Department of Molecular Biology & Biotechnology Protocols and tips in protein purification or How to purify protein in one day Second edition 2018 2 Contents I. Introduction 7 II. General sequence of protein purification procedures 9 Preparation of equipment and reagents 9 Preparation and use of stock solutions 10 Chromatography system 11 Preparation of chromatographic columns 13 Preparation of crude extract (cell free extract or soluble proteins fraction) 17 Pre chromatographic steps 18 Chromatographic steps 18 Sequence of operations during IEC and HIC 18 Ion exchange chromatography (IEC) 19 Hydrophobic interaction chromatography (HIC) 21 Gel filtration (SEC) 22 Affinity chromatography 24 Purification of His-tagged proteins 25 Purification of GST-tagged proteins 26 Purification of MBP-tagged proteins 26 Low affinity chromatography 26 III. “Common sense” strategy in protein purification 27 General principles and tips in “common sense” strategy 27 Algorithm for development of purification protocol for soluble over expressed protein 29 Brief scheme of purification of soluble protein 36 Timing for refined purification protocol of soluble over -expressed protein 37 DNA-binding proteins 38 IV. Protocols 41 1. Preparation of the stock solutions 41 2. Quick and effective cell disruption and preparation of the cell free extract 42 3. Protamin sulphate (PS) treatment 43 4. Analytical ammonium sulphate cut (AM cut) 43 5. Preparative ammonium sulphate cut 43 6. Precipitation of proteins by ammonium sulphate 44 7. Recovery of protein from the ammonium sulphate precipitate 44 8. Analysis of solubility of expression 45 9. Analysis of expression for low expressed His tagged protein 46 10. Bio-Rad protein assay Sveta’s easy protocol 47 11. Protocol for accurate determination of concentration of pure protein 47 12. Calibration plot for gel filtration column 48 13. Ni-chelating cartridges : cleaning and recharging 48 14. SDS PAGE Sveta’s easy protocol 49 15. DTNB reaction 50 16. Refolding protocol 50 3 V. Charts and Tables 51 Ammonium sulphate –Sugar refractometer 53 NaCl-Sugar refractometer 54 Calibration plot for Hi-load Superdex 200 column 55 Calibration plot at various salt concentrations for Superdex200 GL column 56 Ammonium sulphate table 57 Properties of amino acids 58 VI. Appendix 1 59 1.Determination of protein concentration 59 2.How to concentrate proteins 64 3.How to store proteins 67 4.How to estimate purity of a protein preparation 68 VII. Appendix 2 69 1. Prepare your solutions right! 69 2. Gel filtration 70 Separation by molecule’s size 70 Calibration plot 71 Main reasons for “abnormal” mobility of some proteins during 72 gel filtration 3. Time saving tips 73 4. Golden rules in protein purification 74 VIII. Show cases 75 1. Two step purification protocol and a lesson to learn (BPSL 1549 (BLF1)) 75 2. It is easy with the coloured proteins! 79 Single-domain haemoglobin cgb from Campylobacter jejuni 79 Flavohemoprotein HMP from Escherichia coli 81 3. Almost typical three step purification (Glutamate Dehydrogenase from 85 Clostridium symbiosum) 4. Monitoring purification by enzyme activity (Glutamate Dehydrogenase 88 from Clostridium symbiosum) 5. Power and failure of dye pseudo affinity chromatography 91 Phenylalanine Dehydrogenase from Nocardia sp. 91 Glutamate Dehydrogenase from Clostridium symbiosum 97 6. DNA binding proteins 102 Typical Heparin protocol: RuvC from phage bIL67 102 Playing with solubility: LrpC from Bacillus subtilis 104 Heparin and solubility: FrmR from Escherichia coli 105 Real power of Heparin: Human FEN 108 In need of nuclease treatment: SfsA 111 7. Tagged proteins 116 Proper use of a His-tag: Human protein PARP1 expressed in E.coli 116 Removable tag: SAG19 118 Cleaver use of tag: TssA 121 8. Special cases 124 Phosphoglycerate Kinase (PGK) from Escherichia coli 124 Low affinity chromatography: BPSL1958 127 BacM from Myxococcus Xanthus 130 134 Perfect purity: IGPD from Arabidopsis thaliana 4 Abbreviations AA acrylamide APMSF 4-Amidinobenzylsulfonyl fluoride hydrochloride AS ammonium sulphate CBV column bed volume CE crude extract CFE cell free extract CM carboxymethyl DEAE diethylaminoethyl DTNB Dithionitrobenzole 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 PBS phosphate saline buffer PS protamine sulphate PSA ammonium persulphate PAGE polyacrylamide