Preparative Protein Chemistry

Transcription

1 Biochemistry 412 Preparative Protein Chemistry 19 February 2008

2 The Three Eras of Protein Purification 1. The Classical (Pre-Recombinant DNA) Era (pre-1978) - Proteins purified from natural sources only 2. The Recombinant DNA (Pre-Genomic) Era (~ late 90s) - Proteins purified from natural sources* and recombinant cells 3. The Genomic and Post-Genomic Era(s) (late 90s - present) - Nearly all protein purification from recombinant cells, since most information about proteins is now in sequence (and other) databases *Note: purification of proteins from natural sources was often motivated by the need to get protein for amino acid sequencing so that oligonucleotide probes could be designed and used to clone the gene encoding the protein.

3 Purification schemes vary, depending on the source of the protein and its intrinsic biophysical properties... some flow-charts for typical schemes follow.

4 Purification Scheme for Proteins from their Natural Source

5 Purification Scheme for Soluble Recombinant Proteins

6 Purification Scheme for Insoluble Recombinant Proteins

7 Purification Scheme for Membrane-Associated Proteins

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10 Proteins are Amphiphilic Macro-Ions Positively-charged basic residues (K, R, & H) Hydrophobic patch Macromolecular dimensions: ca. 40 Å Ligand binding pocket (active site) Negatively-charged acidic residues (E & D) >>> The charged groups, hydrophobic regions, size, and solvation affect the biophysical properties of the protein and largely determine its purification behavior.

11 Chromatography Sample containing proteins or peptides Liquid flow Liquid flow Separation according to: -molecular weight/ size -charge -hydrophobicity -affinity Time :

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13 Three Phase Strategy: An aid in developing the purification scheme Purity Achieve final purity. Remove trace impurities, structural variants, aggregates, viruses, etc. Remove bulk impurities Polishing Isolate product, concentrate, stabilize Intermediate purification Capture Step 13

14 Sample Preparation General considerations: Select extraction procedure according to source and location of protein Use gentle procedures to minimize acidification and release of proteolytic enzymes Work quickly at sub-ambient temperatures Use buffer to maintain ph, ionic strength Goal: To stabilize sample 14

15 Always Limit the Number of Steps Maximize the Yield at Each Step Yield (%) % / step Number of steps 90% / step 85% / step 80% / step 75% / step 20% overall yield! 15

16 Gel Filtration

17 Gel Filtration (GF) Chromatography

18 The principle of gel filtration -- excluded volume [Note: gel filtration chromatography is also sometimes called size exclusion chromatography ] V o = void volume V t = bed volume V e = elution volume V i = V t - V o

19 Principles of gel chromatography (con d)

20 Gel Filtration Elution Volumes as a Function of Molecular Weight Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

21 Ion Exchange Chromatography

22 Ion Exchange (IEX) Chromatography

23 Ion Exchange Chromatography (con d) Cation exchange column Anion exchange column

24 Some other popular chromatographic methods: Hydrophobic interaction chromatography Affinity chromatography Reverse phase chromatography

25 Hydrophobic Interaction Chromatography (HIC)

26 Affinity Chromatography

27 Reversed Phase Chromatography (RPC) (elution with organic solvents)

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29 Linking Chromatography Techniques It is good to design your purification to have the start conditions of each step match the end conditions of the previous step in order to avoid intervening buffer exchange steps, which add to your losses. Start conditions Technique End conditions Small sample volume GF Diluted sample Buffer change (if required) Low ionic strength IEX High ionic strength or ph change High ionic strength HIC Low ionic strength Specific binding conditions AC Specific elution conditions Note: after IEX, HIC, or AC, sample is concentrated, too. 29

30 In addition, there are non-chromatographic protein purification techniques, e. g.: Ammonium sulfate precipitation Sedimentation (rare) Recombinant gene product over-expression Inclusion body prep (see earlier slide) Detergent extraction Heat treatment (especially for recombinant thermophile proteins expressed in E. coli) Etc.