NIH Public Access
`Author Manuscript
`Curr Protoc Protein Sci. Author manuscript; available in PMC 2012 December 10.
`Published in final edited form as:
`Curr Protoc Protein Sci. 2004 November ; CHAPTER: Unit–6.3. doi:10.1002/0471140864.ps0603s38.
`
`Preparation and Extraction of Insoluble (Inclusion-Body)
`Proteins from Escherichia coli
`
`Ira Palmer and Paul T. Wingfield
`National Institutes of Health Bethesda, Maryland
`
`Abstract
`High-level expression of many recombinant proteins in Escherichia coli leads to the formation of
`highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally
`formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic
`space. Inclusion bodies can be recovered from cell lysates by low speed centrifugation. Following
`preextaction (or washing) protein is extracted from washed pellets using guanidine·HCl. The
`solubilized and unfolded protein is either directly folded as described in UNIT 6.1 or further
`purified by gel filtration in the presence of guanidine·HCl as described here. A support protocol
`describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-
`PAGE.
`
`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 purification 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.
`
`BASIC PROTOCOL 1 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)
`
`Author for correspondence: Paul T. Wingfield, NIH Building 6B, Room IB32. Bethesda. MD 20892-2775. pelpw@helix.nih.gov.
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`Palmer and Wingfield
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`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: http://www.kinematica-inc.com)
`
`French pressure cell (e.g., Thermo Electron Corp: http://www.thermo.com)
`
`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 in lysis 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 EMD Chemicals and Sigma.
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`2
`
`3
`
`4
`
`5
`
`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.
`
`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.
`
`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.
`
`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.
`
`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 between 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
`
`8
`
`9
`
`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.
`
`Repeat step 7 two more times.
`
`If the supernatant is still cloudy or colored, continue washing the pellet
`until the supernatant is clear.
`
`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.
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`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
`extraction 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 g wet 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 determined by measuring the total protein concentration or
`by analyzing the washed pellets via SDS-PAGE (see Support Protocol
`and UNIT 10.1) to determine 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.
`
`Alternative Extraction of protein using a reduced concentration of chaotrope
`Some recombinant protein may be extracted from an inclusion body with a lower
`concentration of chaotrope resulting in fewer contaminants being extracted as well. The
`effectiveness of using a low chaotrope extraction buffer needs to be evaluated on a case by
`case basis. If it is determined to be effective, substitute the extraction buffer with the low
`chaotrope extraction buffer.
`
`11
`
`12
`
`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).
`
`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.
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`13
`
`Use the clarified inclusion body extract for preparing folded protein (UNIT 6.5)
`or purify further by gel filtration (see Basic Protocol 2).
`
`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.
`BASIC PROTOCOL 2 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) in which the denatured protein is folded
`into a native and biologically active structure.
`
`Materials
`
`Gel-filtration medium: Superdex 200 PG (preparative grade; GE Healthcare Life
`Sciences)
`
`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: GE Healthcare Life Sciences XK 16/100, 26/100, or 50/100
`
`Packing reservoir: GE Healthcare Life Sciences RK 16/26 (for 16- and 26-mm-i.d.
`columns) and RK 50 (for 50-mm-i.d. column)
`
`Chromatography pump: GE Healthcare Life Sciences P-50 or P-900
`
`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, monitors,
`and sample loops) listed separately above are supplied as components of the ÄKTAexplorer
`chromatography system (GE Healthcare Life Sciences), 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 ÄKTA-FPLC
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`chromatography system (also from GE Healthcare Life Sciences), which is designed for
`small- to medium-scale work. For further details on this equipment see the manufacturer’s
`literature (e.g., Process Products, GE Healthcare Life Sciences).
`
`NOTE: Perform steps 1 to 11 at room temperature. After the column is packed, equilibrate
`and elute at 4°C.
`
`Pack the column
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`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%.
`
`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).
`
`Fix the chromatography column in an upright position, using a level to adjust the
`position. Attach the packing reservoir.
`
`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.
`
`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.
