`
` H___fi__Jm___c___=__________E__=__=_____
`
`E:
`
`. AVAILABLE
`* ,0" THE we:
`
`mmwwnmacsmm;
`
`1 of 13
`
`Fresenius Kabi
`Exhibit 1015
`
`

`

`BIOTECHNOLOGY
`
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`2 of 13
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`Fresenius Kabi
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`

`...
`
`EDITOR
`JEROME S. SCHULTZ
`Center for Biotechnology
`and Bioengineering
`University of Pittsburgh
`Pittsburgh, Pennsylvania 15219
`Phone: (412) 383-9712
`Fax: (412) 383-9710
`E-mail: jssbio+@pitt.edu
`Editorial Assistant: Anne M. Brumfield
`E-mail: amb@engrng.pitt.edu
`
`ASSOCIATE EDITOR, REVIEWS
`LARRY V. McINTIRE
`Rice University
`Institute of Biosciences and
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`G. R. Brown Hall, Room Wl00D
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`Phone: (713) 527-4903
`Fax: (713) 285-5154
`E-mail: mcintire@rice.edu
`
`ADVISORY BOARD
`Fredric Bader
`Auto Immune
`James Bailey
`ETH, Zurich
`Kenneth Bischoff
`University of Delaware
`Bruce Dale
`Michigan State University
`John Gerlt
`University of Illinois, Urbana- Champaign
`Juan Hong
`University of California-Irvine
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`Rakesh Jain
`Harvard Medical School
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`Genentech, Inc.
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`Purdue University
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`Massachusetts Institute of Technology
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`Cornell University
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`Lehigh University
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`University of California-Davis
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`Cornell University
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`Genentech, Inc.
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`K. Venkat
`Phyton Catalytic, Inc.
`Henry Wang
`University of Michigan
`Howard Weetall
`National Institute of Science and Technology
`
`BIOTECHNOLOGY
`==-=--=-==-=--=-®
`
`JANUARY/FEBRUARY 1998
`VOLUME 14, NUMBER 1
`
`Copyright 1998 by the American Chemical Society
`and the American Institute of Chemical Engineers
`
`BIPRET 14(1) 1-166 (1998)
`ISSN 8756-7938
`
`KURT F. WENDT LIBRARY
`COLLEGE OF ENGINEERING
`
`MAR 31 1998
`
`UW-MADISON, WI 53706
`
`EDITORIAL
`
`1
`
`Biochemical Engineering Fundamentals: The Foundations of Our
`Professiol).
`Wei-Shou Hu, and James R. Swartz
`
`BIOCHEMICAL ENGINEERING(cid:173)
`HISTORICAL PERSPECTIVES
`
`3
`
`Shake Flask to Fermentor: What Have We Learned?
`Arthur Humphrey
`
`8 Mathematical Modeling and Analysis in Biochemical
`Engineering: Past Accomplishments and Future Opportunities
`James E. Bailey
`
`BIOCHEMICAL ENGINEERING FUNDAMENTALS(cid:173)
`QUANTITATIVE PROCESS ANALYSIS
`
`21
`
`Transport Properties of Rolled, Continuous Stationary Phase
`Columns
`Kent Hamaker, Jiyin Liu, Christine M. Ladisch, and
`Michael R. Ladisch*
`
`31 Mass-Transfer Properties of Microbubbles. 1. Experimental
`Studies
`Marshall D. Bredwell and R. Mark Worden*
`
`39 Mass-Transfer Properties of Microbubbles. 2. Analysis Using a
`Dynamic Model
`R. Mark Worden* and Marshall D. Bredwell
`
`47
`
`Oxidative Renaturation of Hen Egg-White Lysozyme. Folding vs
`Aggregation
`Eliana De Bernardez Clark,* Diane Hevehan, Sandra Szela, and
`Jhansi Maachupalli-Reddy
`
`55 Optimal Screening of Surface-Displayed Polypeptide Libraries
`Eric T. Boder and K. Dane Wittrup*
`
`BIOCHEMICAL ENGINEERING FUNDAMENTALS(cid:173)
`MANUFACTURING TECHNOLOGY
`
`Two-Dimensional Fluorescence Spectroscopy: A New Tool for
`On-Line Bioprocess Monitoring
`Stefan Marose, Carsten Lindemann, and Thomas Scheper*
`
`Sniffing Out Trouble: Use of an Electronic Nose in Bioprocesses
`Pradyumna K. Namdev,* Yair Alroy, and Vijay Singh
`
`63
`
`75
`
`3 of 13
`
`Fresenius Kabi
`Exhibit 1015
`
`

