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`In Vivo PROTEIN DEPOSITION
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`[3] Isolating Inclusion Bodies from Bacteria
`
`By GEORGE GEORGIOU and PASCAL V ALAX
`
`Introduction
`
`In 1975 Prouty et al. 1 first described the formation of dense, amorphous
`intracellular granules in Escherichia coli cells grown in the presence of the
`amino acid analog canavanine. These granules, comprised primarily of
`polypeptide chains, could be solubilized by sodium dodecyl sulfate (SDS)
`and were not surrounded by any sort of membrane. For several years this
`observation was considered an aberrant, and rather irrelevant, cellular
`response induced by growth under nonphysiological conditions. It was not
`until much later that it became apparent that protein aggregation in vivo is
`a widespread phenomenon, manifested in cells overexpressing heterologous
`proteins or native proteins beyond a certain level, and in cells exposed to
`thermal or other kinds of physiological stress. In addition, mutations re(cid:173)
`sulting in amino acid substitutions, deletions, or insertions can interfere
`with the folding of a polypeptide to the native state, causing the formation
`of protein aggregates.2
`Intracellular protein aggregates form dense, electron-refracting particles
`that can be distinguished readily from other cell components by electron
`microscopy. For this reason, protein aggregates, at least those observed in
`microorganisms, are usually called inclusion bodies or, less often, refractile
`bodies. However, it should be noted that protein misfolding and self-associa(cid:173)
`tion may occur even when inclusion bodies cannot be detected by electron
`microscopy or following careful cell fractionation (H. G. Gilbert, personal
`communication, 1999). Nonetheless, for practical purposes, it is safe to
`assume that the expression of proteins susceptible to aggregation at a level
`2% or greater of the total cell protein will be accompanied by the appearance
`of readily identifiable inclusion bodies.
`Inclusion bodies have been used extensively as a source of relatively
`pure, albeit misfolded, polypeptide chains that can be renatured to give the
`biologically active soluble protein. The refolding of proteins from inclusion
`bodies has proven of great value for analytical and preparative purposes.
`As of 1998, there have been over 300 reports of mammalian, plant, and
`microbial proteins obtained and renatured from inclusion bodies formed
`in E. coli, Tens of kilograms of biologically active bovine somatotropin are
`
`1 W. Prouty, M. J. Kamovsky, and A. L. Goldberg, J. Biol. Chem. 250, 1112 (1975).
`2 A. L. Fink, Folding Design 3, R9 (1998).
`
`METHODS IN ENZYMOLOGY, VOL 309
`
`Copyright C 1999 by Acadern.ic Press
`All rigMs of reproduction in any fonn reserved.
`0076-6879/99 $30.00
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`produced every year from inclusion bodies and even larger amounts of
`human hemoglobin may eventually be produced by renaturation. Irrespec(cid:173)
`tive of the scale, the recovery of purified active proteins from inclusion
`bodies involves the following steps3 : (1) isolation of inclusion bodies, (2)
`solubilization, (3) removal of protein impurities, and (4) refolding.
`Often the sequence of steps 3 and 4 is interchanged so that the desired
`protein is purified after refolding. Obviously, the success of steps 2-4 de(cid:173)
`pends on the quality of material from step 1. Also, preparing high-quality
`inclusion bodies is important for structural studies.45
`Inclusion bodies formed in cells overexpressing a certain polypeptide
`are generally expected to be comprised predominantly of that polypeptide.
