Protein Expression and Purification 22, 101–107 (2001)
`doi:10.1006/prep.2001.1418, available online at http://www.idealibrary.com on
`
`Refolding and Purification of Yeast Carboxypeptidase Y
`Expressed as Inclusion Bodies in Escherichia coli
`
`Moon Sun Hahm and Bong Hyun Chung1
`Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon 305-600, Korea
`
`Received December 11, 2000, and in revised form February 5, 2001; published online May 7, 2001
`
`The carboxypeptidase Y (CPY)2 from Saccharomyces
`cerevisiae has been extensively studied as a model for
`The genes encoding carboxypeptidase Y (CPY) and
`CPY propeptide (CPYPR) from Saccharomyces cerevis-
`vacuolar protein transport, targeting, and maturation
`iae were cloned and expressed in Escherichia coli. Six
`(1). The gene for CPY encodes an inactive precursor
`consecutive histidine residues were fused to the C-ter-
`(preproenzyme) containing a signal peptide of 20 amino
`minus of the CPYPR for facilitated purification. High-
`acids followed by a propeptide of 91 amino acids and a
`level expression of CPY and CPYPR-His6 was achieved mature form of 421 amino acids. In the endoplasmic
`but most of the expressed proteins were present in the
`reticulum the signal peptide is cleaved off to form pro-
`form of inclusion bodies in the bacterial cytoplasm. CPY which is still inactive. After pro-CPY is targeted
`The CPY and CPYPR-His6 produced as inclusion bodies
`into the vacuole, the pro-CPY is processed and activated
`were separated from the cells and solubilized in 6 and into the mature CPY by cleavage of the N-terminal
`3 M guanidinium chloride, respectively. The denatured proregion (2, 3). In this process, the CPY propeptide
`CPYPR-His6 was refolded by dilution 1:30 into the rena-
`(CPYPR) plays an important role in folding and tar-
`turation buffer (50 mM Tris–HCl containing 0.5 M NaCl
`geting of CPY (4, 5).
`and 3 mM EDTA, pH 8.0), and the refolded CPYPR-
`To date, a few successful attempts have been made
`His6 was further purified to 90% purity by single-step to overproduce CPY via recombinant DNA technology,
`immobilized metal ion affinity chromatography. The mostly using S. cerevisiae as an expression host (6, 7).
`denatured CPY was refolded by dilution 1:60 into the The yeast seems to be the best candidate as a host for
`renaturation buffer containing CPYPR-His6 at various
`the overproduction of mature proteins from inactive
`concentrations. Increasing the molar ratio of CPYPR-
`and propeptide-containing zymogens like pro-CPY, be-
`His6 to CPY resulted in an increase in the CPY refolding
`cause Escherichia coli, the most widely used recombi-
`yield, indicating that the CPYPR-His6 plays a chaper- nant host, does not harness the capability for trans-
`one-like role in in vitro folding of CPY. The refolded forming zymogen into a mature form by in vivo
`CPY was purified to 92% purity by single-step p-amino-
`proteolytic processing. More recently, however, we have
`benzylsuccinic acid affinity chromatography. When reported an interesting study on the production of ac-
`refolding was carried out in the presence of 10 molar
`tive CPY using E. coli (8). Two different pro-CPYs from
`eq CPYPR-His6, the specific activity, N-(2-furanacryl- Hansenula polymorpha and S. cerevisiae were ex-
`oyl)-L-phenylalanyl-L-phenylalanine hydrolysis activ-
`pressed as inclusion bodies in E. coli, and a refolding
`ity per milligram of protein, of purified recombinant
`process was developed to obtain active CPYs from inclu-
`CPY was found to be about 63% of that of native
`sion bodies. In this process, the renatured pro-CPY was
`S. cerevisiae CPY.
`q 2001 Academic Press
`
`1 To whom correspondence should be addressed. Fax: 182-42-860-
`4594. E-mail: chungbh@mail.kribb.re.kr.
`
`1046-5928/01 $35.00
`Copyright q 2001 by Academic Press
`All rights of reproduction in any form reserved.
