Preparation and Extraction of Insoluble
`(Inclusion-Body) Proteins from
`Escherichia coli
`
`UNIT 6.3
`
`High-level expression of many recombinant proteins in Escherichia coli leads to the
`formation of highly aggregated protein commonly referred to as inclusion bodies
`(UNITS 5.1 & 6.1). Inclusion bodies are normally formed in the cytoplasm; alternatively,
`if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies are
`not restricted to E. coli; they can also form in yeast, mammalian, and insect cells.
`
`Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily
`contaminated with E. coli cell wall and outer membrane components. The latter are
`largely removed by selective extraction with detergents and low concentrations of either
`urea or guanidine·HCl to produce so-called washed pellets. These basic steps result in a
`significant purification of the recombinant protein, which usually makes up ∼60% of the
`washed pellet protein. The challenge, therefore, is not to purify the recombinant-derived
`protein, but to solubilize it and then fold it into native and biologically active protein.
`
`Basic Protocol 1 describes preparation of washed pellets and solubilization of the protein
`using guanidine·HCl. The extracted protein, which is unfolded, is either directly folded as
`described in UNIT 6.5 or further purified by gel filtration in the presence of guanidine·HCl
`as in Basic Protocol 2. A Support Protocol describes the removal of guanidine·HCl from
`column fractions so they can be monitored by SDS-PAGE (UNIT 10.1).
`
`Other methods discussed in the Commentary section of this unit include the direct purifi-
`cation of polyhistidine-tagged proteins solubilized in guanidine·HCl, preparative removal
`of guanidine·HCl by reversed-phase chromatography as a prelude to protein folding, and
`the solubilization of inclusion bodies with anionic detergents.
`
`PREPARATION AND EXTRACTION OF INSOLUBLE (INCLUSION-BODY)
`PROTEINS FROM ESCHERICHIA COLI
`Bacterial cells are lysed using a French press, and inclusion bodies in the cell lysate are
`pelleted by low-speed centrifugation. The pellet fraction is washed (preextracted) with
`urea and Triton X-100 to remove E. coli membrane and cell wall material. Guanidine·HCl
`(8 M) and dithiothreitol (DTT) are used to solubilize the washed pellet protein. Extraction
`with the denaturant simultaneously dissociates protein-protein interactions and unfolds
`the protein. As a result, the extracted protein consists (ideally) of unfolded monomers,
`with sulfhydryl groups (if present) in the reduced state.
`
`Materials
`
`E. coli cells from fermentation (UNIT 5.3) containing the protein of interest
`Lysis buffer (see recipe)
`Wash buffer (see recipe), with and without urea and Triton X-100
`Extraction buffer (see recipe)
`250- and 500-ml stainless steel beakers
`Waring blender
`Polytron tissue-grinder homogenizer (Brinkmann)
`French pressure cell (e.g., Thermo Electron Corp.; http://www.thermo.com)
`
`Contributed by Ira Palmer and Paul T. Wingfield
`Current Protocols in Protein Science (2004) 6.3.1-6.3.18
`Copyright C(cid:2) 2004 by John Wiley & Sons, Inc.
`
`BASIC
`PROTOCOL 1
`
`Purification of
`Recombinant
`Proteins
`
`6.3.1
`
`Supplement 38
`
`Page 1
`
`KASHIV EXHIBIT 1021
`IPR2019-00791
`
`