`

`2A
`
`Biotechnol. Prag., 1998, Vol. 14, No. 1
`
`79 Bioprocess Fault Detection by Nonlinear Multivariate Analysis: Application of an Artificial
`Autoassociative Neural Network and Wavelet Filter Bank
`Hiroshi Shimizu, Kouichi Yasuoka, Keiji Uchiyama, and Suteaki Shioya*
`
`88 Genetic Approaches to the Detection of Contaminants in Escherichia coli Fermentations
`James R .. Swartz* and Nancy McFarland
`
`92 Purific'atfon of a; Antigenic Vaccine Protein by Selective Displacement Chromatography
`Abhinav A. Shukla, Robert L. Hopfer, Deb. N. Chakravarti, Eric Bartell, and Steven M. Cramer*
`
`102 Solv~nt Evapor~tion Processes for the Production of Controlled Release Biodegradable Microsphere
`Formulations for Therapeutics and Vaccines
`Jeffrey L.·Cleland*
`
`108 Development and Scale-up of a Microsphere Protein Delivery System
`Mark A. Tracy
`
`BIOCHEMICAL ENGINEERING FUNDAMENTALS(cid:173)
`CREATING KNOWLEDGE AND ADVANCING FRONTIERS
`116 Metabolic Engineering of Propanediol Pathways
`D. C. Cameron,* N. E. Altaras, M. L. Hoffman, and A. J. Shaw
`
`126 Engineering a Human Bone Marrow Model: A Case Study on ex Vivo Erythropoiesis
`Athanassios Mantalaris, Peter Keng, Patricia Bourne, Alex Y. C. Chang, and J. H. David Wu*
`
`134 Development of Technologies Aiding Large-Tissue Engineering
`P. Eiselt, B.-S. Kim, B. Chacko, B. Isenberg, M. C. Peters, K. G. Greene, W. D. Roland, A. B. Loebsack,
`K. J. L. Burg, C. Culberson, C. R. Halberstadt, W. D. Holder, and D. J. Mooney*
`
`141 Flow Through, Immunomagnetic Cell Separation
`Jeffrey J. Chalmers,* Maciej Zborowski,* Liping Sun, and Lee Moore
`
`149 Production and Purification of Two Recombinant Proteins from Transgenic Corn
`Ann R. Kusnadi, Elizabeth E. Hood, Derrick R. Witcher, John A. Howard, and Zivko L. Nikolov*
`
`156 Bioprocessing for Tree Production in the Forest Industry: Conifer Somatic Embryogenesis
`Roger Timmis
`
`There is no Supporting Information for this issue.
`
`* In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about
`the paper should be addressed.
`
`I
`
`4 of 13
`
`Fresenius Kabi
`Exhibit 1015
`
`

