reports (from 2005 or later). HHMI Summer Research Fellowship in Chemical Biology: http://web.mit.edu/cld/hhmi/ Next Laboratory Session: #4 Topics: Affinity Tags for Protein Purification I. Overview of protein expression and general protein purification strategies II. Affinity tags for protein isolation A. Characteristics of tags B. Common affinity tags (GST, FLAG, and His) C. Cleavage of affinity tags III. SDS gel analysis of purified proteins Please note that we will complete our discussion of Quickchange DNA mutagenesis (continued from Lecture #2 notes) in our March 5 th lecture, prior to lab sessions #9-11. I. PROTEIN EXPRESSION AND GENERAL PURIFICATION STRATEGIES Consider our progress so far for H396P Abl(229-511) expression and isolation: Prior to Session 2, BL21(DE3) expression cells co-transformed with 1) an H396P Abl-encoding ______-resistant vector and 2) a YopH Tyr _______________-encoding _______-resistant vector were spread onto a LB-agar plate with antibiotics for colony selection. kan/strep plate Pick a single colony spin down and freeze cell pellet lyse cells and isolate H396 Abl protein grow up overnight starter culture 500-mL culture induce protein overexpression Successful Abl kinase domain expression in _____________ requires co-expression with a phosphatase! (Seeliger, M.A. et al. Protein Sci . 14, 3135-3139 (2005)) Prior to 2005, expression of active Abl kinase domain was carried out in insect cells. Insect cells yield _______gram quantities of protein, but are time consuming and _______________ to maintain. in bacteria with very low yields (______grams). While biochemical studies can be carried out with tiny amounts of protein, milligram quantities are required for biophysical and ______________ studies, such a crystallography and NMR.
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The low yields of soluble Abl in E. coli may be due to the ____________ of Tyr kinase activity in bacteria. Phosphatase co-expression prevents high levels of toxic kinase activity. YopH is a non-selective Tyr phosphatase, meaning it dephosphorylates most phosphotyrosine (____) substrates, regardless of the specific sequence. Yields of purified Abl K domain protein using the co-expression method in BL21-DE3 cells range from ___ to ____ mg/L. PROTEIN PURIFICATION In Session 5 you will lyse (split open) your BL21-DE3 cells and isolate the H396P Abl protein. Crude cell lysate has many components. Your lysate mixture will include Overexpressed ______-tagged H396Abl-kinase domain Overexpressed Yop phosphatase E. Coli proteins, DNA, and metabolites ( = what we want, × = what we don’t want) Strategies for protein purification rely on exploiting unique protein characteristics Solubility (1) Salting in. (2) Salting out
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