Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`A PRACTICAL GUIDE TO PROTEIN EXPRESSION AND REFOLDING
`FROM INCLUSION BODIES
`
`Lisa D. Cabrita, Michelle K.M. Chow and Stephen P. Bottomley
`
`This is an edited version of:
`“Protein Expression and Refolding – a practical guide to getting the most out of inclusion
`bodies”, Cabrita and Bottomley, 2004, Biotechnology Annual Review, 10:31-50.
`
`If referring to anything in this document, please cite the above paper.
`
`Contents
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`1. Introduction
`2. Recombinant protein expression in E.coli
`
`
`
`
`
`
`2.1
`Promotors and fusion partners
`2.2
`Codon bias, disulfide bond formation and E.coli strains
`2.3
`Cell-free expression
`
`
`
`
`
`3. Preparation and solubilization of inclusion bodies
`
`
`4.
`
`
`
`
`
`5. Refolding
`5.1
`Protein concentration during refolding
`5.2
`The refolding buffer
`
`
`
`5.3
`Disulfide bond formation
`
`
`5.4
`Refolding methods
`5.5
`Folding aids
`5.6
`Folding screens
`6. Analysis of refolded protein
`7. Conclusion
`Figure 1
`Table 1
`Table 2
`Table 3
`References
`
`
`
`
`
`
`
`
`
`
`
`1
`
`
`
`
`
`KASHIV EXHIBIT 1072
`IPR2019-00797
`
`Page 1
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`1. INTRODUCTION
`One of most significant aspects of biomedical
`and scientific research in the post-genomic
`era is the ability to rapidly express and purify
`a target protein. With an immense amount of
`sequence data available, all over the world,
`structural genomic projects are being
`undertaken in which a vast array of proteins
`are expressed, purified and structurally
`characterised. In addition to these high-
`throughput approaches, both academic and
`industrial laboratories are expressing proteins
`of
`interest
`for structural,
`functional and
`therapeutic
`investigations. The general
`course for this process involves rapid cloning
`of target genes, followed by recombinant
`expression of the protein, usually in the host
`E.coli.
` Currently, E.coli remains the dominant
`expression host for recombinant proteins,
`due to its advantages such as ease of
`handling, cost effectiveness and high
`success rates. However, it does have some
`disadvantages, for example, the inability to
`perform many post-translational modifications
`and frequent deposition of the expressed
`protein product
`into
`insoluble
`inclusion
`bodies. Inclusion body formation is often an
`undesired event, and a number of
`approaches can be utilized to obtain soluble
`protein. On the other hand, inclusion body
`formation does not necessarily have to be a
`dead-end process. Sometimes harvesting of
`proteins from inclusion bodies can prove to
`be very fruitful and may present a valid option
`for successful purification (Figure 1).
`In
`recent years, there has been an increase in
`products and protocols which are generally
`applicable to the inclusion body issue. New
`strains of E.coli and fusion partners are
`available, which offer a range of options to
`minimize, or maximize,
`inclusion body
`formation. Furthermore, a more
`rational
`approach
`to protein refolding has been
`established, which allows
`the user
`to
`determine very quickly whether refolding from
`inclusion bodies is a viable option.
`
`
`2. RECOMBINANT PROTEIN EXPRESSION
`IN E.COLI
`recombinant proteins
`The expression of
`within E.coli is affected by several factors.
`These include, but are not limited to: plasmid
`copy number, mRNA stability, upstream
`elements, temperature and codon usage. The
`expression of an uncharacterized protein
`often depends largely on a combination of
`these and other factors, and success in
`protein expression is often difficult to predict.
`However, over the years numerous advances
`have been made to improve both expression
`and solubility. This has been made possible
`through the development of novel tags, fusion
`partners, and vector systems, such that the
`choices for recombinant expression in E.coli
`are continually expanding.