`However, this is often not the case. We have found6 that the composition
`of inclusion bodies is a complex function of the mode of expression and
`the growth conditions. For example, purified inclusion bodies from E. coli
`cells expressing ,B-lactamase contain between 35 and 95% intact ,B-lactamase
`polypeptides. The rest are composed of a variety of intracellular proteins,
`some lipids, and a small amount of nucleic acids. Homogeneous inclusion
`bodies [95% (w/w) ,B-lactamase] were obtained by expressing the protein
`without its leader peptide, in which case aggregation occurred within the
`bacterial cytoplasm. This gave rise to large, highly regular inclusion bodies
`that could be separated readily from other particulate matter in cell lysates
`(Fig. 1). As might be expected, the efficiency of ,B-lactamase refolding was
`inversely proportional to the level of contaminants present in the inclusion
`body preparation.7
`"High-quality" inclusion bodies consist primarily of the overexpressed
`recombinant protein with as little contaminating material as possible. The
`quality of inclusion bodies obtained from bacteria overexpressing a desired
`protein depends on two parameters: (1) the degree to which extraneous
`polypeptides, and possibly other macromolecules, are incorporated within
`the aggregate and (2) the ability to separate inclusion bodies from other
`cellular particles having a similar sedimentation coefficient and from mate(cid:173)
`rial, mostly membrane vesicles, that becomes adsorbed onto the surface of
`the protein particles following cell lysis. It is usually difficult to ascertain
`whether extraneous proteins form an integral part of the aggregates or
`represent copurifying contaminants. There is increasing evidence, discussed
`in other articles of this volume, that protein aggregation involves the associ-
`
`3 R. Ruboph and H. Lilie, FASEB J. 50, 49 (1996).
`4 K. Oberg, B. A. Chrunyk, R. B. Wetzel, and A. L. Fink, Biochemistry 33, 2628 (1994).
`5 T. M. Przybycien, J.P. Dunn, P. Valaic, and G. Georgiou. Prat. Engin. 7, 131 (1994).
`6 P. Valax and G. Georgiou, Biotech. Progr. 9, 539 (1993).
`7 P. Valax, " in Vivo and i,1 Vitro Folding and Aggregation of Escherichia coli ,8-Lactamase."
`Ph.D Dissertation, Univ. of Texas at Austin, 1993.
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`FIG. 1. (A) E. coli cells containing inclusion bodies and (B) inclusion bodies isolated by
`sucrose density centrifugation. lnclusion bodies were formed in the cytoplasm by expressing
`/3-lactamase (a normally secreted protein) carrying a deletion of the first 20 amino acids (-20,
`-1) of the leader sequence.
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`ation of subdomains in partially folded intermediates.2 The degree of speci(cid:173)
`ficity of the interactions that lead to protein aggregation varies from protein
`to protein. As a consequence, the extent of incorporation of cellular proteins
`and other extraneous macromolecules into inclusion bodies is protein de(cid:173)
`pendent. Nonetheless, as was shown with ~-lactamase,6·8 it is often possible
`to modify the expression conditions to reduce the amount of extraneous
`material incorporated within the inclusion bodies.
`Often the only step in the preparation of inclusion bodies is low-speed
`centrifugation of cell lysates. The effectiveness of this step depends to a
`large degree on the method of cell disruption. Low-speed centrifugation
`results in the sedimentation of membrane vesicles and cell wall fragments
`together with the inclusion bodies. In addition, other cellular components
`can become adsorbed nonspecifically onto the surface of inclusion bodies
`following cell lysis. The presence of contaminating substances causes a
`number of complications during subsequent solubilization and refolding
`steps: First of all, proteases may copurify with inclusion bodies during high(cid:173)
`speed centrifugation.9•10 Many proteases are active in the presence of high
`concentrations of denaturants used to solubilize the inclusion bodies and
`can rapidly cleave unfolded proteins under these conditions. For example,
`the presence of contaminating E. coli outer membrane protease OmpT was
`shown to cause significant reductions in the recovery of active porcine
`growth hormone11 and creatine kinase9 from inclusion bodies. Second,
`impurities found in inclusion bodies have to be removed eventually either
`prior to, or after, refolding. Third, protein as well as nonproteinaceous
`impurities interfere with refolding. In an interesting study, Maachupalli(cid:173)
`Reddy and co-workers12 examined the effects of typical inclusion body
`contaminants such as DNA, ribosomal RNA, lipids, and other proteins on
`the in vitro refolding of hen egg-white lysozyme. They found that the
`presence of other polypeptides prone to aggregation reduced the refolding
`yield significantly. The effect of RNA, DNA, and phospholipids at concen(cid:173)
`trations up to 30% of that of hen lysozyme did not have a significant
`effect on refolding yields. However, Darby and Creighton13 found that
`nonproteinaceous contaminants had a dramatic effect on the refolding of
`bovine pancreatic trypsin inhibitor (BPTI) mutants from inclusion bodies.
`
`8 G. A. Bowden, A. M . Paredes, and G. Georgiou, Bio/Technology 9, 725 (1991).
