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2 Main headache : Aggregation!! But where can aggregation start? Expression Cell lysis and sample preparation Purification Storage
3 Protein aggregates structure models Precipitated inclusion bodies
4 Mechanisms of Protein Aggregation Philo J. Current Pharmac. Biotech. (2009), 10, Nucleation!! Continuos Progretion
5 How can we identify aggregates? Insoluble aggregates 100μm 1mm or more: Can be detected by eye 1 μm - ~100μm: Can be detected by absorbance (340, 490 or 600nm) = Removed by filtration or centrifugation Soluble aggregates (dimers-oligomers) mau 10nm - ~ 1 μm. Can be detected by GF, 150 light scattering, native gels = Removed by chromatography (mainly GF) & ultrafiltration ml
6 Aggregation = several types of interactions Exposure of hydrophobic surfaces Denaturation or small perturbations Covalent aggregates Incorrect intermolecular disulfide bridges, bityrosine Reversible protein aggregation Weak non-covalent interactions. (Due to changes in solution conditions: protein concentration / change in ph / conductivity / polarity)
7 Soluble Insoluble Covalent Non-covalent Reversible Irreversible Folded Unfolded
8 The Challenge: Intrinsically Disordered Proteins (IDP) Should get even MORE special treatment
9 Intrinsically disordered protein and unstructured domains Also known as: Natively unfolded proteins Protein-protein & domain-domain interactions Structural flexibility that enables binding to a large number of partner ligands Implications for regulation, activity and function
10 Commonly used affinity Tags and fusion proteins Tag Size (aa) Resin His 5-15 Ni/Co Streptag II 8 StrepTactin Chitin-binding domain 52 Chitin SUMO ~100 Ni/Co 6His-Thioredoxin ~115 Ni/Co GST ~200 Glutathion MBP ~400 Amylose 6His-NusA ~500 Ni/Co
11 Lipoyl domain fusion tag (HLT) His Lipoyl domain TEV Target Dr.Mark Allen: MRC-CPE, Cambridge, England Lipoyl domain: N-terminal part of the lipoyl domain in B. Stearothermophilus E2p Extremely soluble and often prevent proteins to form IB Extremely resistant to proteases Known NMR structure Difficult to crystallize 109 amino acids MW: Da Over-expression of IDP NMR studies Theoretical pi: 4.53
12 Additives and Stabilizers Reduce protein-protein interaction Keep protein at lower concentration High salt concentration Protect correct disulfide bonds Chaotropes as Urea or aggregation suppressors as Arginine Protect exposed hydrophobic sites with PEG, citrate, and non-ionic detergents (bellow the CMC) More Additives to stabilize the native state: Glycerol, Sucrose, Sorbitol, Trehalose, Glycine, etc
13 Agents that may promote protein solubility Sarah E. Bondos and Alicia Bicknell Analytical Biochemistry (2003) 316 (2) pg
14 Should TEV protease be cleavage before or after GF separation of soluble aggregates? DefrozeniASSPAnkSH3Superose12 Anal002:1_UV1_280nm DefrozeniASSPAnkSH3Superose12 Anal002:1_UV2_260nm DefrozeniASSPAnkSH3Superose12 Anal002:1_UV3_220nm Load immediately after Ni affinity HLTiASPPAnkSH3Superose12Prep001:1_UV1_280nm HLTiASPPAnkSH3Superose12Prep001:1_Inject HLTiASPPAnkSH3Superose12PrepDelay002:1_UV1_280nm HLTiASPPAnkSH3Superose12Prep001:1_Logbook mau DefrozeniASSPAnkSH3Superose12 Anal002:1_Fractions DefrozeniASSPAnkSH3Superose12 Anal002:1_Inject DefrozeniASSPAnkSH3Superose12 Anal002:1_Logbook mau Load 12h after Ni affinity Load inmediately after affinity 20.0 GF of Defrozen iassp Ank SH Load 12hours after affinity Waste ml 20 0 After TEV cleavage, monomers are stable ml Only monomer undergoes TEV protease cleavage (aggregates can not be cleaved) Try to eliminate your soluble aggregates as soon as possible!!!
15 Possible Strategy to avoid further aggregation Ni column Ni column TEV protease + dialysis GF immediately Negative Ni column TEV protease Concentration Negative Ni column GF and/or Ion exchange REVIEW Nature Methods - 5, (2008) Protein production and purification Structural Genomics Consortium
16 Thanks!! Dr. Tsafi Danieli (Expression Facility Hebrew University) Collaborators: Prof. Assaf Friedler (Hebrew Univ.) Michal Maes Hadar Rafaely Anat Iosub Chen Katz Shahar Rotem Tzvika Hayouka Ronen Gabizon Shahar Sukeinik Academic Committee Wolfson Centre for Applied Structural Biology Prof. Roger Kornberg - Chairperson Prof. Oded Livnah - Director Prof. Etana Padan Prof. Shy Arkin
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