`
`2.1 Promoters and fusion partners
`In regulating protein expression, the choice of
`promoter and/or vector system is important,
`as one system may be more suited to a given
`target
`protein
`than
`another. Several
`promoters are available (Table 1) - probably
`the most well-known variety is the T7-derived
`promoter, as
`found
`in
`the pET vectors
`(Novagen). These IPTG-inducible promoters
`have in the past been associated with “leaky”,
`or premature, expression prior to induction,
`which is problematic for proteins which are
`toxic to cells. However, this issue has been
`addressed with co-transformed plasmids
`pLysS and pLysE which can act as strong
`repressors. Aside from this issue, the T7 is a
`strong promoter which enables high level
`expression of target proteins. Overexpression
`can also lead to the formation of intracellular
`inclusion bodies, sometimes in far greater
`excess
`than
`the soluble protein. This
`insoluble-soluble ratio can sometimes be
`adjusted by altering the expression time,
`induction temperature and/or IPTG levels.
`
`The T7-based vectors have previously
`included affinity tags to facilitate the detection
`and purification of proteins, although they are
`also now often designed to include other
`features. Of interest are ‘fusion partners’ that
`
`2
`
`Page 2
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`in general, ‘fuse’ the protein of interest with
`another, to circumvent the solubility issue and
`also aid in purification. The classic GST
`fusion system has been
`implemented
`successfully for a large number of proteins [1-
`4], however more recently, a number of other
`alternative options have also been developed
`(Table 2 and [5]). One example is the 42 kDa
`maltose binding protein (MBP), which has
`been used successfully
`to
`increase
`the
`solubility of a range of targets [6-8] and has
`been shown
`to have
`little effect during
`crystallization - this is a great advantage, as
`fusion tags generally need to be removed to
`aid in crystallography. A similar tag is Nus A
`(Novagen), a 54 kDa protein, which was
`identified as being the most soluble protein
`out of a pool of almost 4000 E.coli proteins
`[9]. As well as assisting in folding, some tags,
`such as the ~15 amino-acid S-tag (Novagen),
`for example can be used to detect, quantitate
`and purify soluble protein [10].
`
`2.2 Codon bias, disulfide bond formation
`and E.coli strains
`Poor expression of a target protein may also
`be associated with codon bias – that is,
`codons used infrequently in the prokaryotic
`system. The Rosetta (Novagen) and BL21-
`CodonPlus strains (Stratagene) have the
`advantage of co-expressing plasmids that
`code for the rare tRNA’s and have been used
`with success for a number of proteins [11-13].
`Moreover, the development of E.coli that
`accommodates disulfide bond formation can
`also improve the yields of certain targets. The
`AD494(DE3) strain (Novagen), which has a
`single mutation in the thioredoxin reductase
`gene, has enabled the production of a range
`of proteins
`[14-16]. The Origami strain
`(Novagen), however, combines a double
`mutation in both the thioredoxin reductase
`and the glutathione reductase gene, hence
`providing a more
`favourable oxidizing
`environment
`within
`the
`cytoplasm.
`Furthermore, the recent Rosetta-Gami strain
`(Novagen) is a powerful combination of the
`Rosetta and Origami strains, encompassing
`rare codon usage and disulfide bond
`
`formation. This strain may be particularly
`useful for eukaryotic or intracellular proteins
`which
`typically display alternative codon
`usage and require disulfide bond formation,
`respectively.
`
`2.3 Cell-free expression
`Cell free expression (also known as ‘in vitro
`transcription-translation’)
`has
`been
`an
`invaluable tool for many years. It exploits the
`E.coli, wheat germ and rabbit reticulocyte
`systems
`to generate protein and
`is
`increasingly seen as a viable alternative to
`other expression systems
`for recalcitrant
`proteins. The technology itself was developed
`some 30 years ago [17], however, more
`recently been subject to several significant
`developments
`[18,19]. These
`include
`the
`‘continuous-flow’ system whereby amino
`acids and energy sources are supplied into a
`reaction chamber, while synthesized proteins
`and used substrates are removed using an
`ultrafiltration membrane
`[18], and
`the
`“semicontinuous’ method, involving the use of
`two chambers separated by a dialysis
`membrane [19,20]. Roche also now offers a
`transcription-translation
`‘Rapid
`in
`vitro
`Translation System’, which has been
`successful for a number of targets, and can
`also
`incorporate molecular chaperones,
`detergents and other additives to assist in
`folding.