`9 P. C. Babbit, B. L. West, D. D. Buechter, I. D. Kuntz, and G. L. Kenyon, Bio/Technology
`8, 945 (1990).
`10 J.-M. Betton, N. Sa.~son, M. Hofnung, and M. Laurent, J. Biol. Chem. 273, 8897 (1998).
`11 N. K. Puri, M. Cardamone, E. Crivelli, and J. C. Traeger, Prot. Expr. Puri[. 4, 164 (1993).
`ii J. Maachupalli-Reddy, B. D. Kelley, and E. De Bernardez-Clark, Biotech. Prog. 13, 144
`(1997).
`13 N. J. Darby and T. E. Creighton, Nature 344, 715 (1990).
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`They suggested that even under the strongly denaturing conditions used
`to solubilize the inclusion bodies, BPTI is in a tight complex with a substance
`that affects its solubility and folding properties. This substance was found
`to be an acidic polymer, which unfortunately was not characterized further.
`Cardamone et al. 14 reported that the recovery yield of recombinant porcine
`growth hormone from inclusion bodies is lower than what can be achieved
`with the purified protein. They proposed that "morphopoietic factors"
`intrinsic to the inclusion bodies were responsible for preventing the protein
`from following the same folding pathway during renaturation, thus enhanc(cid:173)
`ing aggregation.
`From these results it should be evident that the preparation of inclusion
`bodies suitable for protein recovery requires careful consideration of the
`expression conditions to minimize both the extent of nonspecific protein
`incorporation and the purification protocol. Because the formation of inclu(cid:173)
`sion bodies is protein dependent, it is not possible to develop procedures
`that work for every case. Therefore, the remainder of this article is intended
`more as a set of recommendations that generally give good results in
`our experience.
`
`Protein Expression
`
`The bacterial strain, expression vector, and growth conditions have a
`pronounced effect on inclusion body formation. Expression from a T7
`promoter15 is accompanied by very high rates of protein synthesis, which
`enhance inclusion body formation. Genes placed downstream of a T7 pro(cid:173)
`moter are transcribed by the T7 RNA polymerase. The gene for the latter
`is usually expressed from a relatively weak inducible promoter such as the
`lac promoter. Addition of the inducer IPTG turns on the synthesis of TI
`RNA polymerase, which in tum transcribes the desired gene from the TI
`promoter. For most applications, expression from a T7 promoter is the
`preferred way for inducing the formation of inclusion bodies. Ideally, the
`host strain should be a mutant defective in the gene responsible for the
`transport of the inducer into the cell. In such a host, induction with subsatu(cid:173)
`rating concentrations of inducer can be employed to adjust the level of
`transcription per cell.16 Another promoter that we have found to be favor(cid:173)
`able for inclusion body formation is the PL of bacteriophage ,\. With the
`PL promoter, a very high rate of transcription is achieved by a temperature
`
`14 M. Cardamone, N. K. Puri, and M. R. Brandon, Biochemistry 34, 5773 (1995).
`15 W. F. Stud.ier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, Meth. Enzymol 85,
`61 (1990).
`16 D. A. Siegele and J.C. Hu, Proc. Natl. Acad. Sci. U.S.A. 94, 8168 (1997).
`
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`upshift to 42°, although cold temperature induction can also be employed.17
`At 42° the combination of a higher temperature, which generally favors
`aggregation over folding, and the high rate of protein synthesis favor inclu(cid:173)
`sion body formation. However, exposure of the bacteria to a supraoptimal
`temperature is also accompanied by the increased synthesis of heat shock
`proteases, which may cause degradation of the cloned product.