`

`Biotechnol. Prag., 1998, Vol. 14, No. 1
`
`3A
`
`AUTHOR INDEX
`
`Alroy, Y., 75
`Altaras, N . E., 116
`Bailey, J . E., 8
`Bader, E. T., 55
`Bartell, E., 92
`Bourne, P ., 126
`Bredwell, M. D., 31, 39
`Burg, K. J . L., 134
`Cameron, D. C., 116
`Chacko, B., 134
`Chakravarti, D. N. , 92
`Chalmers, J. J., 141
`Chang, A. Y. C., 126
`Cleland, J. L., 102
`Cramer, S. M., 92
`Culberson, C., 134
`De Bernardez Clark, E.,
`47
`
`Eiselt, P., 134
`
`Greene, K. G., 134
`
`Halberstadt, C. R., 134
`Hamaker, K., 21
`Hevehan, D., 47
`Hoffman, M. L., 116
`Holder, W. D. , 134
`Hood, E. E ., 149
`Hopfer, R. L., 92
`Howard, J. A,., 149
`Hu, W.-S., 1
`Humphrey, A., 3
`
`Isenberg, B., 134
`
`Keng, P ., 126
`Kim, B.-S., 134
`Kusnadi, A. R., 149
`
`Ladisch, C. M., 21
`Ladisch, M. R., 21
`Lindemann, C., 63
`Liu, J., 21
`Loebsack, A. B., 134
`
`Maachupalli-Reddy, J.,
`47
`Mantalaris, A., 126
`Marose, S., 63
`McFarland, N., 88
`Mooney, D. J., 134
`Moore, L., 141
`
`Namdev, P. K., 75
`Nikolov, Z. L. , 149
`
`Peters, M. C., 134
`
`Roland, W. D., 134
`
`Scheper, T., 63
`Shaw, A. J., 116
`Shimizu, H., 79
`Shioya, S., 79
`Shukla, A. A., 92
`Singh, V., 75
`Sun, L. , 141
`Swartz, J. R., 1, 88
`Szela, S., 47
`Timmis, R. , 156
`Tracy, M. A., 108
`Uchiyama, K., 79
`Witcher, D. R., 149
`Wittrup, K. D. , 55
`Worden, R. M., 31, 39
`Wu, J. H. D., 126
`Yasuoka, K., 79
`Zborowski, M., 141
`
`A Publisher Item Identifier (PII) has been adopted by the ACS and several
`other publishers to provide unique identification of individual published
`documents. The PII appears at the bottom of the first page. Include the PII
`in all document delivery requests for copies of the document.
`
`5 of 13
`
`Fresenius Kabi
`Exhibit 1015
`
`

`

`Biotechnol. Prog. 1998, 14, 47−54
`
`47
`
`Oxidative Renaturation of Hen Egg-White Lysozyme. Folding vs
`Aggregation
`
`Eliana De Bernardez Clark,* Diane Hevehan,† Sandra Szela, and
`Jhansi Maachupalli-Reddy‡
`
`Department of Chemical Engineering, Tufts University, Medford, Massachusetts 02155
`
`Since the inception of recombinant DNA technology, different strategies have been
`developed in the isolation, renaturation, and native disulfide bond formation of proteins
`produced as insoluble inclusion bodies in Escherichia coli. One of the major challenges
`in optimizing renaturation processes is to prevent the formation of off-pathway inactive
`and aggregated species. On the basis of a simplified kinetic model describing the
`competition between folding and aggregation, it was possible to analyze the effects of
`denaturant and thiol/disulfide concentrations on this competition. Although higher
`guanidinium chloride (GdmCl) concentrations resulted in higher renaturation yields,
`the folding rate was negatively affected, indicating an optimum range of GdmCl for
`optimum renaturation rates and yields. Similarly, higher total glutathione concentra-
`tions resulted in higher yields but decreased rates, also indicating an optimum total
`glutathione concentration for optimum renaturation rates and yields (6-16 mM), with
`an optimum ratio of reduced to oxidized glutathione between 1 and 3. To characterize
`the nature of aggregates, aggregation experiments were performed under different
`oxidizing/reducing conditions. It is shown that hydrophobic interactions between
`partially folded polypeptide chains are the major cause of aggregation. Aggregation
`is fast and aggregate concentration does not significantly increase beyond the first
`minute of renaturation. Under conditions which promote disulfide bonding, aggregate