`
`2 Abbreviations used: CPY, carboxypeptidase Y; CPYPR, carboxy-
`peptidase Y propeptide; CPYPR-His6, carboxypeptidase Y propeptide
`with six consecutive histidines at the C-terminus; FAPP, N-(2-furana-
`cryloyl)-L-phenylalanyl-L-phenylalanine; GdmCl, guanidinium chlo-
`ride; IMAC, immobilized metal ion affinity chromatography; molar
`isopropyl-b-D-thiogalactopyranoside;
`eq, molar equivalent; IPTG,
`PVDF, polyvinylidene difluoride.
`
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`102
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`HAHM AND CHUNG
`
`resulting plasmids were designated pECPYPRH and
`pECPY, respectively.
`
`digested in vitro by proteinase K, and then CPY was
`activated by the cleaved CPYPR.
`The present study describes a novel method to obtain
`active CPY from CPY inclusion bodies overexpressed Culture and Recovery of Inclusion Bodies
`in E. coli. Here we used the recombinant CPYPR pro-
`The plasmids pECPYPRH and pECPY were trans-
`duced by E. coli to stimulate the CPY refolding and
`formed into E. coli BL21(DE3). The transformants were
`purified the refolded CPY by single-step p-amino-
`grown to 0.6 OD600 at 378C in shake flasks containing
`benzylsuccinic acid affinity chromatography. In addi-
`50 mL of Luria–Bertani medium (0.5% yeast extract,
`tion, the specific activity of the purified recombinant
`1% tryptone, and 1% NaCl) containing 100 mg/mL ampi-
`CPY was compared with that of native CPY from S.
`cillin. Expression was induced by the addition of isopro-
`cerevisiae.
`pyl-b-D-thiogalactopyranoside
`(IPTG)
`(Sigma, St.
`Louis, MO) to a final concentration of 1 mM, and the
`cells were grown for an additional 4 h at 378C. The cells
`were harvested by centrifugation at 6000g for 10 min
`and lysed by sonication. The soluble and insoluble frac-
`tions were then separated by centrifugation at 12,000g
`for 10 min at 48C.
`
`Construction of CPY and CPYPR Expression Vectors
`
`MATERIALS AND METHODS
`Strains, Plasmids, and Enzymes
`E. coli DH5a[F 2 lacZ M15 hsdR17(r 2 m2) gyrA36]
`was used as the host for subcloning and E. coli
`BL21(DE3) (Novagen, Madison, WI) for gene expres-
`sion. The plasmid pBluescript(SK+) (Stratagene, La
`Solubilization of Inclusion Bodies, Refolding, and
`Jolla, CA) was used for subcloning and amplification of
`Purification
`CPYPR and CPY genes and the plasmid pET22b(1)
`(Novagen) was used for expression. Restriction en-
`The transformants were harvested from 500 mL cul-
`zymes and modifying enzymes were purchased from ture medium, resuspended in 50 mM Tris–HCl buffer
`Boehringer-Mannheim (Germany) and used according
`(pH 8.0) and disrupted by sonication. The CPYPR-His6
`to the recommendations of the supplier. Plasmid DNA and CPY inclusion bodies were solubilized in 50 mM
`was purified using a QIAEX II gel extraction kit (Qia- Tris–HCl/3 mM EDTA buffer (pH 8.0) (Buffer A) con-
`gen, Germany). Other DNA manipulation experiments
`taining 3 M and 6 M guanidinium chloride (GdmCl),
`were performed as described by Sambrook et al. (9).
`respectively. The denatured CPYPR-His6 and CPY were
`rapidly diluted into Buffer A containing 0.5 M NaCl to
`give a final GdmCl concentration of 0.1 M. In the refold-
`ing of CPY, 3 molar eq of CPYPR-His6 was added into
`The cDNA encoding CPYPR with six consecutive his-
`the renaturation buffer, unless otherwise specified.