`
`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.
`
`Carefully pour the slurry of medium into the column, introducing material along
`the side of the column to avoid creating air bubbles.
`
`Let the column stand 5 min and then unclamp the bottom of the column.
`
`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.
`
`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.
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`10
`
`11
`
`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.
`
`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.
`
`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 XK 50/100 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 XK 50/100 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 particularly low extinction coefficient, at 230 nm
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`(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
`
`100% and 90% ethanol, 0° to 4°C
`
`1× SDS sample buffer (UNIT 10.1)
`
`Gilson Pipetman (http://www.gilson.com)
`
`1
`
`Additional reagents and equipment for gel electrophoresis (UNIT 10.1)
`Pipet 25 μl sample containing the protein of interest into a 1.5-ml
`microcentrifuge tube.
`Add 225 μl cold (0° to 4°C) 100% ethanol to the sample in the tube.
`The final ethanol concentration is 90% by volume.
`
`2
`
`3
`
`4
`
`5
`
`6
`
`Mix the sample and ethanol by vortexing. Chill 5 to 10 min at
`−20°C or colder (e.g., −80°C).
`
`Do not use a magnetic stirrer, as it could damage the
`medium.
`
`Microcentrifuge the sample 5 min at maximum speed (~15,000 ×
`g), 4°C. Carefully withdraw the supernatant and retain the pellet.
`
`The pellet may be difficult to see. Be careful not to draw
`the pellet out of the microcentrifuge tube with the
`supernatant.
`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.
`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
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`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° to 100°C. Load on an SDS-
`polyacrylamide gel (UNIT 10.1).
`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 mM EDTA
`
`8 M guanidine·HCl (764 g/liter)
`
`5 mM DTT (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, Invitrogen Life Technologies
`—is used; see APPENDIX 3A). Buffer can be stored without DTT for at least 1 month
`at 4°C.
`
`Low Chaotrope Extraction buffer
`50 mM Tris·Cl, pH 7.0
`
`5 mM EDTA
`
`3 M guanidine·HCl (287 g/liter)
`
`5 mM DTT (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, Invitrogen Life Technologies
`—is used; see APPENDIX 3A). Buffer can be stored without DTT for at least 1 month
`at 4°C.
`
`Gel-filtration buffer
`50 mM Tris·Cl, pH 7.5
`
`4 M guanidine·HCl (382 g/liter; ultrapure)
`
`5 mM DTT (770 mg/liter)
`
`Buffer can be stored without DTT at least 1 month at 4°C. Filter (as for 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 mM EDTA
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`5 mM DTT (770 mg/liter)
`
`5 mM benzamidine·HCl (780 mg/liter)
`
`Prepare immediately before use
`
`The Tris·Cl and EDTA are diluted from concentrated stock solutions. The
`other components are added to the diluted buffer before use.
`
`Wash buffer
`
`100 mM Tris·Cl, pH 7.0
`
`5 mM EDTA
`
`5 mM DTT (770 mg/liter)
`
`2 M urea (120 g/liter; ultrapure (Invitrogen Life Technologies)
`
`2% (w/v) Triton X-100 (20 g/liter; Calbiochem EMD Chemicals)
`
`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 insoluble recombinant protein or to put more effort
`into generating soluble protein (e.g., by modifying the expression vector, changing the host
`strain and fermentation conditions, or coexpressing chaperones etc.) is dictated by the nature
`of the protein. Enhancement of soluble protein expression has also been achieved by the use
`of fusion tags (Esposito and Chatterjee, 2006). If protein folding is attempted, a small
`protein (10 to 17 kDa) with, for example, one or two cysteine residues might be expected to
`fold in reasonable yield (>25%) from extracted inclusion bodies. Larger proteins (>25 kDa)
`with many cysteine residues may be more problematic, and lower folding yields (< 20%)
`can normally be expected. In the latter case, if only small amounts of material are needed
`then lower folding yields will still provide adequate for study.