`

`Probe sonicator
`Beckman J2-21M centrifuge with JA-14 rotor (or equivalent)
`Beckman Optima XL-90 ultracentrifuge with 45 Ti rotor (or equivalent)
`0.22-µm syringe filters (e.g., Millex from Millipore)
`20-ml disposable syringe
`Additional equipment for breaking cells, homogenizing cells and pellets and
`centrifuging at low and high speeds (UNIT 6.2)
`
`Break cells and prepare clarified lysate
`1. Place thawed E. coli cells in a stainless steel beaker. Add 4 ml lysis buffer per gram
`wet weight of cells. Keep bacterial cells cool by placing the beaker on ice in an ice
`bucket.
`The cells can be pretreated with lysozyme prior to lysis in the French press. Lysozyme
`treatment involves incubating cells −20 min at 20
`◦
`◦
`to 25
`C inlysis buffer supplemented
`with 200 µg/ml lysozyme, with intermittent homogenization using a tissue grinder. It
`should be emphasized that this optional step is carried out before French press breakage
`and is not simply an alternative method of cell breakage (compare the comments made
`in the annotation to step 4 of UNIT 6.2). Its purpose is to aid removal of the peptidoglycan
`and outer membrane protein contaminants during the washing steps (steps 6 to 9; for
`further details see UNIT 6.1 and Fig. 6.1.5). An example of this approach is given in Basic
`Protocol 1 of UNIT 6.5.
`
`For sensitive proteins, replace benzamidine in the lysis buffer by a protease inhibitor
`cocktail that includes five protease inhibitors with broad specificity for the inhibition of
`aspartic proteases, cysteine proteases, serine proteases, and metalloproteases, as well as
`aminopeptidases. These are supplied by various companies including Calbiochem and
`Sigma.
`
`2. Suspend cells using a Waring blender and homogenize using the Polytron tissue-
`grinder homogenizer until all clumps are disrupted, as described in UNIT 6.2,
`step 3.
`
`3. Lyse cells with two passes through the French pressure cell operated at 16,000 to
`18,000 psi (with the high-ratio setting, pressure gauge readings between 1011 and
`◦
`1135), chilling the cell suspension to 4
`C after each pass, as described in UNIT 6.2,
`steps 2 and 4.
`
`4. Reduce the viscosity of the suspension by sonicating 5 min at full power with 50%
`duty cycle (on for 5 sec, off for 5 sec) using an ultrasonic homogenizer, as described
`in UNIT 6.2, step 5.
`5. Clarify the lysed cell suspension by centrifuging 1 hr at 22,000 × g (12,000 rpm in
`◦
`a JA-14 rotor in a Beckman J2-21M centrifuge), 4
`C.
`
`Unbroken cells, large cellular debris, and the inclusion body protein will be pelleted.
`
`The JA-14 rotor uses 250-ml centrifuge bottles. For processing smaller volumes the
`Beckman JA-20 rotor (or equivalent) with 50-ml tubes can be used, at 13,500 rpm
`(22,000 × g).
`The procedure for dealing with insoluble inclusion-body proteins now diverges from that
`for purifying soluble proteins (UNIT 6.2).
`
`Prepare washed pellets
`6. Carefully decant the supernatant from the pellet. Using a tissue homogenizer, suspend
`the pellet with 4 to 6 ml wash buffer per gram wet weight cells.
`
`Complete homogenization of the pellet is important to wash out soluble proteins and
`cellular components. Removal of cell wall and outer membrane material can be improved
`by increasing the amount of wash solution to 10 ml per gram cells.
`
`Current Protocols in Protein Science
`
`Preparation and
`Extraction of
`Inclusion Bodies
`
`6.3.2
`
`Supplement 38
`
`Page 2
`
`