`size, but not concentration, may increase due to disulfide bond formation, resulting
`in covalently bonded aggregates.
`
`High expression levels of recombinant proteins in
`bacteria often lead to the formation of inactive aggregates
`or inclusion bodies. Formation of inclusion bodies can
`be advantageous because the product is protected from
`proteolytic degradation, and downstream processing is
`facilitated since the product can be easily isolated from
`cellular components by centrifugation or microfiltration.
`The general strategy used to recover inclusion body
`proteins involves: (1) isolation of inclusion bodies after
`disintegration of cells by mechanical forces, followed by
`washing with detergent solutions, such as Triton X-100,
`or low concentration of denaturants, such as 1-2 M urea;
`(2) solubilization of inclusion bodies with 8 M urea or 6-8
`M guanidinium chloride in combination with reducing
`agents, such as dithiothreitol (DTT), dithioerythritol
`(DTE), or (cid:2)-mercaptoethanol ((cid:2)-ME); and (3) removal of
`denaturant to promote folding (Fischer, 1994; Rudolph
`and Lilie, 1996). In the case of disulfide bonded proteins,
`step 3 is perfomed under oxidizing conditions. To be
`acceptable for commercial applications, renaturation
`processes must be fast and inexpensive and must give
`high yields of active product. Folding yields may be
`limited by misfolding as well as aggregation, the latter
`
`* Corresponding author: fax, 617-627-3991; e-mail, edeberna@
`tufts.edu.
`† Current address: Department of Chemical Engineering, North-
`werstern University, Evanston, IL 60208.
`‡ Current address: Claris Corp., 5201 Patrick Henry Dr., Santa
`Clara, CA 95052.
`
`being favored at high protein concentrations (Zettlmeissl
`et al., 1979; Kiefhaber et al., 1991; Goldberg et al., 1991).
`Several methods, including dilution, dialysis, diafil-
`tration, gel filtration, and immobilization onto a solid
`support, may be employed to remove or reduce excess
`denaturing and reducing agents allowing proteins to
`renature. Dilution of the denatured solution directly into
`renaturation buffer is the easiest process (Thatcher and
`Hitchcock, 1994). Since dialysis is based on the diffusion
`of smaller molecules and ions through membranes, it may
`be too slow to be used in commercial-scale production of
`proteins. Diafiltration is a more practical membrane-
`based alternative because the rate of denaturant removal
`is not diffusion limited. However, accumulation of
`denatured protein on the membrane may limit its ap-
`plication. Gel filtration chromatography has been suc-
`cessfully used to renature secretory leukocyte protease
`inhibitor (Hamaker et al., 1996) and lysozyme (Batas and
`Chaudhuri, 1996). However, problems in flow through
`the column may arise due to protein aggregation upon
`buffer exchange. Aggregation in a chromatographic
`column can be prevented by immobilizing individual
`polypeptide chains onto the matrix (Light, 1985; Creigh-
`ton, 1985). Potential complications may arise if folding
`of the protein is inhibited by binding to the solid support,
`which could be prevented by using fusion proteins
`(Stempfer et al., 1996).
`Another factor to be considered in optimizing a refold-
`ing process is protein purity. Inclusion body proteins can
`be contaminated with varying levels of host proteins,
`
`S8756-7938(97)00123-9 CCC: $15.00
`
`© 1998 American Chemical Society and American Institute of Chemical Engineers
`Published on Web 01/06/1998
`
`6 of 13
`
`Fresenius Kabi
`Exhibit 1015
`
`

