path: root/users/kanzure/protein-purification.txt

stability window for Taq polymerase

pH
ionic strength
size
isoelectric point (pI)
hydrophobicity
solubility

* temperature stability - need to work rapidly at lowered temperature
* pH stability - selection of buffers for extraction and purification; selection of conditions for ion exchange, affinity or reversed phase chromatography
* organic solvents stability - selection of conditions for reverse phase chromatography
* detergent requirement - consider effects on chromatographic steps and the need for detergent removal. consider choice of detergent.
* salt (ionic strength) - selection of conditions for precipitation techniques and hydrophobic interaction chromatography
* cofactors for stability and activity - selelection of additives, pH, salts, buffers
* protease sensitivity - need for fast removal of proteases or addition of inhibitors
* sensitivity to metal ions - need to add EDTA or EGTA in buffers
* redox sensitivity - need to add reducing agents
* molecular weight - selection of gel filtration media
* charge properties - selection of ion exchange conditions
* biospecific affinity - selection of ligand for affinity medium
* post translational modifications - selection of group specific affinity medium
* hydrophobicity - selection of medium for hydrophobic interaction chromatography




protein properties used during purification

* charge - ion exchange (IEX)
* size - gel filtration (GF)
* hydrophobicity - hydrophobic interaction (HIC), reversed phase (RPC)
* biorecognition (ligand specifity) - affinity (AC)
* charge, ligand specificity or hydrophobicity - expanded bed adsorption (EBA) follows the principles of AC, IEX or HIC



suitability of purification techniques for the Three Phase Purification Strategy:

IEX:
    main features:
        - high resolution
        - high capacity
        - high speed
    capture: 3stars
    intermediate: 3stars
    polish: 3stars
    sample start condition:
        - low ionic strength
        - sample volume
        - not limiting
    sample end condition:
        - high ionic strength or pH change
        - concentrated sample
HIC:
    main features:
        - good resolution
        - good capacity
        - high speed
    capture: 2stars
    intermediate: 3stars
    polish: 1stars
    sample start condition:
        - high ionic strength
        - sample volume
        - not limiting
    sample end condition:
        - low ionic strength
        - concentrated sample
AC (affinity chromatography):
    main features:
        - high resolution
        - high capacity
        - high speed
    capture: 3stars
    intermediate: 3stars
    polish: 2stars
    sample start condition:
        - specific binding conditions
        - sample volume
        - not limiting
    sample end condition:
        - specific elution conditions
        - concentrated sample
GF (gel filtration):
    main features:
        - high resolution
        - using Superdex(TM) media
    intermediate: 1stars
    polish: 3stars
    sample start condition:
        - limited sample volume (<5% total column volume)
        - limited flow rate range
    sample end condition:
        - buffer exchanged (if required)
        - diluted sample
RPC:
    main features:
        - high resolution
    intermediate: 1stars
    polish: 3stars
    sample start condition:
        - requires organic solvents
    sample end condition:
        - in organic solvent
        - risk loss of biological activity



"Ammonium sulphate precipitation is a common sample clarification and concentration step at laboratory scale and in this situation HlC (which requires high salt to enhance binding to the media) is ideal as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HlC column. Dilution of the fractionated sample or rapid buffer exchange using a Sephadex G-25 desalting column will prepare it for the next IEX or AC step."

"GF is well suited for use after any of the concentrating techniques (IEX, HlC, AC, EBA) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation (gel filtration is a non-binding technique with limited volume capacity and unaffected by buffer conditions)."



Logical combinations of chromatographic steps

crude sample -> GF (desalt mode) -> AC -> ___ -> GF
crude sample -> GF (desalt mode) -> IEX -> ___ -> GF
crude sample -> Hlc (dilution may be needed) -> IEX -> GF
crude sample -> GF (desalt mode) -> IEX -> HlC -> GF

clear or very dilute samples -> AC -> GF or RPC
clear or very dilute samples -> IEX -> GF or RPC
clear or very dilute samples -> IEX -> HlC -> GF
clear or very dilute samples -> precipitation (e.g., in high ionic strength) -> resolubilise -> treat as for sample in high salt concentration

"A sample is purified using a combination of techniques and alternative selectivities. For example, in an IEX-HlC-GF Three Phase Strategy the capture step selects according to differences in charge (IEX), the intermediate purification step according to differences in hydrophobicity (HlC) and the final polishing step according to differences in size (GF)."