`

`The concentration of urea and Triton X-100 in the wash buffer can be varied. The urea
`concentration is usually between 1 and 4 M; higher concentrations may result in partial
`solubilization of the recombinant proteins. The usual detergent concentration is 0.5% to
`5%. Triton X-100 will not solubilize inclusion body proteins; it is included to help extract
`lipid and membrane-associated proteins.
`7. Centrifuge the suspension 30 min at 22,000 × g (12,000 rpm in JA-14), 4
`C. Discard
`supernatant and, using the tissue homogenizer, suspend the pellet in 4 to 6 ml wash
`buffer per gram wet weight of cells.
`
`◦
`
`8. Repeat step 7 two more times.
`
`If the supernatant is still cloudy or colored, continue washing the pellet until the super-
`natant is clear.
`
`9. Suspend the pellet in wash buffer minus the Triton X-100 and urea, using 4 to 6 ml
`buffer per gram wet cells. Centrifuge 30 min at 22,000 × g (12,000 rpm in JA-14),
`◦
`4
`C.
`
`The final wash removes excess Triton X-100 from the pellet.
`If necessary the washed pellets can be stored at −80
`◦
`C. It is better to store material at
`this stage rather than after the extraction stage (see annotation to step 13).
`Extract recombinant protein from washed pellets with guanidine·HCl
`10. Using the tissue homogenizer, suspend the pellet in guanidine·HCl-containing ex-
`traction buffer. Use 0.5 to 1.0 ml buffer per gram wet weight of original cells if the
`extract will be subjected to gel filtration, and 2 to 4 ml buffer if the extract will be
`used in protein folding procedures. Perform this step at room temperature.
`To estimate the amount of recombinant protein in the washed pellets, use the following
`guidelines. (1) An expression level of 1% corresponds to ∼1 mg recombinant protein per
`1 gwet cells. (2) The recovery of highly aggregated recombinant protein in the washed
`pellets is ∼75% that originally present in the cells. (3) About 60% of the total washed
`pellet protein is recombinant-derived. Thus, if 50 g cells is processed and the expression
`level is 5%, the washed pellets contain ∼200 mg recombinant protein.
`The total amount of recombinant-derived protein in washed pellets can be directly deter-
`mined by measuring the total protein concentration or by analyzing the washed pellets
`via SDS-PAGE (see Support Protocol and UNIT 10.1) todetermine the proportions of the
`protein constituents.
`
`For gel-filtration purposes, the pellets from 50 g wet weight E. coli cells are solubilized
`with 40 to 50 ml extraction buffer (see Basic Protocol 2); the concentration of recombinant
`protein in the extract will be 4 to 5 mg/ml. For direct protein folding (UNIT 6.5), the
`pellets are extracted with 100 to 200 ml buffer, and the concentration of recombinant
`protein 1 to 2 mg/ml. If the washed pellet is heavily contaminated with outer cell wall and
`peptidoglycan material, the extract must be diluted further with extraction buffer (usually
`1:1 to 1:3) to reduce the viscosity before it can be used for chromatography.
`11. Centrifuge the suspension 1 hr at 100,000× g (30,000 rpm in 45 Ti rotor in a Beckman
`◦
`Optima XL-90 ultracentrifuge), 4
`C.
`For volumes <250 ml the Beckman 70 Ti rotor (capacity 6 × 39 ml) can be used at 32,000
`rpm (−100,000 × g).
`12. Carefully pour off the supernatant from the pellet. Filter the supernatant through a
`0.22-µm syringe filter attached to a 20-ml disposable syringe.
`
`The filter removes unpelleted large cell wall debris that will clog most chromatography
`columns.
`
`13. Use the clarified inclusion body extract for preparing folded protein (UNIT 6.5) or
`purify further by gel filtration (see Basic Protocol 2).
`
`Current Protocols in Protein Science
`
`Purification of
`Recombinant
`Proteins
`
`6.3.3
`
`Supplement 38
`
`Page 3
`
`

`

`Nickel-chelate affinity chromatography can also be directly used for His-tagged proteins
`(see Commentary in this unit and Basic Protocol 3 in UNIT 6.5).
`The extract can be stored at −80
`◦
`C until required. Freeze in plastic (or polyethylene)
`containers rather than glass. Divide sample into 10- to 20-ml aliquots instead of freezing
`in one large lot and fill containers to only 50% to 75% capacity.
`
`MEDIUM-PRESSURE GEL-FILTRATION CHROMATOGRAPHY IN THE
`PRESENCE OF GUANIDINE HYDROCHLORIDE
`Washed, extracted pellets (see Basic Protocol 1) contain >50% recombinant protein and
`are used as the starting material for purification of the protein of interest by gel-filtration
`chromatography. Superdex 200 gel-filtration medium, which allows high flow rates, is
`◦
`washed and packed into a column. The column is equilibrated at 4
`C and the sample is
`applied.
`
`Assay of column fractions by gel electrophoresis in the presence of SDS is complicated by
`the fact that guanidine·HCl forms a precipitate with SDS. Therefore, preparing samples
`for gel analysis involves selective precipitation of protein from guanidine·HCl prior to
`SDS-PAGE (see Support Protocol). The purified (or partially) purified protein is used as
`the starting material for procedures (e.g., UNIT 6.5) inwhich the denatured protein is folded
`into a native and biologically active structure.
`
`Materials
`
`Gel-filtration medium: Superdex 200 PG (preparative grade; Amersham
`Biosciences)
`5% (v/v) ethanol
`Gel-filtration buffer (see recipe)
`Guanidine·HCl extract of E. coli cells containing the protein of interest (see Basic
`Protocol 1)
`4- to 6-liter plastic beaker
`Chromatography column: Amersham Biosciences XK 16/100, 26/100, or 50/100
`Packing reservoir: Amersham Biosciences RK 16/26 (for 16- and 26-mm-i.d.
`columns) and RK 50 (for 50-mm-i.d. column)
`Chromatography pump: Amersham Biosciences P-6000 or P-500
`Injection valve (to select between sample loop and pump)
`UV monitor and fraction collector
`Sample loop (volume determined by size of column; also see annotation to step 15)
`
`NOTE: The various components of the chromatography system (pumps, valves, mon-
`itors, and sample loops) listed separately above are supplied as components of the
`¨AKTAexplorer chromatography system (Amersham Biosciences), which is used to run
`the XK 50/100 column. The smaller XK columns (2.6 and 2.5 cm i.d.) are run using
`the ¨AKTA-FPLC chromatography system (also from Amersham Biosciences), which is
`designed for small- to medium-scale work. For further details on this equipment see the
`manufacturer’s literature (e.g., Process Products, Amersham Biosciences).
`
`NOTE: Perform steps 1 to 11 at room temperature. After the column is packed, equilibrate
`◦
`and elute at 4
`C.
`
`Pack the column
`1. Wash the gel-filtration medium in a large plastic beaker with 5% ethanol. Let the
`medium settle and adjust the volume of liquid to yield a gel slurry concentration of
`65% to 75%.
`
`Current Protocols in Protein Science
`
`BASIC
`PROTOCOL 2
`
`Preparation and
`Extraction of
`Inclusion Bodies
`
`6.3.4
`
`Supplement 38
`
`Page 4
`
`