`

`48
`
`nucleic acids, and cell membrane components (Thatcher,
`1990; Hart et al., 1990; Valax and Georgiou, 1993). It is
`thought that the presence of these microbial contami-
`nants may induce aggregation, thus reducing renatur-
`ation yields. Maachupalli-Reddy et al. (1997) showed
`that, while nonproteinaceous contaminants have little
`effect on renaturation yields, aggregation of protein
`contaminants can result in significant losses by triggering
`coaggregation of the desired protein.
`The rate-limiting steps in protein folding occur late in
`the pathway, after the rapid formation of compact
`intermediates.
`In proteins containing disulfides, the
`formation of those bonds is the major rate-determining
`step (Hlodan et al., 1991; Fischer, 1994). As the number
`of disulfide bonds in a protein increase, the number of
`possible cysteine combinations upon folding increases
`dramatically. Lysozyme, which contains four disulfide
`bonds, has 105 possible cysteine combinations; while
`BPTI with only three disulfide bonds has only 15
`combinations. It does not seem plausible for an unfolded
`protein to search all possible conformational states within
`the time scale of folding (Levinthal, 1968). Consequently,
`it is generally accepted that proteins follow a finite
`number of pathways (Creighton, 1992). Protein folding
`pathways can be experimentally elucidated by trapping
`intermediates which normally would not accumulate to
`significant levels. Kinetic analysis of disulfide bonded
`proteins is aided by the fact that intermediates contain-
`ing unpaired cysteines can be trapped (Darby and
`Creighton, 1995). The mechanism and order of formation
`of disulfide bonds in BPTI and RNAse have been eluci-
`dated in this manner (Creighton, 1992).
`The most common methods used to promote oxidation
`during refolding are (1) air oxidation, (2) the oxido
`shuffling system, and (3) the use of mixed disulfides
`(Rudolph, 1990). Although air oxidation in the presence
`of trace amounts of metal ions is simple and inexpensive,
`renaturation rates and yields are generally low. Higher
`oxidation rates and yields can be obtained by utilizing
`“oxido shuffling” reagents, low molecular weight thiols
`in reduced and oxidized forms, which allow for both
`formation and reshuffling of disulfide bonds. The most
`common oxido shuffling reagents are reduced and oxi-
`dized glutathione (GSH/GSSG), but the pairs cysteine/
`cystine, cysteamine/cystamine, DTT/oxidized glutathione,
`and DTE/oxidized glutathione have also been utilized.
`Typically a 1-3 mM reduced thiol and a 10:1 to 5:1 ratio
`of reduced to oxidized thiol are used to promote proper
`disulfide bonding (Rudolph and Lilie, 1996). More re-
`cently, Buchner and Rudolph (1992) and Hevehan and
`De Bernardez Clark (1997) showed that optimum rena-
`turation yields are obtained when the ratio of reduced
`to oxidized thiol is anywhere between 1:1 and 3:1.
`Another strategy employed to oxidize proteins during
`folding is the formation of mixed disulfides between
`oxidized glutathione and reduced protein before rena-
`turation (Rudolph, 1990). Formation of mixed disulfides
`increases the solubility of the denatured protein by
`increasing the hydrophilic character of the polypeptide
`chain (Fischer, 1994; Rudolph and Lilie, 1996). Disulfide
`bond formation is then promoted by adding catalytic
`amounts of a reducing agent in the renaturation step.
`Formation of off-pathway species, such as incorrectly
`folded species and aggregates, is the cause of decreased
`renaturation yields.
`Intermediates with hydrophobic
`patches exposed to the solvent play a crucial role in the
`partition between native and aggregated conformations.
`Folding intermediates possess significant elements of the
`secondary structure but little of the native tertiary
`
`Biotechnol. Prog., 1998, Vol. 14, No. 1
`
