Monitoring the purification Quantitation of total protein Assay of enzymatic activity (units) Specific activity = enzymatic activity / protein Enrichment = specific activity of fraction/original specific activity Yield = units after purification step /units in the original preparation x 100% A successful purification step should not only increase the specific activity of the enzyme substantially but the enzyme should also be recovered in good yield. Note that the yield of enzyme after a particular purification procedure may be low not because the procedure is failing to purify that protein, but because it is causing some inactivation of the enzyme. An additional problem sometimes experienced is that the enzyme may have been active immediately subsequent to the purification step but may lose substantial activity if stored for long periods prior to assay.

Assessment of purity Proof that the protein is pure can only really be obtained by protein sequencing. However, considerably simpler analytical procedures can be used to provide a good indication of the degree of purity. SDS-PAGE Analytical Isoelectric focusing Two-dimensional Gel Electrophoresis The detection method used to stain the gels is also important. For example Coomassie Blue staining is much less sensitive than silver staining and it is possible to have what looks like a pure protein on Coomassie Blue that will not look so good when the same gel Silver stained.

Hypothetical Purification Scheme Growth / Induction Harvest cells Lysis of cells by Sonication Centrifuge to remove Precipitation of Nucleic Acids Ammonium Sulfate fraction Dialysis Ion-Exchange Chromatography Concentration Gel Filtration Chromatography Hydrophobic Chromatography Dialysis / Concentration

Methods for the Determination of Protein Concentration Direct Methods - require very pure protein and composition (or sequence) Absorbance 280 nm quick, quite accurate if protein is pure Absorbance 280 nm in Guanidine-HCl best method is protein is pure Absorbance 205 nm accurate,very sensitive to contaminants Amino Acid analysis time consuming, expensive Indirect Method (Dye Binding) - require ‘standard’ for comparison Bradford Assay (BioRad) fastest, easiest, least accurate BCA Assay (Pierce) simple, better than Bradford Lowry Assay (with TCA Precipitation) eliminates buffer problems

Over-expression and Purification of CheR Methyltransferase Problem: All attempts to over-express CheR led to formation of inclusion bodies; only a small fraction of the protein remained soluble and this was not fully active. Attempts to refold the protein were unsuccessful. Solution: Co-expression with a soluble fragment of the Tar chemoreceptor. The co-expression of a ‘substrate’ for CheR increased its solubilty (stochiometrically) dramatically. This led to the development of an efficient purification scheme, that result in high purity, stable and active protein. T7 Promoter ctar gene cheR gene Vector with both genes downstream of a T7 promoter Note this does not produce a fusion protein.

Grow, induce, harvest cells Prepare lysate Protocol Grow, induce, harvest cells Prepare lysate Precipitate CheR-cTar complex with 20% Amonium sulfate Load onto DEAE column wash briefly Connect in series with CM column Wash extensively Elute CheR from CM column with salt gradient Few proteins precipitate at this low concentration of ammonium sulfate. CheR does not bind to DEAE under these conditions, but cTar does and the complex binds through cTar. The column is washed and any CheRthat comes off the complex binds to the CM column. Any proteins that have bound to the DEAE column that may come off will not bind to the CM column

SDS-PAGE of Samples from CheR Purification cTar M 1 2 3 4 5 6 7 1 - cell free lysate 2 - 20% ammonium sulfate ppt 3 - 20% ammonium sulfate supernatant 4 - 20% ammonium sulfate dialysate 5- CM Fraction x 6 -CM Fraction x+2 7 - CM Fraction x+4

Ethanol Precipitation Step in Purification of HU Protein HU is a small non-specific DNA binding protein of E. coli. Purification protocols were giving very low yields (i.e. most of the HU was being lost in early steps in the purification). Addition of a 70% ethanol precipitation step early in the procedure eliminated almost all proteins except a couple of closely related proteins which were easily separated with a couple of columns. (Almost all cellular proteins are precipitated in 70% ethanol but HU remained soluble). 1 - whole cells 2 - lysed cell 3 - low speed supernatant 4 - cell fre lysate 5 - 70% EtOH supernatant 6 - DEAE pooled fraction 7 - Heparin Sepharose pooled fractions 8 - Purified HU protein HU

Affinity Purification of Antibody Against Signaling Domain of Tar Chemoreceptor using His-tagged Signaling Domain Polyclonal sera against the cytoplasmic domain of the Tar chemoreceptor is primarily against regions outside the highly conserved signaling domain (SD) because the flanking regions are very antigenic. His6-SD was bound to a Ni-NTA column and a-cTar polyclonal sera was passed over it, washed and bound IgG was released with 4M MgCl2. The eluted anti-sera was highly specific for the signaling domain. SD SD cTar cytoplasmic domain His6-SD SD Tar Chemoreceptor

Current Protocols in Protein Science Wiley & Sons, NY (the big “blue” book) Guide To Protein Purification, M.P. Deutscher, Ed. Methods in Enzymology Vol. 182 (also other Meth. Enz.) Protein Purification: Principles and Practices, R.K. Scopes Springer-Verlag, New York. (1982). A little dated but probably the best general book. Online Tutorial and Java Based Protein Purification Simulation: http://www.tlsu.leeds.ac.uk/courses/bioc2060/proteinlab102/proteinlab.html