`

`The XK 16/100, 26/100, and 50/100 columns are 100 cm long and have inner diameters
`of 16, 26, and 50 mm, respectively. Hence, for an XK 50/100 column, column volume =
`
`radius (2.5 cm)2 × 3.1416 × bed height (97 cm) ∼= 1900 ml, and ∼2 liters preparative-
`grade Superdex 200 is required. To pack this column, the gel medium is suspended in 5%
`ethanol to give a total volume of 3 liters which corresponds to ∼70% gel slurry (it should
`be noted that the RK 50 reservoir has a capacity of 1 liter, so the 3 liters of gel slurry can
`be poured in a single operation).
`
`2. Fix the chromatography column in an upright position, using a level to adjust the
`position. Attach the packing reservoir.
`
`3. Add sufficient 5% ethanol to displace the air from a few centimeters of the bottom
`of the column. Clamp off the bottom of the column.
`
`4. Gently mix the gel-filtration medium in the plastic beaker using a glass rod or plastic
`paddle to an even slurry of 70% medium suspended in 5% ethanol.
`
`Do not use a magnetic stirrer, as it could damage the medium.
`
`5. Degas the suspension 5 to 10 min using a vacuum flask and laboratory vacuum.
`
`The ethanol is included to reduce the surface tension and density of the solvent, thus
`allowing air bubbles that form to rise to the surface more quickly.
`
`6. Carefully pour the slurry of medium into the column, introducing material along the
`side of the column to avoid creating air bubbles.
`
`7. Let the column stand 5 min and then unclamp the bottom of the column.
`
`8. Attach the chromatography pump to the packing reservoir and pump 5% ethanol
`(degassed) into the column at an appropriate flow rate (based on manufacturer’s
`instructions). Pack the column at a pressure greater than the pressure at which the
`column will be run (up to twice as high), but not greater than the maximum pressure
`rating of the column.
`For example, the XK 50/100 column (rated to 0.5 MPa) is packed at ∼20 to 30 ml/hr and
`∼0.4 MPa.
`9. After the medium has settled, turn off the pump and close the bottom of the column.
`Pipet fluid from the reservoir and remove the reservoir.
`
`Once the column has been packed, be careful to prevent air from entering the column bed.
`Air will disturb the bed and reduce the column separation resolution.
`
`10. Attach the column top adapter to the column. Place the top of the adapter onto the
`top of the packed medium and gently compress the medium.
`
`11. Reattach the pump to the column and wash the column with water at a flow rate that
`will generate the maximum pressure to be used. If the medium continues to settle,
`readjust the top adapter to maintain a firm fit against the gel.
`◦
`
`From this point onward, perform all steps at 4
`
`C.
`
`Equilibrate the column
`◦
`12. Equilibrate the column with at least 1 column volume of 4
`C gel-filtration buffer.
`Although the proteins were extracted with buffer containing 8 M guanidine·HCl (see Basic
`Protocol 1), the gel-filtration buffer contains only 4 M guanidine·HCl. The concentration
`is reduced to allow faster flow rates and for reasons of economy. Most proteins remain
`unfolded at the lower guanidine·HCl concentration. If, however, the protein elutes in an
`anomalous manner (e.g., in more than one peak or at an elution position not consistent
`with its size), and assuming there is adequate reducing agent present, then try increasing
`the guanidine·HCl concentration in the gel-filtration buffer.
`
`Current Protocols in Protein Science
`
`Purification of
`Recombinant
`Proteins
`
`6.3.5
`
`Supplement 38
`
`Page 5
`
`