`tidines at the C-terminus (CPYPR-His6) was amplified
`The refolded CPYPR-His6 was purified by immobi-
`by PCR (Perkin-Elmer 2400, NJ) using S. cerevisiae
`lized metal ion affinity chromatography (IMAC) using
`a His6-tag at the C-terminus. Cellufine chelate resin
`2805 (10) genomic DNA library as template and two
`primers: 58-CTAGGCCATATGATCTCATTGCAAAGA-
`(Amicon, Beverly, MA) (8 mL) was packed into a glass
`CCG-38 (new NdeI site, 58 to start codon) and 58-GTG-
`column (10 3 130 mm, Amicon), and the column was
`GTGCTCGAGGTTGACACGAAGCTGATA-38
`(new
`saturated with Ni(II) ions. The column was washed
`XhoI site). The PCR product was gel purified using a with distilled water and finally with 20 mM Tris–HCl/
`QIAEX II gel extraction kit (Qiagen), digested with
`0.5 M NaCl buffer (pH 8.0). The solution containing
`refolded CPYPR-His6 was loaded onto the column. For
`NdeI and XhoI (Boehringer Mannheim), and inserted
`into a pBluescript(SK+) vector (Stratagene), resulting
`the elution of proteins, a continuous imidazole gradient
`was applied by the gradual mixing of 20 mM Tris–HCl/
`in the plasmid pBCPYPRH.
`The CPY cDNA coding sequence was amplified using
`0.5 M NaCl buffer (pH 8.0) containing 1 M imidazole
`S. cerevisiae 2805 genomic DNA as the template. The with 20 mM Tris–HCl/0.5 M NaCl buffer (pH 8.0).
`primers used were 58-CTTCGTCATATGAAGATTAA-
`The refolded CPY was purified by p-aminobenzylsuc-
`GGACCCTAAAATC-38 with an NdeI site and 58-
`cinic acid affinity chromatography. The preparation of
`CTCGAGTTATAAGGAGAAACCACCGTGGATCCA-
`p-aminobenzylsuccinic acid affinity gel was made as
`38 with a XhoI site. The PCR product (1270 bp) was gel
`follows. Ice-cold 1 M HCl (37 mL) was added to the p-
`purified, digested with NdeI and XhoI, and cloned into
`aminobenzylsuccinic acid (Sigma) solution followed by
`pBluescript (SK+), resulting in the plasmid pBCPY.
`390 mg NaNO2 in 20 mL water. After 30 min of reaction
`at 08C, the diazotized benzylsuccinate was added to 10
`The plasmids pBCPYPRH and pBCPY were digested
`with NdeI and XhoI. The fragments isolated from these mL of Amino Spherilose resin (Isco Inc., Lincoln, NE)
`plasmids were directly ligated into pET22b(1) digested
`in 10 mL of 0.1 M Na2CO3. The pH was adjusted to 9.5.
`with the same restriction enzymes, respectively. The The Amino Spherilose resin slowly turned orange, and
`
`Page 2
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`

`REFOLDING AND PURIFICATION OF YEAST CPY
`
`103
`
`after 6 h of reaction at 08C the gel was washed with
`0.1 M NaHCO3 solution (pH 9.5) and several times with
`water. The affinity gels (8 mL) prepared were packed
`in a glass column (10 3 130 mm, Amicon) and then
`equilibrated with 0.01 M Mes buffer (pH 6.0). The col-
`umn was washed with 0.01 M Mes buffer (pH 6.0) con-
`taining 0.5 M NaCl, and the bound CPY eluted with
`0.1 M succinic acid solution (pH 6.0) containing 0.5
`M NaCl.
`
`SDS–PAGE Analysis and Determination of CPY
`Activity
`The soluble fraction was resuspended in a volume of
`gel loading buffer (0.5 M Tris–HCl, pH 6.8, 2% glycerol,
`10% SDS, 0.1% bromophenol blue) to give a final con-
`centration equivalent to the insoluble fraction so that
`relative soluble/insoluble protein concentration in the
`SDS–PAGE gels represent those in the original culture.
`Cell proteins were separated on a 12% (w/v) SDS–
`polyacrylamide gel and detected with Coomassie bril-
`liant blue R-250 staining. Quantitative protein determi-
`nation was made densitometrically (Bio-Rad GS-700,
`Hercules, CA) by reading the band intensities of Coo-
`massie-stained gels. Total protein concentration was
`determined by the method of Bradford (11). The activity
`of CPY was measured at 378C from the decrease in
`absorbance at 330 nm of 0.2 mM N-(2-furanacryloyl)-
`L-phenylalanyl-L-phenylalanine (FAPP) (Sigma) in 50
`mM Mes buffer (pH 6.0) containing 1 mM EDTA, as
`described by Peterson et al. (12).