`In E. coli, protein aggregation is enhanced by mutations in certain
`global regulatory pathways or in specific chaperone genes. The heat shock
`transcription factor u 32 is responsible for upregulating the expression of a
`number of E. coli chaperones under stressful conditions, including the
`DnaK-DnaJ-GrpE and GroEL-GroES systems. Mutations in the rpoH gene
`encoding u 32 have been shown to induce the complete aggregation of a
`normally soluble recombinant protein.18
`19 However, massive aggregation
`•
`of host proteins also occurs in rpoH mutants and, as a result, the insoluble
`fraction contains, in addition to the desired polypeptide, a number of con(cid:173)
`taminating proteins. Mutations in the dnaK, dnaJ, grpE, groEL, or groES
`genes have also been found to favor the aggregation of recombinant proteins
`while having little effect on host protein solubility.19 Hosts carrying muta(cid:173)
`tions in dnaK, dnaJ, or grpE are particularly useful when it is desired to
`direct a protein into inclusion bodies. The DnaK-DnaJ-GrpE chaperone
`machinery binds newly synthesized polypeptides and mutations that inter(cid:173)
`fere with its function are more likely to lead to rapid aggregation before
`proteolytic degradation can occur. Indeed, a number of unrelated overex(cid:173)
`pressed proteins have been found to aggregate extensively in a grpE280
`mutant, whereas the extensively studied groES30 or groEL140 alleles af(cid:173)
`fected folding of only a limited number of proteins, often at the expense
`of reduced yields (J. Thomas, personal communication).20
`•2 1
`Normally secreted heterologous proteins can be expressed either in
`the cytoplasm or with a prokaryotic leader peptide for targeting to the
`periplasmic space. Inclusion bodies can form in either cellular compartment.
`For secreted proteins containing two or more cysteine residues, the forma(cid:173)
`tion of protein aggregates in the periplasm may be increased by the coex(cid:173)
`pression of E. coli cysteine oxidoreductases (DsbC or DbA).22 Periplasmic
`inclusion bodies are smaller and of irregular shape, most likely because
`
`17 S. C. Macrides. MicrobioL Rev. 60, 512 (1996).
`18 A. I . Gragerov, E. S. Martin, M. A. Krupenko, M. V. Kashlev, and V. G. Nikiforov, FEBS
`Lett. 291(2), 222 (1991).
`19 A. Gragerov, E. Nudler, N. Komissarova, G. A. Gaitanaris, M . E. Gottesman, and V.
`Nikiforov, Proc. Natl. A cad. Sci. U.S.A. 89, 10341 ( 1992).
`20 J. G. Thomas and F. Baneyx, Mo/. Microbial. 21, 1185 (1996).
`21 J. G. Thomas and F. Baneyx, Prot. Express. Puri/ 11, 289 (1997).
`22 J. C. Joly, W. S. Leung, and J. Swartz, Proc. Natl. Acad. Sci. U.S.A. 95, 2773 (1998).
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`In Vivo PROTEIN DEPOSITION
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`topological constraints limit the growth of the aggregate.8 Inclusion bodies
`in the cytoplasm are larger and often highly regular (Fig. 1 ). For this reason,
`they can be separated more readily from ceU debris and may contain lower
`amounts of extraneous polypeptides, phospholipids, and nucleic acids.6 In
`some cases, high levels of expression of secreted proteins that are prone
`to aggregation are accompanied by the accumulation of the precursor form
`(the preprotein) with the leader peptide uncleaved.6•23 The presence of the
`leader peptide renders preproteins highly insoluble and they are found
`either in the membrane fraction or are sequestered within cytoplasmic
`aggregates. These aggregates cofractionate with periplasmic inclusion bod(cid:173)
`ies formed by the mature protein in the periplasm. The formation of a
`mixed aggregate population, consisting of preprotein and mature protein
`inclusion bodies, poses further complications during refolding and should
`be avoided.
`The growth conditions can also be optimized to enhance inclusion body
`formation. As was mentioned earlier, growth at 42° results in decreased
`yields of soluble protein. Aeration, i.e., the concentration of dissolved
`oxygen, has been shown to affect protein aggregation.24 The effect of dis(cid:173)
`solved oxygen on protein aggregation is complex and, in our experience at
`least, growth in a low dissolved oxygen environment can either enhance
`or reduce the formation of inclusion bodies, depending on the promoter
`and plasmid vector employed (unpublished data). The addition of ethanol
`to the growth media at concentrations around 3% (v/v) has been found
`to increase inclusion body formation with some proteins, such as human
`SPARC, but had the reverse effect with others.21 Finally the culture pH,
`carbon source, and growth in minimal versus rich media also affect in vivo
`solubility in a protein-dependent manner.
`
`Inclusion Body Isolation
`
`Obtaining a homogeneous preparation of inclusion bodies requires a
`three-step process involving (1) cell Iysis, (2) fractionation of the ceU lysates
`to resolve the inclusion bodies from the cell debris by taking advantage of
`differences in size and density, and (3) removal of adsorbed contaminants.