IEX-HlC-GF is regarded as the standard if you know nothing about the proteins.


RPC = reversed phase chromatography



Purification step                 Media                                Quantity   Code No.
Capture                           STREAMLINE SP                        300 ml     17-0993-01
Capture                           STREAMLINE DEAE                      300 ml     17-0994-01
Capture                           HiPrepTM 16/10 SP XL                  1 column   17-5093-01
Capture                           HiPrep 16/10 Q XL                    1 column   17-5092-01
Intermediate purification         HiPrep Phenyl (high sub)             1 column   17-5095-01
Polishing                         HiLoadTM 16/60 Superdex 75 prep grade 1 column   17-1068-01
Polishing                         HiLoad 16/60 Superdex 200 prep grade 1 column   17-1069-01
Sample clarification/conditioning Pre-packed PD-10 Column              30 columns 17-0851-01
Sample clarification/conditioning HiTrap Desalting                     5 columns  17-1408-01
Sample clarification/conditioning HiPrep 26/10 Desalting               1 column   17-5087-01



Pre-packed column        Sample volume   Sample volume    Code No.
                         loading per run recovery per run
HiPrep Desalting 26/10   2.5 -15 ml      7.5 - 20 ml      17-5087-01
HiTrap Desalting         0.25 - 1.5 ml   1.0 - 2.0 ml     17-1408-01
Fast Desalting PC 3.2/10 0.05 - 0.2 ml   0.2 - 0.3 ml     17-0774-01
PD-10 Desalting          1.5 - 2.5 ml    2.5 - 3.5 ml     17-0851-01


Table 6. Recommended HiTrap affinity columns for laboratory scale separation.
                                           Code No.   Quantity/                Approximate
Application              HiTrap column
                                                      components               binding capaci-
                                                                               ty per ml gel
                                           17-5079-01 5 x 1 ml                 human
Isolation of             HiTrap rProtein A
                                           17-5080-01 1 x 5 ml                 IgG 50 mg/ml
immunoglobulins
                                           17-5029-02 2 x 1 ml
IgG classes, fragments
and subclasses
                                           17-0402-01 5 x 1 ml                 human
IgG classes, fragments   HiTrap Protein A
                                           17-0402-03 2 x 1 ml                 IgG 20 mg/ml
and subclasses
                                           17-0403-01 1 x 5 ml
                                           17-0404-01 5 x 1 ml                 human
IgG classes, fragments   HiTrap Protein G
                                           17-0404-03 2 x 1 ml                 IgG 25 mg/ml
and subclasses
                                           17-0405-01 1 x 5 ml
including human IgG3
strong affinity
to monoclonal mouse
IgG1 and rat IgG
Monoclonal and poly-     MAbTrapTM GII      17-1128-01 HiTrap Protein G         as above
clonal IgG from ascites                               column (1 ml),
fluid, serum and cell                                 accessories, pre-made
culture supernatant                                   buffers for 10 purifica-
                                                      tions
Mouse recombinant        RPAS Purification 17-1362-01 HiTrap Anti-E column,    0.17 mg
Single chain antibody    Module                       accessories, pre-made    ScFv/5 ml
Fragment variable                                     buffers for 20 purifica-
(ScFv) produced in                                    tions
E.Coli
IgY antibodies from      HiTrap IgY        17-5111-01 1 x 5 ml                 IgY 20 mg/ml
egg yolk                 Purification
IgM                      HiTrap IgM        17-5110-01 5 x 1 ml                 IgM 5 mg/ml
                         Purification
Group Specific Media:    HiTrap Con A      17-5105-01 5 x 1 ml                 transferrin
Glycoproteins or                                                               4 mg/ml
polysaccharides
Specificity: branched
mannoses,
carbohydrates
withterminal mannose
or glucose(a Man>
a Glc> GlcNAc)
Specificity:             HiTrap Lentil     17-5106-01 5 x 1 ml                 thyroglobulin
branched mannoses        Lectin                                                4 mg/ml
with fucose linked
a(1,6) to the N- acetyl-
glucosamine,
(a Man> a Glc>
GlcNAc) N- acetyl-
glucosamine binding
lectins