`
`FIG. 1. Physical map of the plasmids, (A) pECPYPRH and (B)
`pECPY, for expression of CPYPR-His6 and CPY, respectively.
`
`FIG. 2. SDS–PAGE analysis of proteins expressed in E. coli
`BL21(DE3) harboring the plasmids (A) pECPYPRH and (B) pECPY.
`Lane 1, molecular weight marker; lane 2, total cellular fraction, no
`IPTG induction; lane 3, total cellular fraction, IPTG induction; lane
`4, inclusion body fraction; lane 5, soluble fraction; lane 6, purified
`CPYPR-His6.
`
`RESULTS AND DISCUSSION
`Expression of CPYPR-His6 and CPY in E. coli
`The plasmids pECPYPRH and pECPY constructed
`to express CPYPR-His6 and CPY, respectively, are based
`on the T7 expression system of the pET plasmids (Nova-
`gen), and a translational start codon was inserted in
`frame with each of the cDNA sequences encoding
`CPYPR and CPY (Fig. 1). The synthetic gene fragment
`encoding six consecutive histidines was fused to the 38
`end of the CPYPR gene to facilitate purification of the
`encoded product using immobilized metal ion affinity
`chromatography.
`Figure 2 shows the expression of CPYPR-His6 and
`CPY in E. coli. The CPYPR was very stably expressed
`in E. coli, although it is a relatively small polypeptide
`with a molecular weight of 11 kDa (Fig. 2A). Utilizing
`this property, we have developed CPYPR as a fusion
`partner for the efficient expression of small polypep-
`tides in E. coli (13). Densitometry analysis shows that
`the CPYPR-His6 expressed accounts for about 30% of
`the total cellular protein (Fig. 2A, lane 3). Induction
`
`Page 3
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`

`104
`
`HAHM AND CHUNG
`
`of E. coli transformant harboring the plasmid pECPY
`resulted in high-level expression of the protein with a
`molecular weight of approximately 47 kDa, accounting
`for 30% of the total cellular protein (Fig. 2B, lane 3).
`It was also found that most of the CPYPR-His6 and
`CPY expressed existed in the form of inclusion bodies
`in the cytoplasm of E. coli. According to densitometric
`scanning, the CPYPR-His6 and CPY inclusion body
`preparations were 65 and 69% pure, respectively.
`The CPYPR-His6 and CPY proteins separated by
`SDS–PAGE were transferred to a PVDF membrane
`filter, excised, and then subjected to N-terminal amino
`acid sequencing. The first amino acid sequences of
`CPYPR-His6 and CPY were revealed to be Met-Ile-Ser-
`Leu-Gln and Met-Lys-Ile-Lys-Asp, respectively, which
`are in good agreement with the expected amino acid
`sequences.
`
`FIG. 4. Effect of temperature on the refolding yield. The refolding
`was carried out in the renaturation buffer (pH 8.0) containing 0.5 M
`NaCl and 3 molar eq of CPYPR-His6.
`
`Solubilization of CPYPR-His6 Inclusion Bodies,
`Refolding, and Purification
`
`In addition, there is no cysteine in the CPYPR. It ap-
`pears that these properties make the CPYPR inclusion
`After sonication of the transformed cells harboring
`bodies soluble at relatively low GdmCl concentrations.