`structure. Due to the expanded volume of these inter-
`mediates, hydrophobic patches, which are normally
`buried in the native state, are exposed to the solvent.
`When hydrophobic regions on separate polypeptide chains
`interact, intermediates are diverted off the correct folding
`pathway into aggregates. Because aggregation is an
`intermolecular phenomenon, it is highly protein concen-
`tration dependent. The most direct means of minimizing
`aggregation is by decreasing protein concentration. It
`has been suggested that optimum recovery yields can be
`expected if the protein concentration is in the range 10-
`50 μg/mL (Rudolph and Lilie, 1996). Renaturation at
`such low protein concentrations requires large volumes
`of refolding buffer, driving production costs upward. To
`address this problem, Rudolph and Fischer (1990) and
`Fischer et al. (1992) developed a “pulse renaturation” or
`step addition method, which reduces renaturation vol-
`umes by stepwise addition of denatured protein into the
`refolding solution. Enough time is allowed between
`additions for the protein to fold past the early stages in
`the folding pathway, when it is susceptible to aggrega-
`tion. By keeping the protein concentration low in each
`aliquot, high final renaturation yields at high final
`protein concentrations can be obtained (Rudolph and
`Fischer, 1990; Buchner et al., 1992; Fischer et al., 1992).
`A variety of additives have been tested for their ability
`to prevent aggregation. They may act by stabilizing the
`native state, by preferentially destabilizing incorrectly
`folded molecules, by increasing the solubility of folding
`intermediates, or by increasing the solubility of the
`unfolded state. In general, these additives do not seem
`to accelerate the rate of folding, but they do inhibit the
`unwanted aggregation reaction. Among the additives
`tested are sugars, such sucrose (Valax and Georgiou,
`1991) and glycerol (Timasheff and Arakawa, 1989);
`amphiphilic polymers and micelle-forming surfactants,
`such as Triton X-100, CHAPS, poly(vinylpyrrolidone),
`octa(ethylene glycol) monolauryl ether (Wetlaufer and
`Xie, 1995); alkanols, such as n-pentanol, n-hexanol, and
`cyclohexanol (Wetlaufer and Xie, 1993); sulfobetaines
`(Goldberg et al., 1996); poly(ethylene glycol) (Cleland et
`al., 1992); L-arginine (Rudolph, 1990); and low concentra-
`tions of denaturants (Wetlaufer and Xie, 1995; Hevehan
`and De Bernardez Clark, 1997).
`Pioneer work by Goldberg et al. (1991) shed light into
`the nature of interactions responsible for aggregation
`during folding. They showed that incorrect disulfide
`bonding may not be the major cause of aggregation
`because aggregates were formed even when a carboxy-
`methylated protein was folded. By allowing a mixture
`of turkey lysozyme and excess bovine serum albumin to
`simultaneously renature under oxidizing conditions, they
`observed that the lysozyme molecules were trapped in
`heterologous aggregates with BSA, thus showing that
`aggregation is a nonspecific phenomenon. On the other
`hand, Speed et al. (1996) recently reported that in mixed
`folding experiments using the P22 tailspike and coat
`proteins, folding intermediates of the two proteins did
`not coaggregate, but that they rather preferred to self-
`associate, suggesting that aggregation is a specific phe-
`nomenon. Since they only analyzed soluble aggregates,
`Speed et al. (1996) suggested that it is possible that larger
`aggregates could grow by a different mechanism involv-
`ing nonspecific interactions. The specific nature of the
`aggregation phenomenon had been originally proposed
`by London et al. (1974), who showed that foreign proteins
`did not interfere with the refolding of tryptophanase.
`More recently, Maachupalli-Reddy et al. (1997) provided
`new evidence of the nonspecific nature of the aggregation
`
`7 of 13
`
`Fresenius Kabi
`Exhibit 1015
`
`