`
`Although not as efficient as the E.coli
`system, the cell free system based upon
`wheatgerm has also been used [21-23].
`Being a eukaryotic system, it has been
`explored as an alternative, particularly for the
`production of proteins that may not otherwise
`express in E.coli. Cell-free protein expression
`has been limited in the past by low efficiency,
`membrane
`clogging
`and
`consequent
`questionable reproducibility. However, in light
`of recent advancements in the technology,
`continued
`improvements may encourage
`more frequent use and in the future it may
`emerge as an increasingly viable alternative
`to the traditional E.coli expression system.
`
`
`3
`
`Page 3
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`3. PREPARATION AND SOLUBILIZATION
`OF INCLUSION BODIES
`Inclusion bodies are dense amorphous
`aggregates of misfolded protein. They are
`generally formed if a protein has a high
`propensity to misfold and aggregate, or if the
`cellular protein production machinery
`is
`overwhelmed and unable
`to operate
`efficiently. The formation of inclusion bodies
`can sometimes be especially advantageous,
`especially if the protein is toxic to the cell, or
`susceptible to proteolytic attack.
`
`Inclusion bodies are easily identifiable,
`with a morphology similar
`to strings or
`clusters [24], and their isolation from other
`cellular components can be easily achieved.
`Inclusion bodies are contained within the
`insoluble fraction yielded from cell lysis and
`subsequent centrifugation, and it is generally
`accepted that they may be composed of 40-
`90% target protein. Successful renaturation
`depends on the purity of inclusion bodies, as
`proteinaceous contaminants can decrease
`the efficiency of renaturation, presumably as
`such
`contaminants
`can promote
`co-
`aggregation
`[25].
`Inclusion bodies are
`therefore best washed and centrifuged a
`several times in buffer containing detergents
`such as Triton X-100 (0.1-4% (v/v)), sodium
`deoxycholate (2% (w/v)), sarkosyl, or even
`low concentrations (0.5-1 M) of denaturants
`such as guanidine hydrochloride (GdnHCl) or
`urea. Such additives
`remove
`cellular
`contaminants
`that
`adsorb
`onto
`the
`hydrophobic inclusion bodies.
` Following washing, inclusion bodies are
`then solubilized, usually with 4-6 M GdnHCl
`or 8 M urea. GdnHCl
`is a stronger
`denaturant,
`while
`during
`prolonged
`incubations at alkaline pH urea can suffer
`from the formation of isocyanate ions which
`can modify amino acid side chains [26,27].
`Aside
`from denaturants, other unfolding
`alternatives
`include detergents such as
`sarkosyl [28,29], SDS [30] and alkaline pH
`[31,32]. It is important that during unfolding
`any disulfide bonds present are also reduced
`–
`this
`is usually achieved with β-
`mercaptoethanol or DTT (5-100 mM). The
`
`is
`reduction
`for disulfide bond
`need
`highlighted by a pivotal study which
`demonstrated that these bonds persist in high
`denaturant concentrations in the absence of
`reducing agents [33]. Usually solubilization
`can be performed in any buffer that is
`compatible with the protein of interested, for
`example Tris, Hepes or phosphate. Generally
`a neutral pH
`(7-8)
`is
`suitable
`for
`solubilization.
` Upon solubilization of inclusion bodies,
`ample incubation time should be allowed for
`complete unfolding to occur. Incubation at
`either room temperature or 30°C for 1-4
`hours is generally sufficient, whilst some
`studies have opted for 16-24 hours at 4°C.
`Sometimes inclusion bodies may be difficult
`to solubilize and therefore some agitation or
`increased temperature may be required.
`
`Sometimes inclusion bodies are purified
`further by column chromatography such as
`ion exchange, size exclusion or metal affinity
`conducted under denaturing conditions [34-
`36]. This has been seen to enrich the
`proportion of monodispered protein which
`can increase the yield of the refolded target
`protein [37]. More recently, it has been shown
`that inclusion bodies could be directly purified
`by gel filtration only using a macroporous
`medium (eg. 4% agarose) coupled with a
`French press to reduce cell debris [38].