`

`13. Measure the actual flow rate while running the column at a flow rate that generates
`a back pressure about one-half of that generated when packing the column (step 8).
`
`For an XK50/100 column packed using Superdex 200 at 0.4 MPa, a running pressure of
`∼0.2 MPa is used, which generates flow rates of 5 to 10 ml/min that are equivalent to linear
`flow rates of 15.3 to 30.6 cm/hr. The linear flow rate equals the flow rate (ml/hr)/cross-
`sectional area (cm2). At these flow rates it takes between 3 and 6 hr to complete the
`chromatography.
`
`14. Connect tubing from the end of the column to the UV monitor and the fraction
`collector.
`
`Apply the sample
`15. Load the sample loop with the guanidine·HCl extract to be separated.
`Avoid loading a sample volume >5% of the total column volume; the optimum sample size
`is 2% (∼40 ml for the XK 50/100 column). The sample consists of washed pellets extracted
`with guanidine·HCl (see Basic Protocol 1). A sample size of 40 to 50 ml is usually derived
`from ∼50 g wet weight cells. With smaller sample sizes, use columns with proportionally
`smaller diameters (e.g., XK 16/100 or 26/100 columns). If purchase of only one column
`is possible, a 2.5 × 100–cm size is a good compromise for variable sample loading.
`16. Monitor column effluent with the UV monitor and collect fractions with the fraction
`collector.
`For an XK50/100 column, collect 15- to 20-ml fractions in 16 × 20–mm culture tubes.
`The eluent from the column is usually monitored at 280 nm or, if the protein has a par-
`ticularly low extinction coefficient, at 230 nm (guanidine·HCl strongly absorbs below
`225 nm). For an XK 50/100 column, fractions need only be collected after ∼500 ml of
`elution. The excluded volume (void volume) is ∼570 ml. Run a total of one column volume
`(1900 ml) to ensure all of the load material is eluted from the column.
`
`17. Prepare the fractions to be assayed for SDS-PAGE (see Support Protocol and UNIT 10.1).
`
`SUPPORT
`PROTOCOL
`
`PREPARATION OF SAMPLES CONTAINING GUANIDINE
`HYDROCHLORIDE FOR SDS-PAGE
`Because guanidine·HCl forms a precipitate with SDS, it is necessary to remove the
`former before carrying out SDS-PAGE. Protein in column fractions is separated from
`guanidine·HCl by precipitation using 90% ethanol (Pepinsky, 1991).
`
`Materials
`
`Sample containing the protein of interest
`◦
`◦
`to 4
`C
`100% and 90% ethanol, 0
`1× SDS sample buffer (UNIT 10.1)
`Gilson Pipetman (Rainin Instrument)
`Additional reagents and equipment for gel electrophoresis (UNIT 10.1)
`
`1. Pipet 25 µl sample containing the protein of interest into a 1.5-ml microcentrifuge
`tube.
`
`to 4
`
`C) 100% ethanol to the sample in the tube.
`
`◦
`2. Add 225 µl cold (0
`
`◦
`
`Preparation and
`Extraction of
`Inclusion Bodies
`
`6.3.6
`
`Supplement 38
`
`The final ethanol concentration is 90% by volume.
`3. Mix the sample and ethanol by vortexing. Chill 5 to 10 min at −20
`◦
`−80
`◦
`C).
`Do not use a magnetic stirrer, as it could damage the medium.
`
`C orcolder (e.g.,
`
`Current Protocols in Protein Science
`
`Page 6
`
`