`

`Biotechnol. Prog., 1998, Vol. 14, No. 1
`
`reaction by conducting mixed renaturation studies with
`hen egg-white lysozyme and three foreign proteins:
`(cid:2)-galactosidase, bovine serum albumin (BSA), and ribo-
`nuclease A (RNAse A). They found that foreign proteins
`which have a tendency to aggregate when folded in
`isolation, such as (cid:2)-galactosidase and BSA, significantly
`decreased lysozyme renaturation yields by promoting
`aggregation. On the other hand, the presence of refolding
`RNAse A, which does not significantly aggregate upon
`folding in isolation, did not affect lysozyme renaturation
`yields.
`
`Materials and Methods
`Materials. Hen egg-white lysozyme (Lot No. 111H-
`7010), three-times crystallized, dialyzed, and lyophilized,
`was obtained from Sigma Chemical Co. Dithiothreitol
`(DTT) and Micrococcus lysodeikticus cells were also from
`Sigma. Solutions of reduced DTT were prepared im-
`mediately prior to each experiment to minimize air
`oxidation. To avoid artifacts, GdmCl of the ultrapure
`quality was purchased from ICN Biomedicals Inc. HPLC
`grade oxidized glutathione (GSSG) was purchased from
`Calbiochem-Novabiochem Co. All other chemicals were
`reagent grade. The composition of TE buffer was 50 mM
`tris and 1 mM EDTA, with a final pH of 8.
`Denaturation/Reduction. Lysozyme was denatured
`and reduced in a solution of 8 M GdmCl containing 16-
`96 mM DTT, in TE buffer. The resulting protein solu-
`tions were incubated for 1 h at 37 °C. After reaching
`room temperature, concentrations of denatured lysozyme
`were determined spectrophotometrically as described
`below.
`Renaturation/Oxidation. Following denaturation
`and reduction, renaturation was initiated by a rapid
`8-fold or 16-fold dilution of the denatured lysozyme into
`renaturation buffer consisting of TE buffer with various
`amounts of GSSG. Final protein concentration was 1 mg/
`mL unless otherwise indicated. Disulfide bond formation
`during folding was stopped by quenching with iodoacetic
`acid, as previously described (Hevehan and De Bernardez
`Clark, 1997). Aggregation during folding was monitored
`using turbidity measurements. Undiluted protein samples
`were analyzed for light scattering at 600 nm using a
`Hewlett-Packard 8452A photodiode array spectropho-
`tometer.
`Aggregation Experiments. Each time point in a
`typical aggregation experiment was obtained in the
`following manner: 100 μL of 16 mg/mL lysozyme in 8 M
`GdmCl, TE buffer, pH 8, was added to 1500 μL of
`renaturation buffer containing either 1.067, 4.267, or
`13.867 mM GSSG in TE buffer, pH 8, in a 2000 μL
`microcentrifuge tube. After the desired time had elapsed
`(15 s to 10 min), disulfide bonding was stopped by adding
`160 μL of a 0.5 M iodoacetic acid solution in 0.5 M
`potassium hydroxide and 0.5 M tris-HCl, pH 7 (Darby
`and Creighton, 1995). Immediately after quenching, the
`refolding mixture was centrifuged at 14 000 rpm and 4
`°C for 14 min. The supernatant was analyzed for
`enzymatic activity and protein concentration as described
`below. The pellet was washed twice with TE buffer, pH
`8, and was allowed to dissolve in 8 M GdmCl, TE buffer,
`pH 8, for 1 h at 37 °C with vigorous agitation. The
`redissolved fraction of the pellet (non-disulfide-bonded
`aggregates) was separated from the insoluble fraction
`(disulfide-bonded aggregates) by centrifugation at 14 000
`rpm and 4 °C for 14 min. The supernatant was analyzed
`for protein concentration as described below. The pellet
`was washed twice with TE buffer, pH 8, and solubilized
`
`100
`
`80
`
`~ 60
`j::
`Si!
`~ 40
`
`20
`
`0
`
`Jj
`
`49
`
`•- - - -.
`
`1.2
`
`~ 0.9
`
`....
`
`C
`JJ
`gi
`0.6 0
`:;!
`
`0.3
`
`0
`1.2
`
`0
`
`0.2
`
`0.8
`0.6
`0.4
`[protein] (mg/ml)
`Figure 1. Effect of final lysozyme concentration of renaturation
`yield and turbidity measured 3 h after folding was initiated.
`Folding conditions: 0.5 M GdmCl, 2 mM DTT, 5 mM GSSG, 50
`mM tris, 1 mM EDTA, pH 8, 22 °C.
`
`with 8 M GdmCl, 32 mM DTT, TE buffer, pH 8. The
`concentration of solubilized protein was measured as
`described below.
`Enzyme Assay. Enzymatic activity was used to
`measure the concentration of native protein. The lysozyme
`activity assay was a modification of the method used by
`Jolles (1962), as described by Hevehan and De Bernardez
`Clark (1997).
`Protein Concentration. Protein concentrations were
`determined by measuring absorbance at 280 nm with the
`appropriate blank, using extinction coefficients of 2.63
`and 2.37 ((cm mg)/mL)-1 for native and denatured
`lysozyme, respectively (Wetlaufer et al., 1974). Absor-
`bance measurements were conducted with a Hewlett-
`Packard 8452A photodiode array spectrophotometer.
`
`Results
`Kinetic Competition between Folding and Ag-
`gregation. To test the effect of final protein concentra-
`tion on the competition between folding and aggregation,
`0.8-16 mg/mL of denatured reduced lysozyme were
`diluted 16-fold into renaturation buffer obtaining solu-
`tions of variable protein concentration (0.05 to 1 mg/mL)
`in 0.5 M GdmCl, 2 mM DTT, 5 mM GSSG, TE buffer,
`pH 8, 22 °C. Samples were incubated for 3 h atroom
`temperature and assayed for activity. The results are
`shown in Figure 1 where % activity represents the
`conversion of denatured to native protein and turbidity
`qualitatively describes the accumulation of aggregates.
`Figure 1 shows that, as final protein concentration
`increases, renaturation yield decreases while turbidity
`increases, indicating the strong protein concentration
`dependence of the aggregation pathway.
`To determine if lysozyme concentration in the dena-
`tured state influences renaturation yields, solutions of
`denatured reduced lysozyme ranging from 1.6 to 30 mg/
`mL were prepared and then diluted (16-300-fold, ac-
`cordingly) into renaturation buffer to identical refolding
`conditions at 0.1 mg/mL (Table 1). The renaturation
`buffer was supplemented with GdmCl and DTT to
`maintain similar refolding conditions: 0.5 M GdmCl, 2
`mM DTT, 5 mM GSSG, TE buffer, pH 8, 22 °C. Samples
`were incubated for 3 h atroom temperature and assayed
`for activity. Table 1 shows that the renaturation