                           HiTrap column        Code No.        Quantity/             Approximate
Application
                                                                components            binding capa-
                                                                                      city per ml gel
                           HiTrap Peanut        17-5108-01      5 x 1 ml              asialofetuin
Specificity: Terminal ß
                           Lectin                                                     3 mg/ml
-galactose, (Gal ß 1,3
GalNAc > a and ß Gal)
                           HiTrap Wheat         17-5107-01      5 x 1 ml              ovomuroid
Specificity: chitobiose
                           Germ Lectin                                                4 mg /ml
core of N-linked oligo-
saccharides, [GlcNAc
(ß 1,4GlcNAc) 1- 2 >
ß GlcNAc]
                           HiTrap Lectin Test   17-5109-01      4 x 1 ml columns of   as listed above
Specificity: as listed for
                           Kit                                  HiTrap Con A
each column
                                                                HiTrap Lentil Lectin
                                                                HiTrap Peanut Lectin
                                                                HiTrap Wheat Germ
                                                                Lectin
Group Specific Media       HiTrap Blue          17-0412-01      5 x 1 ml              HSA 20 mg/ml
Various Nucleotide-                             17-0413-01      1 x 5 ml
requiring enzymes,
coagulation factors,
DNA binding proteins,
a2-macro-globulin
Proteins and peptides      HiTrap Chelating     17-0408-01      5 x 1 ml              (His)6-tagged
with exposed amino                              17-0409-01      1 x 5 ml              protein (27.6
acids: His (Cys, Trp)                                                                 kD) 12 mg /ml
e.g. a-2-macro-globu-
lin and interferon
Histidine-tagged fusion    HisTrapTM             17-1880-01      HiTrap Chelating      as above
proteins                                                        column (1 ml), acces-
                                                                sories, pre-made
                                                                buffers
Biotin and biotinylated    HiTrap               17-5112-01      5 x 1 ml              biotinylated
substances                 Streptavidin                                               BSA 6 mg/ml
Coagulation factors,       HiTrap Heparin       17-0406-01      5 x 1 ml              ATIII (bovine)
lipoprotein lipases,                            17-0407-01      1 x 5 ml              3 mg/ml
steroid receptors,
hormones, DNA
binding proteins,
interferon, protein
syntheses factors
Matrix for prepara-        HiTrap               17-0716-01      5 x 1 ml              ligand specific
tion of affinity media     NHS-activated        17-0717-01      1 x 5 ml
Coupling of primary
amines
Recommended separation conditions
All HiTrap columns are supplied with a detailed protocol to ensure optimum results
Maximum flow rates: HiTrap 1 ml column: up to 4 ml/min
HiTrap 5 ml column: up to 20 ml/min


Table 7. Volatile buffer systems.
               Volatile buffer systems used in ion exchange chromatography
pH range       Buffer system                          Counter-ion    pK-values for buffering ions
2.0            Formic acid                            H+             3.75
2.3-3.5        Pyridine/formic acid                   HCOO-          3.75; 5.25
3.0-5.0        Trimethylamine/formic acid             HCOO-          3.75; 9.25
3.0-6.0        Pyridine/acetic acid                   CH3COO-        4.76; 5.25
4.0-6.0        Trimethylamine/acetic acid             CH3COO-        4.76; 9.25
6.8-8.8        Trimethylamine/HCl                     Cl-            9.25
7.0-8.5        Ammonia/formic acid                    HCOO-          3.75; 9.25
8.5-10.0       Ammonia/acetic acid                    CH3COO-        4.76; 9.25
7.0-12.0       Trimethylamine/carbonate               CO32-          6.50; 9.25
7.9            Ammonium bicarbonate                   HCO3-          6.50; 9.25
8.0-9.5        Ammonium carbonate/ammonia             CO32-          6.50; 9.25
8.5-10.5       Ethanolamine/HCl                       Cl-            10.0
8.5            Ammonium carbonate                     CO32-          6.50; 9.25