`
`4. REFOLDING
`The renaturation process aims to effectively
`remove the denaturant and thiol reagents and
`allow the protein to refold. The refolding
`process
`is a competing
`reaction with
`misfolding and aggregation events and its
`success depends on a number of factors.
`Studies have shown that for many proteins,
`especially those larger than 150 amino acids,
`folding
`involves
`the
`formation of an
`intermediate species, often resembling a
`‘molten globule’. The molten globule
`ensemble can represents a branch point in
`the folding pathway where it may also lead to
`misfolding and aggregation, as this species
`contains some secondary structure, but little
`
`4
`
`Page 4
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`tertiary structure and hydrophobic patches
`normally buried within
`the protein are
`exposed to solvent [39]. Under appropriate
`conditions
`these
`regions can promote
`aberrant
`interactions
`that may
`lead
`to
`aggregation. Therefore
`it
`is important to
`minimize the aggregation reaction. Several
`variables play important roles in this process,
`including final concentration of the protein to
`be refolded, the components of the refolding
`buffer and the method of refolding.
`
`4.1 Protein concentration during refolding
`The amount of protein refolded will impact the
`yield of protein obtained. Folding competes
`with aggregation, and therefore it is generally
`accepted
`that
`refolding at
`low protein
`concentrations (10-100µg/ml) is the most
`successful approach. There have been
`situations where proteins have been refolded
`in high concentrations of up to 5 mg/ml [40-
`42], albeit
`in
`low
`concentrations of
`denaturant, however, generally speaking, the
`lower the final protein concentration during
`refolding, the greater the efficiency of the
`process.
`
`4.2 The refolding buffer
`Components of a refolding buffer vary widely,
`depending on
`the protein of
`interest.
`Parameters such as pH, ionic strength, redox
`conditions and the presence of ligands can all
`influence the outcome of refolding. Most
`commonly Tris or Hepes-based buffers at
`neutral pH with 50-500 mM NaCl are used,
`however again this depends on the target
`protein. Numerous additives can also be
`included, with varying success with numerous
`targets. These
`include detergents, polar
`additives, weak chaotrophs, osmolytes and
`cations (Table 3). Such additives may either
`act as stabilizers (which stabilize the native
`state or solubilize intermediate forms), or
`promote correct
`folding by preventing
`aggregation. If the target protein is prone to
`proteolysis, a common approach is to include
`proteinase inhibitors (eg. PMSF, aprotinin,
`leupeptin) in the refolding buffer.
`
`4.3 Disulfide bond formation
`is
`A
`redox system during
`renaturation
`required for the correct formation of proteins
`with native disulfide bonds. The combination
`of reduced and oxidized forms of redox
`agents are generally used in molar ratios
`from
`1:1
`up
`to
`10:1. Glutathione
`(GSH/GSSG) is a common reagent, however,
`other alternatives may include a combination
`of cysteine and cystine, or DTT/oxidized
`glutathione. While molecular oxygen in the air
`is suitable and sufficient to promote disulfide
`bond formation, a redox system accelerates
`the shuffling of disulfide bonds. Trace
`amounts of metal ions and slightly alkaline
`pH (8-9) may also be included to catalyze the
`redox reaction. EDTA can be beneficial to
`minimize the effects of oxygen oxidizing
`thiols. The time required for disulfide bond
`shuffling varies between proteins, anywhere
`from 2 hours to 150 hours. 16-48 hours is
`common
`for efficient disulfide shuffling,
`however, the exact time must be determined
`empirically.
`
`4.4 Refolding methods
`Several different methods can be used to
`refold proteins. These
`include dialysis,
`dilution
`and
`column
`chromatography
`techniques. The method selected depends on
`the propensity of the protein to aggregate and
`the kinetics of refolding. The temperature at
`which refolding
`is performed may vary,
`although generally,
`in order minimize
`aggregation, 4°C is best.
`
`Dilution
`Refolding by dilution can be described as
`‘rapid’ or
`‘slow’.
