GENERAL STRATEGIES

By G. KARAK

CONTENTS 1 GENERAL STRATEGIES 2 PREPARATION OF EXTRACTS FROM ANIMAL TISSUES 3 PROTEIN EXTRACTION FROM PLANT TISSUES 4 EXTRACTION OF RECOMBINANT PROTEIN FROM BACTERIA 5 PROTEIN EXTRACTION FROM FUNGI 6 SUBECLLULAR FRACTIONATION OF ANIMAL TISSUES 7 SUBCELLULAR FRACTIONATION OF PLANT TISSUES ISOLATION OF PLASTIDS AND MITOCHONDRIA 8 THE EXTRACTION OF ENZYMES FROM PLANT TISSUES RICH IN PHENOLIC COMPOUNDS 9 AVOIDANCE OF PROTEOLYSIS IN EXTRACTS 10 CONCENTRATION OF EXTRACTS 11 MAKING AND CHANGING BUFFERS 12 PURIFICATION AND CONCENTRATION BY ULTRAFILTRATION 13 BULK PURIFICATION BY FRACTIONAL PRECIPITATION 14 ION-EXCHANGE CHROMATOGRAPHY 15 HYDROPHOBIC INTERACTION CHROMATOGRAPHY 16 AFFINITY CHROMATOGRAPHY 17 DYE-LIGAND AFFINITY CHROMATOGRAPHY 18 LECTIN AFFINITY CHROMATOGRAPHY 19 IMMUNOAFFINITY CHROMATOGRAPHY 20 IMMOBILIZED METAL-ION AFFINITY CHROMATOGRAPHY 21 CHROMATOGRAPHY ON HYDROXYAPATITE 22 THIOPHILIC AFFINITY CHROMATOGRAPHY AND RELATED METHODS 23 AFFINITY PRECIPITATION METHODS 24 ISOELECTRIC FOCUSING 25 CHROMATOFOCUSING 26 SIZE-EXCLUSION CHROMATOGRAPHY 27 FAST PROTEIN LIQUID CHROMATOGRAPHY 28 REVERSED-PHASE CHROMATOGRAPHY OF PROTEINS 29 EXTRACTION OF MEMBRANE PROTEINS 30 REMOVAL OF DETERGENT FROM PROTEIN FRACTIONS 31 PURIFICATION OF MEMBRANE PROTEINS 32 LYOPHILIZATION OF PROTEINS 33 STORAGE OF PURE PROTEINS 34 ELECTROELUTION OF PROTEINS FROM POLYACRYLAMIDE GELS 35 ELECTROBLOTTING OF PROTEINS FROM POLYACRYLAMIDE GELS 36 TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS FOR PROTEOME ANALYSES 37 MICROSCALE SOLUTION ISOELECTROFOCUSING A SAMPLE PREFRACTIONATION METHOD FOR COMPREHENSIVE PROTEOME ANALYSIS 38 PRACTICAL COLUMN CHROMATOGRAPHY 39 DETECTION METHODS 40 PEPTIDE PROTEOMICS 41 MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY OF PROTEINS 42 MASS SPECTROMETRY 43 PURIFICATION OF THERAPEUTIC PROTEINS 44 PURIFICATION PROCESS SCALE-UP GENERAL STRATEGIES 1 DE?NING THE PROBLEM THE CHAPTERS THAT FOLLOW IN THIS VOLUME GIVE DETAILED INSTRUCTIONS ON HOW TO USE THE VARIOUS METHODS THAT ARE AVAILABLE FOR PURI?CATION OF PROTEINS THE QUESTION ARISES, HOW- EVER, OF WHICH OF THESE METHODS TO USE AND IN WHICH ORDER TO USE THEM TO ACHIEVE PU- RI?CATION IN ANY PARTICULAR CASE; THAT IS, THE PURI?CATION PROBLEM MUST BE CLEARLY DE?NED WHAT FOLLOWS OUTLINES THE SORTS OF QUESTION THAT NEED TO BE ASKED AS PART OF THAT DE?NI- TION AND HOW THE ANSWERS AFFECT THE APPROACH THAT MIGHT BE TAKEN TO DEVELOPING A PU- RI?CATION SCHEDULE IT SHOULD BE NOTED HERE THAT THE DISCUSSION CONCENTRATES MAINLY ON LABORATORY-SCALE ISOLATION OF PROTEINS SPECIAL CASES OF PURI?CATION OF THERAPEUTIC PRO- TEINS AND ISOLATION AT INDUSTRIAL SCALE ARE COVERED IN CHAPTERS 43 AND 44 (1–5) 1 1 HOW MUCH DO I NEED? THE ANSWER TO THIS QUESTION DEPENDS ON THE PURPOSE FOR WHICH THE PROTEIN IS REQUIRED FOR EXAMPLE, TO CARRY OUT A FULL CHEMICAL AND PHYSICAL ANALYSIS OF A PROTEIN MAY REQUIRE SEVERAL HUNDREDS OF MILLIGRAMS OF PURI?ED MATERIAL, WHEREAS A KINETIC ANALYSIS OF THE RE- ACTION CATALYZED BY AN ENZYME COULD PERHAPS BE DONE WITH A FEW MILLIGRAMS AND LESS THAN 1 MG WOULD BE REQUIRED TO RAISE A POLYCLONAL ANTIBODY AT THE EXTREME END OF THE SCALE, IF THE OBJECTIVE IS TO OBTAIN LIMITED SEQUENCE INFORMATION FROM THE N-TERMINUS OF A PROTEIN AS A PRELIMINARY TO THE DESIGN OF AN OLIGONUCLEOTIDE PROBE FOR CLONE SCREENING, THEN USING MODERN MICROSEQUENCING TECHNIQUES, A FEW MICROGRAMS WILL BE SUFFICIENT IN THE ?ELD OF PROTEOMICS, PREVIOUSLY ANALYTICAL TECHNIQUES HAVE BECOME PREPARATIVE WITH MASS SPEC- TROMETRY COMMONPLACE FOR SENSITIVE PROTEIN CHARACTERIZATION FROM SPOTS ON GELS CHAP- TERS 36 AND 40–42 DESCRIBE THESE METHODOLOGIES THESE DIFFERENT REQUIREMENTS FOR QUAN- TITY MAY WELL DICTATE THE SOURCE OF THE PROTEIN CHOSEN (SEE SUBHEADING 1 4 ) AND WILL CERTAINLY IN?UENCE THE APPROACH TO PURI?CATION PURI?CATION OF LARGE QUANTITIES OF PROTEIN REQUIRES USE OF TECHNIQUES, AT LEAST IN THE EARLY STAGES, THAT HAVE A HIGH CAPACITY BUT LOW RESOLVING POWER, SUCH AS FRACTIONAL PRECIPITATION WITH SALT OR ORGANIC SOLVENTS (SEE CHAP- TER 13) PROCESS ONLY WHEN THE VOLUME AND PROTEIN CONTENT OF THE EXTRACT HAS BEEN REDUCED TO MANAGEABLE LEVELS, METHODS OF MEDIUM RESOLUTION AND CAPACITY, SUCH AS ION-EXCHANGE CHROMATOGRAPHY (SEE CHAPTER 14) CAN BE USED LEADING ON, IF NECESSARY, TO HIGH-RESOLUTION