Protocols and tips in protein purification or How to purify protein in one day Contents I. Introduction 4 II. General sequence of protein purification procedures 5 Preparation of equipment and reagents 6 Preparation of the stock solutions 7 Preparation of the chromatographic columns 8 Preparation of crude extract (cell free extract or 9 soluble proteins fraction) Pre chromatographic steps 10 Chromatographic steps 11 III. “Common sense” strategy in protein purification 18 General principles and tips in “common sense” strategy 19 Algorithm for development of purification protocol for soluble over expressed protein 22 Brief scheme of purification of soluble protein 29 Timing for refined purification protocol of soluble over -expressed protein 30 DNA-binding proteins 31 IV. Protocols 33 Preparation of the stock solutions 34 Quick and effective cell disruption and preparation of the crude extract 35 Protamin sulphate (PS) treatment 36 Analytical ammonium sulphate cut (AM cut) 36 Preparative ammonium sulphate cut 37 Precipitation of proteins by ammonium sulphate 37 Recovery of protein from the ammonium sulphate precipitate 38 Preparation of samples for analysis of solubility of expression 38 Bio-Rad protein assay Sveta’s easy protocol 40 Protocol for accurate determination of concentration of pure protein 41 SDS PAGE Sveta’s easy protocol 42 V. Charts and Tables 43 Ammonium sulphate –Sugar refractometer 44 NaCl-Sugar refractometer 45 Calibration plot at various salt concentrations for Superdex200 GL column 46 Calibration plot for Hi-load Superdex 200 column 47 Ammonium sulphate table 48 VI. Appendix 49 Determination of protein concentration 50 How to concentrate proteins 54 How to store proteins 58 Time saving tips 59 Golden rules in protein purification 60 Contact details 60 2 Abbreviations AA acrylamide AS ammonium sulphate CBV column bed volume CE crude extract CM carboxymethyl DEAE diethylaminoethyl DTT dithiothreitol EDTA ethylenediaminetetraacetic acid FF fast flow HEPES N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) HIC hydrophobic interaction chromatography IEC ion exchange chromatography MES 2-morpholinoethanesulfonic acid MW molecular weight MWCO molecular weight cut off PS protamine sulphate PSA ammonium persulphate PAGE polyacrylamide gel electrophoresis Q quaternary ammonium S methyl sulphanate SDS sodium dodecyl sulphate SEC size exlusive chromatography or gel filtration SP sulphopropyl TEMED N,N,N',N' tetramethylethylenediamine TP target protein UV ultraviolet 3 I. Introduction Why do we need to purify protein in one day? After 25 years and 180 proteins it has become clear to me that for some proteins even one extra day under conditions normally used for protein purification can detrimentally affect their activity and crystallisation ability. Whilst the majority of proteins can survive long purification and can be kept and stored at 4ºC for days and even weeks with little loss in activity, it does them no harm to be purified in one day either. To achieve the goal of complete protein purification in one day you should move fast and choose appropriate protocols, avoiding long procedures such as dialysis, long centrifugations and slow chromatography. Below are some protocols and tips which help me to achieve this goal. My approach is based on classical chromatography for natively (no tags) over- expressed proteins. Nowadays the high throughput approach dictates increasing use of tags in protein purification and sometimes classical methods are considered to be out of fashion. This is not the case. Very often tagged proteins cannot be purified to desirable purity by using just one tag affinity chromatography and second purification step is required anyway. In many cases the protein cannot be expressed and correctly folded with the tag attached, so expression of a native protein is still required. My approach does not differ much from tagged purification with respect to time, especially if tag removal is necessary. Please note that this brochure is not a complete guide to protein purification and you should still read serious books on the theory of chromatography to become familiar with the subject. 