Extraction process           Typical conditions           Protein source             Comment
Gentle                                                    erythrocytes E.coli
                             2 volumes water to                                      lower product yield but
                             1 volume packed              periplasm: intracellular   reduced protease
Cell lysis (osmotic shock)   pre-washed cells             proteins                   release
Enzymatic digestion          lysozyme 0.2 mg/ml,          bacteria: intracellular    lab scale only, often
                             37 °C, 15 mins.              proteins                   combined with
                                                                                     mechanical disruption
Hand homogenisation          follow equipment             liver tissue
                             instructions
Mincing (grinding)           "                            muscle
Moderate                     follow equipment             muscle tissue, most
                             instructions                 animal tissues, plant
Blade homogeniser                                         tissues
Grinding with abrasive       "                            bacteria, plant tissues
e.g. sand
Vigorous                     follow equipment             cell suspensions:          small scale, release of
Ultrasonication              instructions                 intracellular proteins in  nucleic acids may cause
or                                                        cytoplasm, periplasm,      viscosity problems
bead milling                                              inclusion bodies           inclusion
                                                                                     bodies must be
                                                                                     resolubilised
Manton-Gaulin                follow equipment             cell suspensions           large scale only
homogeniser                  instructions
French press                 follow equipment             bacteria, plant cells
                             instructions
Fractional precipitation     see section on fractional    extracellular: secreted    precipitates must be
                             precipitation                recombinants,              resolubilised
                                                          monoclonal antibodies,
                                                          cell lysates
Details from Protein Purification, Principles and Practice, R.K. Scopes and other sources.


Table 9. Common substances used in sample preparation.
                                     Typical conditions for use         Purpose
Buffer components                    20 mM, pH7.4                       maintain pH minimise
Tris                                                                    acidification caused by
                                                                        lysosomal disruption
NaCl                                 100 mM                             maintain ionic strength of
                                                                        medium
EDTA                                 10 mM                              reduce oxidation damage,
                                                                        chelate metal ions
Sucrose or glucose                   25 mM                              stabilise lysosomal membra-
                                                                        nes, reduce protease release
Detergents                           See Table 10                       extraction and purification of
lonic or non-ionic detergents                                           integral membrane proteins
                                                                        solubilisation of poorly soluble
                                                                        proteins
DNAse and RNAse                      1 μg/ml                            degradation of nucleic acids,
                                                                        reduce viscosity of sample
                                                                        solution
Protease inhibitors*                                                    Inhibits
                                     0.5 - 1 mM
PMSF                                                                    serine proteases
                                     0.4 - 4 mM
APMSF                                                                   serine proteases
                                     0.2 mM
Benzamidine-HCl                                                         serine proteases
                                     1 μM
Pepstatin                                                               aspartic proteases
                                     10 - 100 μM
Leupeptin                                                               cysteine and serine proteases
                                     10 - 100 μM
Chymostatin                                                             chymotrypsin, papain,
                                                                        cysteine proteases
                                     1 - 100 μM
Antipain-HCl                                                            papain, cysteine and serine
                                                                        proteases
                                     2 - 10 mM
EDTA                                                                    metal dependent proteases,
                                                                        zinc and iron
                                     2 - 10 mM
EGTA                                                                    metal dependent proteases
                                                                        e.g. calcium
Reducing agents                      1 - 10 mM                          keep cysteine residues
1, 4 dithiothreitol, DTT                                                reduced
                                     1 - 10 mM
1, 4 dithioerythritol, DTE
                                                                        "
                                     0.05%
Mercaptoethanol
                                                                        "
Others                               5 - 10%                            for stabilisation, up to 50% can
Glycerol                                                                be used if required
PMSF - Phenylmethylsulfonyl fluoride
APMSF - 4-Aminophenyl-methylsulfonyl fluoride
PMSF is a hazardous chemical. Half-life time in aqueous solution is 35 min. PMSF is usually stored
as 10 mM or 100 mM stock solution (1.74 or 17.4 mg/ml in isopropanol) at - 20° C.
* Protease inhibitors are available in pre-made mixes from several suppliers.
Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer.,
Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L.
Rydén, 1998, 2nd ed. Wiley Inc. and other sources.


