Reading instructions to partitioning in aqueous two-phase systems

Transcription

1 Reading instructions to partitioning in aqueous two-phase systems Copy from Separation Processes in Biotechnology (ed. Asenjo) Aqueous two-phase separations, Albertsson, Johansson och Tjerneld OH-pictures Aqueous two-phase systems - Examples of phase systems - Phase diagrams - Partitioning of proteins - Partitioning of solutes and particles in phase systems - Enhancement of protein partitioning - Application examples 1

2 Aqueous two-phase systems - high water content, up to 95% - compatible with biological systems - preparative work - Processes operations - Analytical tool Aqueous two-phase systems System type thermo-separating polymer/h 2 0 thermo-separating polymer /salt/h 2 O polymer 1/polymer 2/H 2 O Example of system Ethyleneoxide (EO)- Proylene oxide (PO) copolymer/h 2 0 Polyethylene glycol (PEG)/K-phosphate/H 2 O PEG/Dextran/H 2 O detergent (micells)/h 2 0 C 12 EO 5 /H 2 0 detergent/polymer/h 2 O C 12 EO 5 /dextran/h 2 0 2

3 Poly(etylenglykol) [PEG] [Poly(etylenoxid)] H[OCH 2 CH 2 ] n OH Linear synthetic polymer Molecular weights eg. 1000g/mol, 1500g/mol, 4000g/mol, 6000g/mol, g/mol, g/mol) Water soluble (suggested: 2 molecules H 2 O/ether oxygen) Thermo separating polymer Salt as phase forming component SO 4 2- > PO 4 2- > F - > Cl - > NO 3 - ClO 3 - > I - > SCN - Li + > Na + > K + >NH 4 + > Mg 2+ Decreasing capability to raise surface tension of water Decreasing effectiveness of salting out proteins Decreasing effectiveness of clouding of polymers 3

4 Dextran Leuconostoc sp. 95% α 1-6 and 5% α 1-3 glycosidic bonds Work up of crude dextran (partial hydrolysis, fractionation) gives preparations of different MW-range (eg g/mol, , ) Aqueous two-phase formation Polymer 1 / Polymer 2 / H 2 O G mix = H mix - T S mix For polymers Hmix dominates over S mix G mix H mix Hmix determined by the interaction between polymer 1 - polymer 2 Interaction attractive repulsive "neutral"!h mix < 0 > 0 " 0!G mix < 0 > 0 " 0 Result complex coacervation phase formation single phase 4

5 Phase diagram Centrifugation top phase volume, V t bottom phase volume, V b Protein Polymer 1 Polymer 2 or salt a Two phase region b One phase region Polymer 2 or salt, % (w/w) Critical point Tie-lines Binodial c Partition coefficient K = C t /C b = e λm/kt Phase volume ratio R = V t /V b bc/ab Yield in top phase Y t (%) = 100/(1 + 1/KR) Partitioning of proteins effects of polymer size effects of protein size effects of type salt ion, charge, ph effects of hydrophobicity log K = log K polymer size + log K protein size + log K charge + log K hydropho 5

6 Charge and salt effect on partitioning Polymer 1/Polymer 2/H 2 O + Low conc. of salt ( 100 mm) ln K salt (ion+ ion-) ln K - /K + electrostatic Z p ln K p, salt potential (U t - U b ) < 0 < 0 < 0 > ln K p, 0 > 0 < ln K p, 0! 0! 0! ln K p, 0! ln K p, 0 > 0 > 0 < 0 < ln K p, 0 > 0 > ln K p, 0 U t - U b = (RT/(Z + + Z - )F) (ln K - /K + ) ln K p, salt = ln K p, 0 + (FZ p /RT)(U t - U b ) Salt and charge effect on protein partitioning 7% (w/w) PEG 8000/ 7% (w/w) dextran 500 aqueous two-phase systerm U t U b = (interfacial) electrostatic potential Salt ln K - /K + U t U b KSCN >0 >0 KCl >0 >0 K 2 SO 4 <0 <0 U t - U b = (RT/(Z + + Z - )F) (ln K - /K + ) ln K p, salt = ln K p, 0 + (FZ p /RT)(U t - U b ) ribonucleas 6