`the plasmid pECPYPRH, the inclusion bodies were sep-
`The denatured CPYPR-His6 was refolded by dilution
`arated by centrifugation and washed with 50 mM Tris–
`1:30 into Buffer A containing 0.5 M NaCl to give a
`HCl buffer (pH 8.0). The inclusion bodies harvested
`final CPYPR-His6 concentration of 500 mg/mL. Upon
`were solubilized in Buffer A containing 3 M GdmCl. We
`dilution, no aggregates were observed, indicating effi-
`investigated the degree of solubilization of CPYPR-His6
`cient refolding of CPYPR-His6. The diluted solution was
`inclusion bodies in Buffer A containing GdmCl at vary-
`subjected to the IMAC for purification of the refolded
`ing concentrations. As a result, the CPYPR-His6 inclu- CPYPR-His6. The refolded CPYPR-His6 was purified to
`sion bodies were found to be completely soluble at 3 M 90% purity by single-step IMAC (Fig. 2A, lane 6). The
`GdmCl which is much lower concentration than the CPYPR-His6 was dialyzed against 50 mM Tris–HCl
`commonly used GdmCl concentration (6 M) for inclu-
`buffer (pH 8.0) and then lyophilized to dryness. These
`sion body solubilization. From inspection of the primary
`recombinant CPYPR-His6 preparations were used for
`sequence of the CPYPR it has been speculated that the
`activating the inactive CPY produced by E. coli.
`frequency of charged amino acids is relatively high (14).
`
`Solubilization of CPY Inclusion Bodies and
`Refolding
`After sonication of the transformed cells harboring
`the plasmid pECPY, the inclusion bodies were prepared
`as described above. The inclusion bodies harvested were
`solubilized in Buffer A containing 6 M GdmCl and re-
`folded by dilution 1:60 into Buffer A to give a final CPY
`concentration of 20 mg/mL. In contrast to the CPYPR-
`His6 inclusion bodies, the CPY inclusion bodies were
`not solubilized at 3 M GdmCl, and a very low CPY
`concentration in the renaturation buffer was required
`to reduce protein aggregation upon dilution. The refold-
`ing yield was estimated from the specific FAPP hydroly-
`sis activity of the refolded CPY relative to that of native
`S. cerevisiae CPY (sequencing grade, Sigma). As ex-
`pected, refolding of CPY in the absence of CPYPR-His6
`resulted in a very low refolding yield of 3%.
`One of the major roles of the CPYPR is to mediate
`correct folding of CPY by acting as a cotranslational
`
`FIG. 3. Time course of CPY refolding in the presence of varying
`molar ratios of CPYPR-His6 to CPY. Molar ratio 0, M; 1, m; 3, m; 5,
`v; 10l.
`
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`

`REFOLDING AND PURIFICATION OF YEAST CPY
`
`105
`
`has a chaperone-like function to stimulate the CPY
`folding.
`The protein refolding efficiency is often dependent
`on the environmental parameters such as temperature
`and pH of renaturation buffer. Figures 4 and 5 show
`the effects of temperature and pH on the CPY refolding
`yield in the absence and presence of CPYPR-His6, re-
`spectively. In the absence of CPYPR-His6, the refolding
`yield was not greatly affected by pH and temperature.
`In contrast, the optimal pH and temperature showing
`the maximum refolding yield clearly exist when the
`CPYPR-His6 is present in the renaturation buffer. The
`maximum refolding yield was observed at pH 8 and
`248C.
`FIG. 5. Effect of pH on the refolding yield. The refolding was carried
`A partial renaturation of CPY has been achieved in
`out in the renaturation at different pH values at 248C in the presence high concentrations of various salts in the absence of
`(m) and absence (m) of 3 molar eq of CPYPR-His6.
`CPYPR (14). The refolding yield of about 10% was ob-
`tained in the presence of 0.9 M (NH4)2SO4, 0.7 M
`Na2SO4, and 1.5 M NaCl. This refolding yield is lower
`than that (16.5%) obtained in the presence of 10 molar
`eq of CPYPR-His6. In this work, 0.5 M NaCl was added
`into the renaturation buffer. The refolding yield of ,3%
`was achieved at 0.5 M NaCl in the absence of CPYPR-
`His6. This indicates that the effect of 0.5 M NaCl on
`CPY refolding is negligible.
`The mature CPY contains five disulfide bonds. At
`first, therefore, the CPY inclusion bodies were solubi-
`lized in the solubilization buffer containing the reduc-
`ing agents such as 100 mM b-mercaptoethanol and 300
`
`chaperone (14, 15). It has been shown that in vivo fold-
`ing of CPY is strictly dependent on the presence of
`CPYPR (4). Therefore, the CPYPR-His6 prepared as
`described above was added to the renaturation buffer
`to promote the in vitro folding of CPY. The refolding
`yield was increased from 3% in the absence of CPYPR
`to 4.5 and 16.5% in the presence of 1 and 10 molar
`eq of CPYPR-His6, respectively (Fig. 3). The maximum
`refolding yield was attained after 120 min, after which
`the refolding yield remained constant for 180 min.