4 II. General sequence of protein purification procedures 5 Preparation of equipment and reagents For protein purification you need the equipment and reagents listed below: Sonicator or French Press Centrifuge, medium speed (30-70Kg) (e.g. J-20, Avanti J-25), Appropriate centrifugation tubes Chromatographic system comprising of, as a minimum, pump and fraction collector (normally system also includes UV monitor and recorder) Gradient mixer Spare pump or second chromatographic system Chromatographic columns packed with different types of matrixes Spectrophotometer VIS (340-800nm) or UV/VIS (190-800nm) Bio-Rad Protein Assay Reagent and Plastic cuvettes 1.6 ml Stock solutions of buffer components Concentrators (VivaSpin 20 and 6, microcone) and low speed refrigerated centrifuge Filter Device such as Filter Holder or Stericup Filter Unit. Apparatus and solutions for SDS-PAGE Refractometer (a pocket one for sucrose is fine) Pippetors, tips, eppendorf tubes, tubes for fraction collector 6 Preparation of the stock solutions It is very convenient to have stock solutions of the main salts and buffers used during purification. Use of the correctly and carefully prepared stock solutions can noticeably improve accuracy and reproducibility of the purification procedures. By having stock solutions you can prepare any buffer for protein purification in seconds. Simply pour required volumes of stock solutions in to a Duran bottle and adjust the volume with ultra pure water to the top mark on the bottle. For example, to prepare buffer 50mM tris pH 8, 100 mM NaCl, 2mM EDTA: pour 50 ml of 1M tris, 20 ml of 5M NaCl and 10 ml of 0.2M EDTA in to a 1 litre bottle and add ultra pure water to 1 litre mark. The most useful stock solutions in protein purification are: 5M NaCl, 4 M (NH4)2SO4, 1M Tris-HCl, pH 8.0. Other stock solutions: You may need EDTA stock solution. Due to low solubility of this compound concentration of stock solution is 0.2M - 0.25M. You should adjust pH of the solution to 6.5 - 7 with 5M NaOH before you adjust it to required volume. You may also need: 1M HEPES-NaOH pH 7.0 or 7.5 1M MES-NaOH pH 6.0 or 6.5 Those buffers belong to so called "Good’s buffers". Unlike tris buffer they are not temperature sensitive so there is no need to control temperature during their preparation (see Protocols section). It is best to keep stocks of buffers in the fridge. Preparation of 1M Tris-HCl pH 8.0 To prepare this solution you need to adjust the pH to 8.0 with concentrated HCl. The problem here is that tris buffer is temperature sensitive. Raising the temperature by three degrees makes pH fall by 0.1. A lot of heat is produced during titration of 1M Tris solution with the concentrated acid so to make reproducible stock solution you need to titrate it on a water-ice bath with a thermometer placed in the solution. The standard temperature is 20oC, so you should make pH 8.0 at 20oC. Titration should be performed before you adjust volume. Because a significant volume of concentrated HCl is required to adjust pH, tris powder should be dissolved in 60-70% of the final volume of solution. Please note that dilution leads to a decrease in buffer pH. Normally we use 50 mM solution for purification (made from the 1M stock) and the actual pH for tris, MES or HEPES buffer is about 0.3 pH units lower then in 1M buffer. For 50mM tris buffer prepared from 1M stock pH 8.0 actual pH at 20oC is about 7.7. However if we use it at 4oC the pH is at about 8. When preparing stock solutions the sequence of procedure is as follow: Weighing powder – dissolving it in water – titration (for buffers and EDTA only) – adjusting volume - filtration (see Protocols section for details) 7 Preparation of the chromatographic columns For the basic purification protocol you need a set of 3 columns packed with DEAE-Sepharose Fast Flow (weak anion exchanger) Phenyl-Toyopearl 630S (hydrophobic matrix) Superdex-200 (gel filtration) The additional set includes: Q-Sepharose Fast Flow (strong anion exchanger) CM-Sepharose fast flow (weak cation exchanger) DEAE-Toyopearl 650S ( weak anion exchanger) SP-Toyopearl 650S (strong cation exchanger) Butyl-Toyopearl 650S (strong hydrophobic matrix) Ethyl-Toyopearl 650S (weak hydrophobic matrix) Heparin-Sepharose Fast Flow (pseudo-affinity) Sets of HPLC (FPLC) columns (if you possess the appropriate system) You may need other types of matrices, such as Hydroxylapatite, Dye matrix, etc. You can buy ready-made columns from GE Healthcare (formerly Amersham) or Bio-Rad. For our scale we need columns 10-30 ml in size. We prefer to buy empty columns and pack them in the lab as this is cheaper and makes them re-packable. In the lab we mainly use Amersham –Pharmacia (GE Healthcare) empty columns C10/10, C10/20, C16/20 and XK16/20 with adapters. To prepare any column (except the gel filtration one): Check that all parts of column are in place (according to specification) Fix empty column on a stand in vertical position Fill bottom outlet with a few ml of ultra pure water Prepare slurry of any matrix taking 1 part sediment matrix and two parts water, warm to room temperature if matrix has been kept in the fridge. De-gassing under vacuum is useful but not overly crucial. Having the bottom outlet of column open, pour the slurry into the column, adding the slurry portion by portion, the matrix should settle at the bottom of the column and the water should run through. When all matrix is in the column continue to add water until matrix has settled, then close the bottom outlet of the column.