`In
`rapid dilution,
`the
`denatured protein is delivered to the refolding
`buffer such that in a very short time period,
`the concentrations of both the protein and
`denaturant decrease rapidly. For example, if
`the protein was denatured in 8 M urea and
`diluted 10-fold
`into buffer,
`the
`final
`concentration of denaturant is 0.08 M. The
`aim of dilution is to remove the denaturant, so
`its final concentration should be low. There
`
`5
`
`Page 5
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`are instances, however, where refolding into
`low molar concentrations of denaturant (1-2
`M) aids in the solubility of the protein.
` Rapid dilution has been used successfully
`for a range of proteins [43-46], however, the
`time period used dilution may be detrimental
`for some proteins, especially if they usually
`refold over a relatively long time period of
`minutes to hours. Forcing the protein to adopt
`its conformation in a short time frame may
`increase
`its chances of misfolding and
`aggregation. Slow dilution is an alternative
`method, as it is a more gentle approach and
`dramatically
`decreases
`the
`effective
`concentration of the refolding protein. The
`‘dropwise’ or ‘pulsed dilution’ method involves
`the solubilized
`inclusion bodies being
`delivered very slowly to the refolding buffer
`(using a pump) and has been successfully
`used for some proteins [36,47].
`
`Dialysis
`In this method, the concentrated denatured
`protein is dialyzed against a refolding buffer,
`such that the concentration of denaturant
`decreases with buffer exchange. This slow
`removal of denaturant allows refolding to
`occur. Unfortunately, the slow removal of
`denaturant often results in the formation and
`exposure of long lived intermediate species
`over a time period and hence there may be
`an increased propensity for the protein to
`aggregate. A variation of one-step dialysis,
`where protein is dialyzed against refolding
`buffer containing no denaturant, is the use of
`a series of refolding buffers with decreasing
`denaturant concentrations. By dialyzing in a
`step-wise fashion (usually 1-3 progressive
`lower denaturant concentrations), this allows
`an equilibrium to be established. Once again,
`whilst refolding is more controlled in this
`environment, long-live intermediate species
`can still be problematic.
`
`On-column refolding
`On-column refolding has been applied to
`several proteins with success and offers an
`alternative where other methods may not be
`
`fruitful. If a protein has a hexa-histidine tag, it
`can be immobilized on a Ni2+ affinity column
`in its denatured state. By applying buffers
`with
`a
`decreasing
`concentration
`of
`denaturant, either step-wise or by using a
`gradient, the protein can be refolded and then
`eluted [48,49].
` Gel filtration is an alternative technique,
`whereby the denatured protein is loaded onto
`the column and refolds as it is passed
`through with buffer [50,51]. A variation of this
`is to equilibrate the column with a linear
`gradient
`with
`decreasing
`denaturant
`concentrations, such that the protein refolds
`gradually
`[52]. Flow
`rates
`required
`for
`successful on-column refolding vary – for
`some proteins, slower flow rates improves
`recovery [38], in other cases faster flow rates
`are better [53].
` On-column refolding methods have also
`been
`improved by
`immobilizing common
`‘foldases’ onto
`the column matrix and
`exploiting them as a folding ‘platform’. This
`has been demonstrated successfully with
`GroEL
`[54,55]and DsbA/DsbC
`[56]. One
`associated concern with on-column refolding
`is
`the clogging of
`filters by aggregated
`protein, however, carefully
`filtering or
`centrifuging samples prior to loading onto the
`column, and sensible choice of column matrix
`can minimize these problems.
`
`Other refolding techniques
`Whilst a majority of proteins can be refolded
`by classic dilution, dialysis or on-column
`techniques,
`the potential development of
`other refolding techniques is constantly being
`explored by researchers. One recent foray
`has been the use of reverse micelles, in
`which the protein is trapped in a water-
`sodium
`bis-2-ethylhexyl
`sulfosuccinate-
`isooctane reverse micellar system [57]. This
`was shown
`to result
`in
`the successful
`recovery of monomeric protein where other
`techniques had failed. Variation in the size of
`the micelles enabled manipulation of the
`degree of oligomerisation of the protein.
`
`
`
`6
`
`Page 6
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`4.5 Folding aids
`Several molecular chaperones can be
`included either in vivo or in vitro to aid folding.