`

`4. Microcentrifuge the sample 5 min at maximum speed (∼15,000 × g), 4
`◦
`withdraw the supernatant and retain the pellet.
`
`C. Carefully
`
`The pellet may be difficult to see. Be careful not to draw the pellet out of the microcentrifuge
`tube with the supernatant.
`
`5. Suspend the pellet in 250 µl cold 90% (v/v) ethanol. Mix thoroughly using a vortex
`mixer.
`
`The 90% ethanol is prepared by mixing 225 µl ethanol and 25 µl H2O.
`◦
`6. Microcentrifuge the sample 5 min at maximum speed, 4
`C. Carefully pipet off the
`supernatant and suspend the pellet in 25 µl of 1× SDS sample buffer.
`Some proteins are more difficult than others to suspend from an ethanol precipitate.
`Electrophoresis sample buffer containing 8 M urea is helpful for such proteins (UNIT 10.1).
`Sonication with a microtip probe can also be used to disperse the sample. A volume of
`sample buffer >25 µl may be required in this case (e.g., 50 µl), and great care must be
`taken to prevent foaming of the sample caused by excessive sonication power.
`◦
`◦
`
`7. Heat the sample 3 to 5 min at 90
`(UNIT 10.1).
`
`to 100
`
`C. Load on an SDS-polyacrylamide gel
`
`REAGENTS AND SOLUTIONS
`Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock
`solutions, see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.
`
`Extraction buffer
`50 mM Tris·Cl, pH 7.0
`5 mMEDTA
`8 Mguanidine·HCl (764 g/liter)
`5 mMDTT (770 mg/liter)
`If the buffer is cloudy, filter through a 0.45- to 0.5-µm filter (the solution
`should be clear if high-quality guanidine·HCl—e.g., ultrapure grade, ICN
`Biomedicals—is used; see APPENDIX 3A). Buffer can be stored without DTT for
`◦
`C.
`at least 1 month at 4
`
`Gel-filtration buffer
`50 mM Tris·Cl, pH 7.5
`4 Mguanidine·HCl (382 g/liter; ultrapure, Invitrogen Life Technologies)
`5 mMDTT (770 mg/liter)
`◦
`C. Filter (as for
`Buffer can be stored without DTT at least 1 month at 4
`extraction buffer; see recipe) and degas before use.
`Higher concentrations of guanidine·HCl (up to 8 M) may be required for some proteins
`(see annotation at step 12 of Basic Protocol 2).
`
`Lysis buffer
`100 mM Tris·Cl, pH 7.0
`5 mMEDTA
`5 mMDTT (770 mg/liter)
`5 mMbenzamidine·HCl (780 mg/liter)
`Prepare immediately before use
`The Tris·Cl and EDTA are diluted from concentrated stock solutions. The other compo-
`nents are added to the diluted buffer before use.
`
`Current Protocols in Protein Science
`
`Purification of
`Recombinant
`Proteins
`
`6.3.7
`
`Supplement 38
`
`Page 7
`
`