`Although incubation of intact E. coli with denaturants has been used
`for the in situ solubilization of aggregated proteins, such as IGF-1,25 in
`general it is necessary to first lyse the bacteria. Laboratory methods for
`
`23 N, Sriubolmas, W. Panbangred, S. Sriurairatana, and V. Meevootisom, Appl. Microbiol.
`Biotechrwl. 41, 373 (1997).
`u S. D. Betts, T. M. Hachigian, and E . Picbersky, Plant. MoL Biol. 26, 117 (1994).
`25 R.H. Hart, P. M. Lester, D. H. Reifsnyder, and J. R. Ogez, Bio/Tech110/ogy 12, 1113 (1994).
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`rupturing bacteria include repeated freeze-thaw cycles, lysozyme-EDT A
`treatment, sonication, and high-pressure homogenization using a French
`pressure cell. The method of lysis determines the size of cell debris that is
`generated. Because inclusion bodies are separated from the debris on the
`basis of size and density, it is an important parameter in recovery. High(cid:173)
`pressure homogenization is the recommended method for obtaining a high
`degree of eel] disruption (90-98% after one pass)26 and smaller size debris.
`Multiple passes have been shown to further reduce the debris size down
`to a median diameter of around 0.3 µ,m. 26 For comparison, the size of
`inclusion bodies ranges between 0.3 and 1.2 µ,m. We have found that
`pretreatment with lysozyme-EDTA further improves inclusion body isola(cid:173)
`tion.6 In gram-negative bacteria, the outer membrane is linked covalently
`to the cell wall and therefore hydrolysis of the peptidoglycan by lysozyme
`may be necessary to facilitate the formation of smaller size outer membrane
`vesicles. As a rule, three passes through either a French press at 20,000 psi
`or equivalent conditions for industrial scale high-pressure homgenization
`28
`equipment are adequate for good separation.27·
`Inclusion bodies are recovered from the cell lysate by centrifugation,
`usually at 15,000-30,000g in the laboratory (up to 20,000g for industrial
`scale). In addition to the aggregated protein, the pelleted fraction contains
`extraneous polypeptides and phospholipids. An appreciable amount of
`nucleic acids is also found. 29 The insoluble fraction obtained after centrifu(cid:173)
`gation contains two prominent bands of molecular mass approximately 34
`and 36 kDa (their migration may vary somewhat depending on the way
`the samples are treated prior to loading on the gel). These correspond to
`the major outer membrane proteins OmpA and OmpC/F, respectively,
`indicating that outer membrane fragments represent a major source of
`contaminating material. Outer membrane proteins are found in both cyto(cid:173)
`plasmic and periplasmic inclusion body preparations. Ribosomal proteins
`have also been found in inclusion bodies.30
`Cosedimenting ribosomes and outer membrane vesicles can be sepa(cid:173)
`rated from inclusion bodies by density gradient centrifugation.8.31 Inclusion
`bodies have a density comparable to that of proteins (1.3-1.4 g/ml), whereas
`the density of outer membrane vesicles is 1.22 g/ml and that of ribosomes
`
`26 H. H. Wong, B. K. O' Neill, and A. P. J. Midd.Jeberg, Biotech. Bioeng. 55, 556 (1997).
`n E. A. Burks, and B. L.Iverson, Biorech. Progr. ll, 112 (1995).
`28 M. E. Gustafson, K. D. Junger, B. A. Foy, J. A. Baez, B. F. Bishop, S. H. Rangwala,
`M. L. Michener, R. M. Leimgruber, K. A. Houseman, R. A. Mueller. B. K. Matthews,
`P. 0 . Olins, R. W. Grabner, and A. Hershman, Pror. Expres. Puri/ 6, 5)2 (1995).
`29 K. E. Langley, T. F. Berg, and T. W. Strickland, Eur. J. Biochem. 163, 313 (1987).
`30 U. Rinas and J. Bailey, Appl. Microbial. Biorechnol. 37, 609 (1992).
`31 L. A. Classen, B. Ahn, H.-S. Koo. and L. Grossman, J. Biol Chem. 266, 11380 (1991).