`
`It should be emphasized that, unless proven otherwise, a protein folded from insoluble
`inclusion bodies will have the same structural and conformational integrity as the same
`protein directly purified from soluble extracts (also see UNIT 6.1). It is similarly true that a
`purified soluble protein can be denatured and renatured (reversible denaturation) without
`structural or conformational modifications (reviewed by Anfinson, 1973; Fersht, 1998; Pain,
`2001, Tsumoto et al., 2003). The main danger of working with an unfolded protein is that it
`vulnerable to chemical modification, especially oxidations of methionine and cysteine and
`also is very sensitive to any contaminating protease activity.
`
`Basic Protocol 1 describes the preparation of inclusion bodies suitable for extraction
`purposes. The insoluble protein is washed in buffers containing various additives by
`suspension and low-speed centrifugation. The resultant washed pellets (inclusion bodies),
`although highly enriched in the recombinant protein (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 directly used in folding scenarios, there may be an
`advantage in further purifying the inclusion bodies, as the bacterial contaminants, especially
`the nonproteinacous ones, may influence protein folding (Darby and Creighton, 1990;
`
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`Maachupalli-Reddy et al., 1997). Inclusion bodies have also been purified from bacterial
`lysates by sucrose step-gradient centrifugation (Georgiou and Valax, 1999).
`
`How inclusion bodies are formed—Inclusion bodies are noncrystalline, amorphous
`structures; however, there is some evidence that the constituent densely packed proteins may
`have native-like secondary structures (Oberg et al., 1994). This suggests that the aggregates
`are formed by the association of partially folded protein or misfolded protein. Folded
`subdomains could presumably associate with complementary binding surfaces in an
`intermolecular manner to form oligomers and eventually aggregates (Fink, 1998). During in
`vitro folding studies with a mixture of proteins, folding intermediates do not coaggregate
`with each other, but only with themselves. This indicates that aggregation occurs by specific
`interaction of certain conformations of folding intermediates rather than by nonspecific
`coaggregation, providing a rationale for recovering relatively pure protein from the inclusion
`body state (Speed et al., 1996).
`
`The propensity of a protein to form inclusion bodies does not appear to be directly related 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 reduced protein (Langley et al., 1987). The reduction
`of disulfides in native proteins can dramatically reduce their solubility and even result in
`unfolding. Hence the reducing environment of E. coli may contribute to the instability 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.
`
`Some of the other factors that may contribute 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 folding intermediates.
`
`Although inclusion body formation and its prevention are of great academic and commercial
`interest, the pragmatic situation is clearly summarized by Seckler and Jaenicke (1992), who
`state: “That inclusion bodies are aggregates of otherwise intact polypeptides in nonnativelike
`conformations has been proven repeatedly by the successful refolding of active proteins
`after dissociation of the aggregates by chemical dissociation.” It must be noted that the
`aforementioned findings of Oberg et al. (1994) do not invalidate this statement. It is simply a
`matter of terminology: the protein in aggregates may have nativelike secondary structures,
`but compared with soluble folded protein it must still be considered nonnative. For an
`updated discussion of inclusion bodies formation and their potential application in
`nanotechnology see García-Fruitós et al., (2012).
`
`Extracting Inclusion Body Protein—Proteins are extracted from inclusion bodies 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,
`proteins 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.
`
`2. Organic solvents: Examples of organic solvents include ethanol and propanol. Generally,
`proteins are not completely unfolded in these solvents. Organic solvents are infrequently
`used for the primary extraction of inclusion bodies but can be useful cosolvents to enhance
`protein folding (see Table 1 in De Bernardez Clark, et al., 1999 and references cited therein).
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`3. Organic solutes: Examples of organic solutes include guanidine·HCl (used at 6 to 8 M)
`and urea (used at 6 to 9 M). The effectiveness of urea is modulated by pH and ionic strength,
`whereas that of guanidine is not. Both solutes are more effective at elevated temperatures
`(see additional discussion of temperature 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 increases in the order Cl− < Br− < I− < SCN−.
`Arginine (0.1–1M) has long been used to facilitate refolding of recombinant prote