`

`Wash buffer
`100 mM Tris·Cl, pH 7.0
`5 mMEDTA
`5 mMDTT (770 mg/liter)
`2 Murea (120 g/liter; ultrapure, Invitrogen Life Technologies)
`2% (w/v) Triton X-100 (20 g/liter; Calbiochem)
`
`Add DTT, urea, and Triton X-100 to the other components immediately before use.
`Prepare this buffer in two forms: one with and one without the urea and Triton
`X-100 (the latter for use in Basic Protocol 1, step 9).
`
`COMMENTARY
`Background Information
`The decision of whether to work with in-
`soluble recombinant protein or to put more ef-
`fort into generating soluble protein (e.g., by
`modifying the expression vector, changing the
`host strain and fermentation conditions, or co-
`expressing chaperones) can be dictated by the
`nature of the protein. A small protein (10 to
`17 kDa) with only one or two cysteine residues
`might be expected to fold in reasonable yield
`from extracted inclusion bodies. Larger pro-
`teins (>25 kDa) with many cysteine residues
`may be more problematic, and lower folding
`yields can normally be expected. In the lat-
`ter case, if only small amounts of material are
`needed then yield is not such an important
`issue.
`It should be emphasized that, unless proved
`otherwise, a protein folded from insoluble in-
`clusion bodies can be expected to have the
`same structural and conformational integrity
`as the same protein directly purified from sol-
`uble extracts (also see UNIT 6.1). It is similarly
`true that a purified soluble protein can be dena-
`tured and renatured (reversible denaturation)
`without structural or conformational modifi-
`cations (reviewed by Anfinson, 1973; Fersht,
`1999; and Pain, 2001).
`Basic Protocol 1 describes the prepara-
`tion of inclusion bodies suitable for extrac-
`tion purposes. The insoluble protein is washed
`in buffers containing various additives by sus-
`pension and low-speed centrifugation. The re-
`sultant washed pellets (inclusion bodies), al-
`though highly enriched in the recombinant pro-
`tein (Figure 6.3.1), are still contaminated with
`bacterial proteins, lipids, and nucleic acids.
`This is not an issue if the protein is going
`to be partially purified, e.g., by gel filtration
`(Basic Protocol 2) prior to folding. However,
`if the solubilized protein is going to be di-
`rectly used in folding scenarios, there may
`be an advantage in further purifying the in-
`clusion bodies, as the bacterial contaminants,
`
`especially the nonproteinacous ones, may in-
`fluence protein folding (Darby and Creighton,
`1990; Maachupalli-Reddy et al., 1997). Inclu-
`sion bodies have also been purified from bac-
`terial lysates by sucrose step-gradient centrifu-
`gation (Georgiou and Valax, 1999).
`
`How inclusion bodies are formed
`Inclusion bodies are noncrystalline, amor-
`phous structures; however, there is some ev-
`idence that the constituent densely packed
`proteins may have native-like secondary struc-
`tures (Oberg et al., 1994). This suggests that
`the aggregates are formed by the association
`of partially folded protein or misfolded pro-
`tein. Folded subdomains could presumably as-
`sociate with complementary binding surfaces
`in an intermolecular manner to form oligomers
`and eventually aggregates (Fink, 1998). Dur-
`ing in vitro folding studies with a mixture of
`proteins, folding intermediates do not coaggre-
`gate with each other, but only with themselves.
`This indicates that aggregation occurs by spe-
`cific interaction of certain conformations of
`folding intermediates rather than by nonspe-
`cific coaggregation, providing a rationale for
`recovering relatively pure protein from the in-
`clusion body state (Speed et al.,1996).
`The propensity of a protein to form inclu-
`sion bodies does not appear to be directly re-
`lated to the presence of sulfhydryl residues,
`as proteins without sulfhydryls still form such
`aggregates. It has been found that aggregates
`derived from proteins that in their native state
`contain disulfides consisting mainly of re-
`duced protein (Langley et al., 1987). The re-
`duction of disulfides in native proteins can
`dramatically reduce their solubility and even
`result in unfolding. Hence the reducing envi-
`ronment of E. coli may contribute to the in-
`stability and low solubility of these types of
`protein. Once the protein has been solubilized,
`reducing agents must be included to prevent
`the formation of nonnative disulfide bonds.
`
`Current Protocols in Protein Science
`
`Preparation and
`Extraction of
`Inclusion Bodies
`
`6.3.8
`
`Supplement 38
`
`Page 8
`
`

`

`Figure 6.3.1 Analysis by SDS-PAGE of fractions from low-speed centrifugation of E. coli cell
`lysates containing aggregated bovine growth hormone. A 12.5% acrylamide gel of dimensions
`12 cm × 16 cm × 1.5 mm was used with the Laemmli buffer system (UNIT 10.1). Lanes a and g
`contain molecular weight standards (low-range standards, Bio-Rad) in order of increasing migration
`distance: phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg white ovalbumin
`(45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and hen egg
`white lysozyme (14.4 kDa). After low-speed centrifugation of the clarified lysate and of the washed
`pellet homogenate (see Basic Protocol 1, steps 5 and 7), the supernatants will be cloudy (lane f)
`and the pellets usually consist of two layers (see Fig. 6.1.5). The bottom layer is inclusion body
`protein plus unbroken cells (lanes b and c) and the top layer consists of outer membrane and
`peptidoglycan fragments (lanes d and e). The outer membrane proteins OmpA (35 kDa) and
`OmpF/C (38 kDa) are indicated by ; and o, respectively. After the washing steps, the growth
`hormone (marked β, 21kDa) is the major constituent (lanes h and i) together, in this example, with
`another plasmid-encoded protein, namely kanamycin phosphotransferase (marked α, 30.8 kDa),
`the product of the gene conferring resistance to the antibiotic kanamycin.
`
`Some of the other factors that may con-
`tribute to aggregate formation include: lack
`of post-translational modification (especially
`glycosylation), lack of access to chaperones,
`and enzymes catalyzing folding (e.g., cis-trans
`isomerase) and the very high concentration of
`protein coupled with limited solubility of fold-
`ing intermediates.
`Although inclusion body formation and its
`prevention are of great academic and commer-
`cial interest, the pragmatic situation is clearly
`summarized by Seckler and Jaenicke (1992),
`who state: “That inclusion bodies are aggre-
`gates of otherwise intact polypeptides in non-
`nativelike conformations has been proven re-
`peatedly by the successful refolding of active
`proteins after dissociation of the aggregates by
`chemical dissociation.” It must be noted that
`the work of Oberg et al. (1994) does not in-
`
`validate this statement. It is simply a matter
`of terminology: the protein in aggregates may
`have nativelike secondary structures, but com-
`pared with soluble folded protein it must still
`be considered nonnative.
`
`Extracting inclusion body protein
`Proteins are extracted from inclusion bod-
`ies using strong protein denaturants. Protein
`denaturation can be induced by the following
`solvent conditions or reagents.
`1. pH. Protein denaturation occurs because
`of the ionization of side chains. Generally, pro-
`teins retain less residual structure (are more
`denatured) when exposed to high pH (e.g.,
`>10.5) compared to low pH (<4.5). Acidic
`or basic pH may be used in conjunction with
`urea or inorganic salts. An example of acidic
`pH extraction is given in UNIT 6.5.
`
`Current Protocols in Protein Science
`
`Purification of
`Recombinant
`Proteins
`
`6.3.9
`
`Supplement 38
`
`Page 9
`
`