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`around 1.5 g/ml. In a sucrose step gradient, inclusion bodies become focused
`into a relatively tight and visible band that can be distinguished clearly
`from membrane vesicles that are lighter and form a separate band. Good
`results have been obtained with a variety of inclusion body proteins using
`a 40-53- 67% (w/w) sucrose step gradient, but slightly different conditions
`have also been used successfully.30 The inclusion body band is found close
`to or at the interface of the 53 and 67% (w/w) sucrose layers and is collected
`with a Pasteur pipette. The inclusion bodies are then resuspended in buffer
`and precipitated by low-speed centrifugation several times to remove the
`sucrose. Analysis of the purified inclusion bodies by SOS-PAGE should
`show a clear reduction in the amount of outer membrane protein. The
`amount of lipid in the preparation is reduced by up to 8-fold, although in
`many cases a substantial amount of lipid still remains.6 The nucleic acid
`content is also reduced significantly, typically by 10- to 20-fold over the
`amount in the inclusion body pellet prior to purification.
`If the density of the inclusion bodies is low, the respective band is
`not well resolved from the cell membrane material. In that case, further
`purification can be obtained using a second sucrose density gradient centrif(cid:173)
`ugation step.6 Either an identical step gradient or a flotation gradient can
`be used at this stage.32 For the latter, material is applied to the bottom
`of the tube and floats up a 60-40% (w/w) gradient until it reaches its
`buoyant density.
`For large-scale protein recovery, cross-flow filtration has been used as
`an alternative to centrifugation. Cross-flow filtration is a separation tech(cid:173)
`nique in which the suspension is flowed parallel to the membrane in order
`to avoid the accumulation of material on the filter surface and to improve
`the filtration rate. Particles above a certain size cutoff are retained by a
`membrane and are collected continuously. With cross-flow filtration, inclu(cid:173)
`sion body concentration and washing can be performed continuously. Pro(cid:173)
`tein recovery yields comparable to those obtained by centrifugation have
`been reported.33
`After separation from cell debris, a series of wash/extraction steps is
`used to further remove contaminating material that had cosedimented or
`was adsorbed on the inclusion bodies.734 Membrane material associated
`with the inclusion bodies is typically removed using mild detergents. An
`appropriate detergent should allow efficient extraction of impurities without
`solubilizing the inclusion bodies or irreversibly binding to the aggregated
`
`32 1. Poquet, M. G. Konnacher, and A. P. Pugsley, Mo/. Microbial. 9, 1061 (1993).
`33 M. M . Meagher, R. T. Barlett, V. R. Rai, and F. R. Khan, Biotech. Bioeng. 43, 969 (1994).
`34 F. A 0 . Marston, in " DNA Cloning: A Practical Approach" (D. M. Glover, ed.), Vol. 3,
`p. 59. IRL Press, Oxford, 1987.
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`polypeptide chains. Price can also be a consideration as some detergents
`are very costly. Several detergents have been used in the literature, including
`Triton X-100, deoxycholate, Berol 185, and octylglucoside.7
`35 Nonspecifi(cid:173)
`·
`cally adsorbed material can also be removed using low concentrations of
`chaotropic agents such as urea or guanidine hydrochloride. Because these
`agents are generally used for the solubilization of aggregated proteins, their
`concentration must be carefully optimized in order to efficiently remove
`contaminating material without significantly solubilizing the inclusion bod(cid:173)
`ies. Alternatively, inclusion bodies may be treated with a combination of
`detergents and low concentrations of chaotropic agents. For example, hu(cid:173)
`man cathepsin B inclusion bodies were purified by extraction in 0.1 % Triton
`X-100 once and 2 M urea twice,36 whereas Belew et al.35 used 0.1 % Bero!
`185 and 0.5 M urea to remove impurities from recombinant human granulo(cid:173)
`cyte-macrophage colony-stimulating factor.
`The remainder of this article describes in detail a general procedure
`we have found useful for inclusion body isolation.
`
`Cell Lysis
`
`The conditions for E. coli growth and protein synthesis have to be
`optimized first, depending on the expression system. At the appropriate
`time after induction, the cells are harvested by centrifugation at 8000g for
`10 min at 4° and washed once in buffer. The cell pellet is resuspended in 10
`mMTris-HCl, pH 7.5, containing 0.75 M sucrose and 0.2 mg/ml lysozyme. It
`is recommended to resuspend the cell pellet from a 50-ml culture of 1.0
`OD600 into 1 ml of buffer. After a 10-min incubation at room temperature,
`a 3 mM EDTA solution is added at a 2: 1 (v/v) ratio and transferred to
`ice for approximately 5 min. Subsequently, the cells are lysed by passing
`through a French press three times at 20,000 psi.