`The most well known E.coli chaperones
`include GroEL-GroES, DnaK-DnaJ-GrpE
`(Hsp70) and also ClpA/ClpB (Hsp100) which
`have been used successfully in refolding
`studies [58-61]. Whilst molecular chaperones
`promote correct
`folding,
`foldases can
`accelerate the process. The three known
`types of
`foldases are 1) peptidyl prolyl
`cis/trans isomerases (PPI’s), which arrange
`prolines into their correct orientation [62], 2)
`disulfide oxidoreductase (DsbA) and disulfide
`isomerase (DsbC), which are found in E.coli
`and promote disulfide bonds [63,64] and 3)
`protein
`disulfide
`isomerase
`(PDI),
`a
`eukaryotic protein which catalyses oxidation
`and isomeration [65].
`
`4.6 Folding screens
`As finding the right conditions to refold a
`protein can be a time-consuming process,
`several commercial screens are available to
`focus on potential conditions that return the
`best yield of monomeric protein. One such kit
`is Hampton Research’s FoldIt screen, which
`employs a variety of additives,
`redox
`conditions and pH. Similar kits are also
`available
`through Novagen and Sigma-
`Aldrich. These kits are the result of sparse
`screens which have proven successful for the
`refolding of a number of proteins [66-68]. The
`commercially prepared kits enable a number
`of
`refolding conditions
`to be sampled
`simultaneously on a small scale, the aim
`being to find suitable conditions which can be
`up-scaled for batch refolding and purification.
`
`An elegant example of using factorial
`screens is demonstrated by a study involving
`procathepsin S and cathepsin S [69]. An
`initial screen used
`to
`identify
`folding
`conditions targeted L-arginine and pH as two
`key factors. While pH was important for both
`proteins, arginine was more beneficial for
`procathepsin only. Once these conditions
`were established, a second screen based on
`these findings was performeds to improve
`
`yields. It was found that while procathepsin
`folded best in the presence of detergent,
`arginine and a redox system, cathepsin
`required only glycerol and the redox couple.
`This illustrates the powerful nature of the
`factorial screen and also demonstrates how
`seemingly similar proteins may have very
`different requirements for refolding.
`
`5. ANALYSIS OF REFOLDED PROTEIN
`Once refolding is completed, it is necessary
`to determine whether the process has yielded
`folded or aggregated protein, and also
`whether disulfide bonds (if any) have formed
`correctly. Perhaps the simplest way to assess
`the quality of the protein is by using a known
`activity assay. While this is possible for some
`proteins, a number of targets will have no
`known
`function or structure, especially
`considering
`the growing
`level of high
`throughput production of proteins
`from
`different genomes. Therefore, it is important
`to develop other techniques to analyse the
`nature of the protein.
`
`Such techniques commonly include size-
`exclusion
`chromatography
`and
`non-
`denaturing PAGE, as well as circular
`dichroism (CD) which reports on secondary
`structure. Distinct features of a typical CD
`scan report on the presence of α-helical, β-
`sheet or random coil structure. Proteins will
`generally be a combination of α-helical and β-
`sheet content, while aggregated material may
`produce a spectrum of predominately random
`coil. Fluorescence
`techniques such as
`dynamic
`light
`scattering
`[70], which
`determines the average diameter of particles
`in solution and lateral turbidimetry [6] can
`also report the presence of aggregates [71],
`as can ultracentrifugation [72] and electron
`microscopy [73].
`
`Assessing the nature of disulfide bonds
`can be achieved by a number of methods.
`Perhaps the most straightforward method is
`the use of reducing and non-reducing gel
`electrophoresis. In the absence of reducing
`agent, there should be a defined ‘band shift’,
`accompanied with any additional bands that
`
`7
`
`Page 7
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`were record of linked domains. For a single
`domain protein, a ‘laddering’ effect is a tell-
`tale sign of intermolecular disulfide bond
`formation. The use of thiol specific dyes
`(eg.DTNB (Ellman’s reagent)) [74], mass
`spectrometry [75] and gel electrophoresis
`(‘cysteine counting’) [76] are all common
`techniques that are also employed.