`

`Table 6.3.1 Gel-Filtration Matrices Suitable for Use with Solutions Containing Guanidine
`Hydrochloride
`
`Matrixa
`
`Mass range (kDa)
`Native proteins Unfolded proteinsb
`
`Reference
`
`Mann and Fish (1972)
`1-80
`10-4,000
`Sepharose CL-6B
`Mann and Fish (1972)
`1-80
`10-5,000
`Bio-Gel A-5m
`Mann and Fish (1972)
`10-300
`60-20,000
`Sepharose CL-4B
`<1-30 c
`1-100
`Sephacryl S-100 HR
`—
`Belew et al. (1978)
`1-50
`5-250
`Sephacryl S-200 HR
`1-100 c
`10-1,500
`Sephacryl S-300 HR
`—
`1->100 c
`20-8,000
`Sephacryl S-400 HR
`—
`I.P. and P.T.W. (unpub. observ.)
`<1-25
`3-70
`Superdex 75
`I.P. and P.T.W. (unpub. observ.)
`1-80
`10-600
`Superdex 200
`aAll resins are from Amersham Biosciences except Bio-Gel A-5m, which is from Bio-Rad. The Sepharose and Bio-Gel
`matrices are normally run under low pressure; all other resins can be run under low or medium pressure. Medium pressure
`is achieved using one of the chromatography pumps indicated in Basic Protocol 2; the pumps are normally included in
`the Amersham Biosciences ¨AKTA-FPLC or ¨AKTAexplorer systems.
`bData on the fractionation range in the unfolded state refer to proteins unfolded with guanidine·HCl; however,the
`guidelines also apply to proteins unfolded and eluted with urea (assuming they are random coils).
`cEstimates based on fractionation range for native proteins.
`
`2. Organic solvents. Examples of organic
`solvents include ethanol and propanol. Gen-
`erally, proteins are not completely unfolded
`in these solvents. Organic solvents are infre-
`quently used for the primary extraction of in-
`clusion bodies but can be useful cosolvents to
`enhance protein folding (see Table 1 in De
`Bernardez Clark, et al., 1999 and references
`cited therein).
`3. Organic solutes. Examples of organic so-
`lutes include guanidine·HCl (used at 6 to 8
`M) and urea (used at 6 to 9 M). The effec-
`tiveness of urea is modulated by pH and ionic
`strength, whereas that of guanidine is not. Both
`solutes are more effective at elevated temper-
`atures (see additional discussion of tempera-
`ture under point 6, below). Organic solutes are
`the most versatile and most commonly used
`denaturants for solubilizing inclusion bodies.
`The denaturing power of guanidinium salts in-
`−− < Br− < I− < SCN
`
`
`
`creases in the order Cl
`.
`4. Detergents. The most common ex-
`amples of protein-denaturing detergents are
`sodium dodecyl sulfate (SDS, an anionic deter-
`gent) and cetyltrimethylammonium bromide
`(CTAB, a cationic detergent). SDS is a very ef-
`fective protein denaturant, as it directly binds
`to the protein However, it should be used with
`caution to solubilize inclusion bodies, as it
`can interfere with the recovery folded pro-
`tein in reasonable yields (see, e.g., van Kim-
`menade et al., 1988). Solubilization with the
`
`anionic detergent N-lauroylsarcosine (Sarko-
`syl) has been used to effectively extract inclu-
`sion body protein with subsequent successful
`protein folding (Nguyen et al., 1993; Burgess
`and Knuth, 1996). Sarkosyl, with a critical
`micelle concentration