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.His-tagAffinity 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-tagOur 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-expressionSuccessful 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)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 Prior to 2005, expression of active Abl kinase domain was carried out • in insect cells. Insect cells yield milligram quantities of protein, but are time consuming and expensive to maintain. • in bacteria with very low yields (micrograms). 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.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 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. • YopH is a non-selective Tyr phosphatase, meaning it dephosphorylates most phosphotyrosine (pY) substrates, regardless of the specific sequence. • Yields of purified Abl K domain protein using the co-expression method in BL21-DE3 cells range from 5 to 15 mg/L.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 mixture will include • Overexpressed His-tagged H396Abl-kinase domain * • Overexpressed Yop phosphatase x • E. Coli proteins, DNA, and metabolites x * what we want x what we don’t wantStrategies for protein purification Ionic Charge Solubili ty 1. Io n exchange chro matography 1. Salting in 2. Electrophoresis 2. Salting out 3. Isoelectric focusing Protein characterist ics Size 1. Dialysis Polarity 2. Gel elect rophoresis 1. Adsorption chromat ography 3. Size exclusion 2. Hydrophobic interaction chromatography chromatography Binding Specificity 1. Affinity ChromatographyAffinity 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  His-tag C. Cleavage of affinity tags III. SDS gel analysis of purified proteinsAffinity Chromatography Chromatography involves a mobile phase (ie. cell lysate with overexpressed protein) and a stationary phase (the column with ligand-bound beads) Three steps in column chromatography: 1) Binding the desired protein to the affinity column 2) Washing away unwanted proteins, DNA etc. 3) Eluting the desired proteinAffinity Chromatography Examples of protein/ligand pairs • Avidin/ or streptavidin --- biotin • Glutathione-S-transferase --- glutathione • Dihydrofolate reductase --- methotrexate However, • Not all proteins have known binding partners • Known binding interactions might not be sufficiently strong towithstand washing steps. • Raising antibodies for all proteins?—no way. Is there a general purification strategy that lends itself to adiverse set of proteins?Affinity Tags In affinity tag purification, the protein of interest (POI) is expressed with a polypeptide affinity “tag” on the N- or C-terminus of the protein. Purification is based on the highly specific interaction between the polypeptide affinity tag and a binding counterpart that is immobilized on solid support POI = Protein of interest POI Tag Column Tags are introduced at the DNA level, either supplied by theexpression vector or using primers.Summary of sequence, size, matrices, and elution conditions of commonly-used affinity tags Figure removed due to copyright restrictions. See tables 1 and 2 from Terpe, K. “Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems.” Appl Microbio Biotech 60 (2003): 523-533. Terpe, K. Appl. Microbio. Biotech. 2003, 60, 523-533An ideal affinity tag: • requires a simple purification process that is free ofharsh reagents. • does not affect (or only minimally affects) the tertiarystructure and biological activity of the POI. • can be easily and specifically removed from the POI following purification to produce native protein. • can be used with diverse types of proteins. Some tags confer additional benefits, such as increasing protein solubility and increasing the yield of recombinant