`
`6. CONCLUSION
`Handling inclusion bodies can be a time-
`consuming and often
`frustrating
`trial-and
`error process. Whilst fundamental principles
`underpin the process, there are no clear rules
`which apply to all proteins, and an approach
`suitable
`for one protein maybe equally
`inappropriate
`for another. Despite
`this,
`working with
`inclusion bodies can be
`beneficial, especially when it is impossible to
`express reasonable quantities of soluble
`protein. As more proteins are successfully
`refolded, trends and patterns may become
`clearer. This will aid the progression and
`further development of biotechnological tools.
`
`8
`
`Page 8
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`Figure 1: A schematic outline of the procedure for purification and refolding of proteins
`from inclusion bodies
``
`
`
`Cell Lysis
`(eg.sonication, French press)
`
`Centrifugation
`
`Pellet (insoluble fraction) retained
`
`Purification of Inclusion Bodies
`Resuspension and centrifugation
`Chromatography (denaturing conditions)
`Eg. Triton X-100, high salt, EDTA
`Eg. Gel Filtration, Ni-NTA
`
`Solubilization of Inclusion Bodies
`Example denaturants:
`4-6 M GdnHCl 6-8 M Urea 0.5-2% SDS
`
`Dialysis
`(direct or step)
`
`Refolding
`On-column refolding
`(linear or step-wise gradient)
`
`Dilution
`(step or drop-wise)
`
`Post-refolding Analysis
`Activity assay
`Circular Dichroism
`Non-denaturing PAGE
`Size Exclusion Chromatography
`Light-scattering
`
`Ultracentrifugation
`
`Eg.
`
`
`
`9
`
`Page 9
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`
`Table 1: Examples of promoters often used in E.coli expression
`
`Example vector
`
`Promotor type
`
`Inducing agent
`
`T7
`T5
`trc
`tac
`lac
`Trp
`araB
`PL (λ)
`phoA
`PLtetO-1
`
`IPTG
`IPTG
`IPTG
`IPTG
`IPTG
`Tryptophan
`L-arabinose
`Temperature shift (42°C)
`Phosphate
`tetracycline
`
`pET (Novagen)
`pQE (Qiagen)
`pTrcHis (Invitrogen)
`pMAL (New England Biolabs)
`pTrip1Ex2 (Clontech)
`pLEX (Invitrogen)
`PBAD (Invitrogen)
`pKC30
`pBKIGF2B-A
`plP-PROTete-6xHN (Clontech)
`
`
`
`
`
`
`
`Table 2: Examples of fusion partners and tags which facilitate solubility and tracking
`protein expression
`
`
`
`Tag
`
`Size
`
`Location in relation to target
`protein
`
`Chloramphenicol acetyltransferase
`Glutathione S-transferase (GST)
`Maltose binding protein (MBP)
`NusA
`S-Tag
`Thioredoxin
`Ubiquitin
`Z-domain (derived from Protein A)
`
`N terminus
`N terminus
`N or C terminus
`N terminus
`N or C terminus, or internal
`N terminus
`N terminus
`N Terminus
`
`24 kDa
`26 kDa
`40 kDa
`54 kDa
`15 residues
`11 kDa
`76 residues
`58 residues
`
`
`10
`
`Page 10
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`Table 3: Examples of buffer additives which may be used to facilitate protein refolding
`
`
`Additive
`
`CHAPS
`EDTA
`Glycerol
`Guanidine HCl
`L-arginine
`Lauroylsarcosine (sarkosyl)
`Lauryl maltoside
`MgCl2/CaCl2
`NaCl/Ammonium sulfate
`Non-detergent sulfobetaine (NDSB) 256/201
`PEG 3350
`SDS
`Sodium citrate/sulfate
`Sucrose/glucose
`TMAO
`Tris
`Triton X-100
`Tween – 80
`Urea
`
`Concentration used
`
`30 mM
`20 mM
`10-50%
`0.1-1 M
`0.4-0.5M
`Up to 4 M
`0.3 mM
`2-10 mM
`0.2-0.5 M
`Up to 1M
`Up to 0.05% (w/v)
`0.1 %
`0.2-0.5 M
`0.4 M
`1-3 M
`0.4-1 M
`0.1-1 %
`0.01 %
`0.1-2 M
`
`Effect
`
`Detergent
`Chelator
`Stabilizer
`Chaotroph
`Stabilizer
`Detergent
`Detergent
`Cation
`Salt
`Solubilizer
`Osmolyte
`Detergent
`Salt
`Stabilizer
`Osmolyte
`Buffer
`Detergent
`Detergent
`Chaotroph
`
`
`
`11
`
`Page 11
`
`

`

`Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
`
`[1]
`
`[2]
`
`[3]
`
`[4]
`
`[5]
`
`[6]
`
`[7]
`
`[8]
`
`[9]
`
`[10]
`
`[11]
`
`REFERENCES
`
`M. Heidari, K.L. Rice, U.R. Kees and W.K. Greene, Expression and purification of the human
`homeodomain oncoprotein HOX11, Protein Expr Purif 25 (2002) 313-318.
