Introduction to Protein Purification

Transcription

1 Introduction to Protein Purification 1

2 Day 1) Introduction to Protein Purification. Input for Purification Protocol Development - Guidelines for Protein Purification Day 2) Sample Preparation before Chromatography - Cell disruption considerations. Centrifugation. Filtration - Chromatography: General Considerations Day 3 & 4) Basic principles on protein expression. Advantages and disadvantages of different expression systems Day 5) Ion Exchange: Main stages in chromatography. Basis for selectivity. Operational considerations. Determination of start conditions. Parameters for optimization. Examples. Day 6) Hydrophobic Exchange Chromatography: Basis for selectivity. Determination of start conditions. Operational considerations. Parameters for optimization. Examples. Other Purification Techniques: Hydroxyapatite, Reverse Phase, Multimode Resins (HIC + IEX & GF + IEX) Day 7) Gel Filtration Chromatography and desalting: Main stages in chromatography. Basis for selectivity. Operational considerations. Determination of start conditions. Parameters for optimization. Examples. SEC-MALS 2

3 Day 8) Affinity chromatography: Main stages. Advantages and Disadvantages. Type of Affinities. Designing and preparing an affinity gel. Day 9) Affinity chromatography: Genetically engineered Tags, recombinant proteins. Cleavage Sites. Parameters for optimization. Examples Day 10) Selection and combination of purification techniques. Examples. Storage. Day 11) How to solve aggregation problems. Mechanism of aggregation. Methods for screening solubility. Considerations when selecting buffer components. Additives that increase yield and prevent aggregation and/or stabilize proteins Day 12) Refolding: Major steps. Refolding methods. Screening. Strategies and examples. Day 13) Principles on protein characterization. Major requirements for purification of proteins for structural studies: crystallography, NMR, others.??) The challenge of Membrane protein purification. Main features, use and removal of detergents. Day 14/15 : Students seminars (~20min each student) 3

4 PURIFICATION STRATEGY I The Protein Purification Facility Protein Purification: aims, purity Pipeline for Purification Overview: separation techniques 4

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6 Educational and Research Center for Protein Expression & Purification Users: Hebrew University Academic Institutes Biotech/Pharma industry Mode of Operation 1. Expression and purification services 2. Individual training and supervision of projects 3. Annual workshop and course 4. Repository of specialized reagents, vectors and cells 6

7 Large and small scale projects Large-scale high purity Structure analysis Animal studies Vaccines Ab production Small scale Enzymatic assays Cellular systems Analysis Protein-Protein Interaction 7

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15 Protein purification is diverse Basic purification is the same strategy change Several types of target protein Different objectives Several scales Various equipment Recombinant Functional studies From natural source Structural studies Affinity tagged Use as biochemical Non-tagged reagent Membrane protein Therapy Soluble protein Protein complex 15

16 Applications of Protein Purification In vitro Activity assays Antibody development / production Protein:protein interaction assays Cell-based activity assays Ligand-binding assays Mass-spectrometric analysis Structural analysis In vivo activity assay For each application you need: different quantities different protein purity start material is different different strategy Post-translational modification tests N-terminal sequencing Electromobility shift assay (band shift) DNA footprinting Protein cross-linking studies Vaccine development/production Probes for protein arrays/chips Expression library screening Other Each purification project must be adapted to your start material and your final needs Don t waste clear thinking on dirty or not healthy proteins!!!!

17 Some Situations Demand Higher Purity Laboratory Scale Raising antibodies or Biochemical studies: > 90-95% Structural (X-Ray, NMR) or Functional Studies > 95% Production Scale Authority regulated e.g. FDA Impurities to check: DNA Endotoxins Host cell proteins Modified forms Dimers Misfolded forms Need economical and robust proceses Validation is important Characterization: > 99% 17

18 Protein Purification Strategy Simple Purification FUSION PROTEIN ONE STEP Affinity 70-95% Purity (not always) For higher purity Multi-Step Purification EXPRESSION Capture Intermediate Purification Polishing NON-FUSION PROTEIN 18

19 Protein Purification - Aims Sufficient purity and quantity Maintained biological activity Economical use of reagents/apparatus Goal to Success: Selection or optimization of the best source Selection and optimization of the most appropriate technique for each step 19

20 PURIFICATION STRATEGY I The Protein Purification Facility Protein Purification: aims, purity Pipeline for Purification Overview: separation techniques 20

21 Protein Production Pipeline Target Selection Target Optimization Gene Cloning Selection of Expression Vector Selection of Expression Host Expression Analysis Solubility Analysis Scaling Up Fermentation Purification Purification Optimization Characterization Pharmaceutical Studies Concentration & Storage Structural Studies: Crystallization NMR- etc Biochemical Studies

22 Sequence of Events - Protein Purification Buffer choice Cell disruption and clarification method Low Scale Purification (Test tube, batch purification) First protein characterization Medium Scale Purification Capture: Affinity columns Intermediate Purification: Ion (or Hydrophobic) exchange chromatography Final polishing : Size-exclusion chromatography Concentration Storage Final Protein characterization Scale-Up 22

23 Spin columns with filter Batch binding Gravity Desalting Columns FPLC columns 23

24 The principles of chromatography techniques Affinity Chromatography (AC) Ion exchange Chromatography (IEX) Hydrophobic interaction Chromatography (HIC) Size exclusion Chromatography (SEC) Bind elute principle Requires specific elution conditions Concentrating effect Diffusion no binding Any elution conditions Diluting effect

25 Overview: separation techniques Technique Parameter Based on for separation Gel filtration Size/Shape MW, Shape, and oligomeric state of the molecule Ion exchange/ Hydroxyapatite/ Charge interaction Asp, Glu, Lys, Arg, His Chromatofocusing Hydrophobic/ Hydrophobic sites Trp, Phe, Ile, Leu, Tyr, Pro Reversed phase interaction Met, Val, Ala Affinity Biological function eg: antibody antigen Metal chelate Affinity for metals poly His Covalent Covalent interaction Uses SH groups (Cys) Multimodal Mixture Hydrophobic + Ionic Interaction 25

26 Linking Chromatography Techniques into a Purification Protocol - General Rules Combine techniques with complementary selectivity's (e.g. IEX, HIC and GF) Minimize sample handling between purification steps (like concentration, dialysis, long assays, nonworking days, etc.) 26

27 CAPTURE - Anion Exchange BeUnWa M R M R RAS 500ml culture after lysis and sonication. Q-Sepharose FF 100x16mm (~20ml) in 25mM TrisHCl ph8.0 buffer + additives. WASH: 7cv 70mM NaCl ELUTION: gradient 10cv mM NaCl + 5cv 0.2-1M NaCl 27

28 POLISH - Size Exclusion R MW bef aft ultr MW R RAS 60 OD280nm (8ml) RAS after Q-Seph. - Load Sephacryl S x26cm - Flow 2.5ml/min - Pool RAS after GF: 36.8 OD280nm 28