gel electrophoresis Q quaternary ammonium S methyl sulphonate SDS sodium dodecyl sulphate SEC size exclusion chromatography or gel filtration SP sulphopropyl TEMED N,N,N',N' tetramethylethylenediamine TP target protein UV ultraviolet Contact details Dr. Svetlana Sedelnikova Department of Molecular Biology and Biotechnology The University of Sheffield Sheffield S10 2TN Please, contact me by e-mail [email protected] All your comments and questions are very welcome 5 With greatest respect and gratitude to my teachers Prof. Alexander Sergeevich Spirin and Prof. Marina Borisovna Garber and people of Institute of Protein Research, Pushchino and also to my employers Prof. David W. Rice and Department of Molecular Biology and Biotechnology of the University of Sheffield 6 I. Introduction Why do we need to purify protein in one day? After 25 years and 360 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 combination of ion-exchange, hydrophobic and size-exclusion 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 defunct. This is not the case. The most justified use of tags is for difficult expressions, such as expression of eukaryotic proteins in E.coli, expression of the domains of the proteins, toxic proteins, in the cases when tags help polypeptide chains to fold, in the cases when correct fold occurs only occasionally and so expression yield is hardly detectable. In such the cases tagged proteins often cannot be purified to the desirable purity by using just one tag affinity chromatography, so more purification steps are required anyway. We also have to remember that tag as well as being often useful in some cases it may be an obstacle that prevents correct proteins folding. I consider the recent trend to do all expressions in a tagged form is misleading . For most of enzymes and similar cytoplasmic proteins the chance to be successfully expressed is greater in the absence of the tag. My recent experience also revealed that if the fusion protein or the tag has to be removed from the target protein you may face a great problem either with protease underperforming or unspecific proteolysis leading to damage of the target protein. In this second edition I have included my recent experience with purification of the tagged proteins. 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. 7 SDS-PAGE equipment 8 II. General sequence of protein purification procedures 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 or 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 (optional) Chromatographic columns packed with different types of matrixes Spectrophotometer VIS (340-800nm) or better UV/VIS (190-800nm) Bio-Rad Protein Assay Reagent Plastic cuvettes (1.6 ml) Stock solutions of salts and buffer components Concentrators (VivaSpin 20, 6 and 0.5ml) Low speed refrigerated centrifuge Filtration Device (such as Filter Holder or Stericup Filter Unit) Apparatus and solutions for SDS-PAGE (ideally precast gels) Refractometer (a pocket one for sucrose is fine) Pippetors and tips Eppendorf tubes Tubes for fraction collector 9 Preparation and use of stock solutions It is very convenient to have stock solutions of the main salts and buffers used during purification. Correctly and carefully prepared stock solutions can noticeably improve accuracy and reproducibility of the purification procedures. Stock solution preparation sequence When preparing stock solutions, follow the sequence below: Weigh powder → dissolve in about 80% of final required volume of ultra pure water→ adjust pH (for buffers and EDTA only) → adjust to final volume with ultra pure water → filter (also see poster in Apendix2) The most useful stock solutions in protein purification are: 5M NaCl 4M (NH4)2SO4 1M Tris-HCl, pH 8.0 o To prepare this solution you need to adjust the pH to 8.0 with concentrated HCl, this titration generates heat. However Tris buffer is temperature sensitive: raising the temperature by 3°C decreases pH by 0.1. To make a reproducible stock solution it is best to adjust pH on a water-ice bath with a thermometer placed in the solution, you are aiming for pH 8.0 at 20oC. A significant volume of concentrated HCl is required to adjust pH, therefore Tris powder should be dissolved in 60-70% of the final volume, pH adjusted to 8.0, then water added to final volume. Other stock solutions: 0.2M - 0.25M EDTA.