`S.M. Park, H.Y. Jung, K.C. Chung, H. Rhim, J.H. Park and J. Kim, Stress-induced aggregation
`profiles of GST-alpha-synuclein fusion proteins: role of the C-terminal acidic tail of alpha-synuclein in
`protein thermosolubility and stability, Biochemistry 41 (2002) 4137-4146.
`D.B. Smith and K.S. Johnson, Single-step purification of polypeptides expressed in Escherichia coli
`as fusions with glutathione S-transferase, Gene 67 (1988) 31-40.
`F. Lipari, G.A. McGibbon, E. Wardrop and M.G. Cordingley, Purification and biophysical
`characterization of a minimal functional domain and of an N-terminal Zn2+-binding fragment from the
`human papillomavirus type 16 E6 protein, Biochemistry 40 (2001) 1196-1204.
`K. Terpe, Overview of tag protein fusions: from molecular and biochemical fundamentals to
`commercial systems, Appl Microbiol Biotechnol 60 (2003) 523-533.
`Y. Nomine, T. Ristriani, C. Laurent, J.F. Lefevre, E. Weiss and G. Trave, Formation of soluble
`inclusion bodies by hpv e6 oncoprotein fused to maltose-binding protein, Protein Expr Purif 23
`(2001) 22-32.
`D. Sachdev and J.M. Chirgwin, Solubility of proteins isolated from inclusion bodies is enhanced by
`fusion to maltose-binding protein or thioredoxin, Protein Expr Purif 12 (1998) 122-132.
`C. di Guan, P. Li, P.D. Riggs and H. Inouye, Vectors that facilitate the expression and purification of
`foreign peptides in Escherichia coli by fusion to maltose-binding protein, Gene 67 (1988) 21-30.
`G.D. Davis, C. Elisee, D.M. Newham and R.G. Harrison, New fusion protein systems designed to
`give soluble expression in Escherichia coli, Biotechnol Bioeng 65 (1999) 382-388.
`J.S. Kim and R.T. Raines, Ribonuclease S-peptide as a carrier in fusion proteins, Protein Sci 2
`(1993) 348-356.
`E. Rajashekhara, M. Kitaoka, Y.K. Kim and K. Hayashi, Characterization of a cellobiose
`phosphorylase from a hyperthermophilic eubacterium, Thermotoga maritima MSB8, Biosci
`Biotechnol Biochem 66 (2002) 2578-2586.
`L.M. Chen, T. Omiya, S. Hata and K. Izui, Molecular characterization of a phosphoenolpyruvate
`carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric
`properties, Plant Cell Physiol 43 (2002) 159-169.
`[13] M. Ubeidat and C.L. Rutherford, Expression and one-step purification of a developmentally regulated
`protein from Dictyostelium discoideum, Protein Expr Purif 25 (2002) 472-480.
`I. Simonovic and P.A. Patston, The native metastable fold of C1-inhibitor is stabilized by disulfide
`bonds, Biochim Biophys Acta 1481 (2000) 97-102.
`J.M. Vinetz, J.G. Valenzuela, C.A. Specht, L. Aravind, R.C. Langer, J.M. Ribeiro and D.C. Kaslow,
`Chitinases of the avian malaria parasite Plasmodium gallinaceum, a class of enzymes necessary for
`p