7 Effect of polymer size on protein partitioning PEG / dextran 500 aqueous two-phase systerm Pullulanase partitioning Hustetedt al, Biotechnol. Bioeng Enhancement of protein partition a. System selection (polymer, MW, salt, ph) b. Phase polymer modification, e.g. affinity partition c. Protein modification Affinity ligand Peptide/ Protein domain 7

8 Affinity protein partitioning from yeast extract Ligand: Cibacron Blue F3GA Glyceraldehyde DH:ase Glucose 6P DH:ase 3 phosphoglycerate kinase Total protein Alcohol DH:ase 5% (w/w) PEG 6000/ 7% (w/w) dextran 500, 25 mm phosphate buffer, ph7 Yeast extract 10% of total system Amino acid partitioning 5% (w/w) PEG 8000/ potassium phosphate, ph 5.5 Eiteman et al, Biotechnol. Progress, 10,

9 Extraction of tag-modified protein (ZZcutinas-WP 4 ) from E.coli-cell homogenate System composition: PEG 4000: 10% NaKPO 4 [15]: 9.4% Cell concentration: 25 g/l DW Number of amino acids: Z Z cutinase Tag WPWPWPWP (WP) 4 ZZ-cutinase ZZ-cutinase-(WP) 4 K ZZ-cutinase K total protein Yield (Y T ) 31% 87% Purification factor Concentration Extraction combined with chromatography of tag-modified protein (ZZ-cutinase-(WP) 4 ) IgG-purified ZZ-cutinase-wt E.coli disintegrate Top phase (PEG4000) Imac-eluate HIC Octyl-eluata 9

10 E. coli partitioning Albertsson and Baird, Exp. Cell Research 28, 1962 PEG 6000/ dextran 48, 10 mm K-phosphate, ph 7 top phase (PEG rich) bottom phase (Dextran rich) interface DNA partitioning 10

11 Temp. induced phase system EO/PO copolymer and water TRANSFER TOP PHASE HEAT Starch polymer and water Protein and water = Target protein Top phase = Contaminants Recycling of EO/PO copolymer EO/PO copolymer * When the amount of propylenoxid (PO) is increased the temperature for the cloud point is decreased in the second temperature induced step: 10% EO20PO80 gives a cloud point of 30 C 10% EO30PO70 gives a cloud point of 40 C 10% Breox (EO50PO50) gives a cloud point of 50 C Partitioning of a plasmid DNA (Ultrafiltrated Lysate) in a Thermoseparating Two-Phase System Plasmid DNA RNA S T B TT TB S = Ultrafiltrated lysate T = Top phase B = Bottom phase TT = Thermoseparated top phase TB = Thermoseparated bottom phase System composition: 2.9% (w/w) EO 50 PO 50, 9.0% (w/w) Dextran T mm Na 2 HPO 4 Results Plasmid Protein RNA DNA Yield in TT (%) Concentration (times) 3-4 Ref: Cecilia Kepka, Department of Biochemistry,Lund University 11

12 Affinity extraction of E. coli poly(histidine)-tagged membrane protein cytochrome bo3 ubiquinol oxidase in polymer induced micelle extraction systems (PIME) Procedure Step 1: Smart solubilization and crude partition(system e.g. C 12 EO 5 -dextran) Step 2-4: Washing steps Step 5: Affinity PIME system (System e.g. C 12 EO 5 - allyldextranidacu(ii)) Result Pur. Fact. = 5.1 Ref: Ulf Sivars, Biochemistry Deparment, Lund University, Yield (%) = 40 Typical K-values of solutes and particles in phase systems K- values salt ions amino acids biomolecules peptides proteins DNA RNA particles membranes organelles extreme/interface cells cell debris 12