Table 10. Examples of ionic and non-ionic detergents.
Sodium dodecyl sulphate            0.1 - 0.5%                 denatures proteins, used for SDS-PAGE
                                                              use non-ionic detergents to avoid
                                                              denaturation
Triton-X-100                       0.1 %                      non-ionic detergent for membrane
                                                              solubilisation.
                                                              Note: may absorb strongly at 280 nm!
Nonidet-P-40                       0.05 - 2%                  "
Dodecyl b D maltoside              1%                         "
Octyl b D glucoside                1 - 1.5%                   "
For further information on detergents: Protein Purification, Principles, High Resolution Methods and
Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc.


Pre-packed column        Sample volume   Sample volume    Code No.
                         loading per run recovery per run
HiPrep Desalting 26/10   2.5 -15 ml      7.5 - 20 ml      17-5087-01
HiTrap Desalting         0.25 - 1.5 ml   1.0 - 2.0 ml     17-1408-01
Fast Desalting PC 3.2/10 0.05 - 0.2 ml   0.2 - 0.3 ml     17-0774-01
PD-10 Desalting          1.5 - 2.5 ml    2.5 - 3.5 ml     17-0851-01


Precipitation agent       Typical conditions for use     Sample type             Comment
Ammonium sulphate         as described                   >1mg/ml proteins        stabilizes proteins, no
                                                         especially immuno-      denaturation, super-
                                                         globulins               natant can go directly
                                                                                 to HIC
Dextran sulphate          as described                   samples with high       precipitates lipoprotein
                                                         levels of lipoprotein
                                                         e.g ascites
Polyvinylpyrrolidine      Add 3% (w/v), stir 4 hours,                            alternative to dextran
                                                         "
                          centrifuge, discard pellet                             sulphate
Polyethylene glycol       up to 20% wt/vol               plasma proteins         no denaturation super-
(PEG, M.W. >4000)                                                                natant goes direct to
                                                                                 IEX or AC. Complete
                                                                                 removal may be difficult
Acetone                   up to 80% vol/vol at 0 °C      useful for peptide      may denature protein
                                                         precipitation or con-   irreversibly
                                                         centration of sample
                                                         for electrophoresis
Polyethyleneimine         0.1% w/v                                               precipitates aggregated
                                                                                 nucleoproteins
Protamine sulphate        1%
                                                                                 "
Streptomycin sulphate     1%                                                     precipitation of nucleic
                                                                                 acids
Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer.,
Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L.
Rydén, 1998, 2nd ed. Wiley Inc.and other sources


Table 13. Examples of denaturing agents.
Denaturing agent                Typical conditions for use     Removal/comment
Urea                            2 - 8M                         remove using Sephadex G25
Guanidine hydrochloride         3 - 8M                         remove using Sephadex G25 or
                                                               during IEX
Triton X-100 with               2%                             ”
 Sarcosyl                       1.5%                           ”
N-octyl glucoside               2%                             ”
Sodium dodecyl sulphate         0.1 - 0.5%                     exchange for non-ionic detergent during
                                                               first chromatographic step, avoid anion
                                                               exchange chromatography
alkaline pH                     > pH 9, NaOH                   may need to adjust pH during
                                                               chromatography to maintain solubility
Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer.,
Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and
L. Rydén, 1998, 2nd ed. Wiley Inc.and other sources