`
`Inclusion Body Isolation
`
`Following cell disruption, the lysate is centrifuged at 12,000g for 30 min
`at 4° and the pellet, which contains the inclusion bodies, is resuspended in
`10 mM Tris-HCI buffer, pH 8.0, containing 0.25 M sucrose, 1 mM EDT A ,
`and 0.1 % sodium azide (1.25 ml of buffer per 50 ml of 1.0 OD600). A
`tissue homogenizer may be used to resuspend the pellet thoroughly. The
`resuspended pellet is layered on the top of a sucrose step gradient [40, 53,
`and 67% (w/w)] in 1 mM Tris-HCI buffer, pH 8.0, containing 0.1 % sodium
`azide and 1 mM EDTA. The sucrose gradient is prepared by carefully
`
`35 M. Belew, Y. Zhou, W. Wang, L.-E. Nystrom, and J.-C. Janson, Chroma1ogr. A 679,67 (1994).
`36 R. Kuhelj, M. Dolinar. M. Pugercar, and V. Turk, Eur. 1. Biochem. 229, 533 (1995).
`
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`
`layering the sucrose solutions, with the more dense one at the bottom of
`the tube. Centrifugation is performed at 108,000g for 90 min at 4°.8 The
`inclusion bodies become focused in a band at the interface between the 53
`and 67% sucrose layers and are recovered and resuspended in either water
`or a suitable buffer, as required for further refolding steps. Several washes,
`followed by reprecipitation of the inclusion bodies by centrifugation at
`12,000g for 30 min, are required to remove the sucrose. Alternatively, the
`sucrose can be removed by dialysis. At this point the aggregated protein
`should be examined by SDS-PAGE to evaluate the degree of purity. If
`the desired polypeptide constitutes less than 70% of the insoluble protein,
`then additional purification is recommended: The inclusion bodies are resus(cid:173)
`pended in 50 mM KH2PO4 , pH 7.0, containing 50 mM octylglucoside and
`incubated for 15 min at room temperature. Following centrifugation at
`12,000g for 20 min, the pellet is resuspended in 0.25 M sucrose solution
`and applied to a second sucrose gradient, as described previously.
`
`[4] Isolation of Amyloid Deposits from Brain
`By ALEx E. ROHER and Yu-MIN Kuo
`
`Introduction
`
`The profuse deposition of insoluble amyloid-/3 (A/3) fibrils in the paren(cid:173)
`chymal and vascular extracellular spaces of the cerebral cortex and lepto(cid:173)
`meningeal vessels is one of the main histopathological lesions of Alzhei(cid:173)
`mer's disease (AD). Fibrillar deposits of amyloid concentrate at the center
`of the senile plaques, usually surrounded by dystropbic neurites, or accumu(cid:173)
`late around cerebral blood vessels, leading to the death of vascular myo(cid:173)
`cytes.1 Nonfibrillar amyloid also deposits in the cerebral cortex in the form
`of diffuse plaques, which are apparently devoid of neuritic pathology.2 The
`40 to 42 amino acid A/3 peptides result from the proteolytic degradation
`of the transmembrane /3-amyloid precursor protein.3 Soluble monomeric
`and oligomeric forms of these peptides are normally present in the human
`
`1 H . M. Wisniewski, C. Baucher, M. Barcikowska, G. Y. Wen, andJ. Currie. Acta Neuropathol.
`78, 337 (1989).
`2 H. Yamaguchi, Y. Nakazato, S. Hirai, M . Shoji. and Y. Harigaya, Am. J. Pathol. l35,
`593 (1989).
`3 D . J. Selkoe, Annu. Rev. Neurosci. 17, 489 (1994).
`
`METHODS IN ENZYMOLOGY, VOL. 309
`
`Copyright O 1999 by Academic Press
`AU righrs of reproduction in any form reserved.
`0076.f,879/99 $30.00
`
`11 of 11
`
`Fresenius Kabi
`Exhibit 1014
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