`These results clearly demonstrate that CPYPR-His6
`
`FIG. 6. Purification of refolded CPY by p-aminobenzylsuccinic acid affinity chromatography. The arrow indicates the CPY-containing
`fractions. The detailed procedure is described under Materials and Methods.
`
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`

`106
`
`HAHM AND CHUNG
`
`mM dithiothreitol, and then refolding was carried out
`as described above. However, the CPY inclusion bodies
`were fully solubilized in 6 M GdmCl-containing buffer
`without the reducing agents. Furthermore, the refold-
`ing yield was not increased by the addition of such
`reducing agents.
`
`Purification of Refolded CPY
`
`After refolding of the denatured CPY in the presence
`of 10 molar eq CPYPR-His6, the refolded CPY was puri-
`fied using p-aminobenzylsuccinic acid affinity chroma-
`tography. The p-aminobenzylsuccinic acid affinity chro-
`matography has been used to purify CPY from baker’s
`yeast (16). Therefore, the affinity gel coupled with p-
`aminobenzylsuccinic acid, a strong inhibitor of CPY,
`was prepared in our laboratory for purification of the
`FIG. 7. Hydrolysis of FAPP by the purified recombinant CPY (m)
`and native CPY (v) at the same CPY protein concentration (0.05
`refolded CPY. The CPY, specifically bound with p- mg/mL). The protein concentration was measured by the Bradford
`aminobenzylsuccinic acid, eluted with 0.1 M succinic method as described under Materials and Methods. As shown in
`acid solution (pH 6.0) containing 0.5 M NaCl (Fig. 6).
`SDS–PAGE analysis, an apparent molecular weight of native CPY
`The CPY fraction collected was dialyzed against 50 mM (nCPY) is greater than that of recombinant CPY (rCPY) due to the
`Mes buffer (pH 6.0) containing 1 mM EDTA and then
`glycosylation. Therefore, the molar concentration, considering a gly-
`cosylation moiety, of rCPY is approximately 1.3 times that of nCPY.
`stored at 48C.
`The purification results are summarized in Table 1.
`The final purity and the recovery yield are 92 and 13%, with recombinant CPY (16.5%) (Fig. 8). From this re-
`respectively. The specific activity of the purified recom-
`sult, it can be reasoned that the glycosylation of CPY
`does not greatly affect its enzymatic activity. In a previ-
`binant CPY corresponded to about 63% of that of the
`native S. cerevisiae CPY (Sigma). As shown on SDS–
`ous study on the functional roles of glycosylation in
`yeast CPY, it has also been revealed that glycosylation
`PAGE in Fig. 7, an apparent molecular weight of native
`CPY is approximately 61 kDa which much greater than
`is required for efficient intracellular transport of CPY,
`but not for vacuolar sorting, in vivo stability, or activity
`that of recombinant CPY (47 kDa). This is because the
`native CPY is a glycoprotein, whereas the recombinant
`(17). Therefore, it appears that the lower specific activ-
`ity shown in the purified recombinant CPY lacking a
`CPY produced from E. coli is not glycosylated.
`To investigate whether a glycosylation moiety of CPY
`glycosylation moiety is caused by the presence of soluble
`affects the enzyme activity or not, the denaturation and misfolded and/or partially folded CPY.
`refolding experiment was carried out with native CPY
`In this study, we have developed a novel method that
`can produce active yeast CPY from inactive inclusion
`as previously done with recombinant CPY. The se-
`quence grade native CPY purchased from Sigma was
`bodies expressed in E. coli. A number of proteases are
`synthesized as zymogens that are only rendered active
`also denatured in Buffer A containing 6 M GdmCl and
`refolded by dilution 1: 60 into Buffer A containing 0.5
`upon proteolytic removal of the propeptide. In most
`cases, one of the main functions of the propeptide in
`M NaCl and 10 molar eq of CPYPR. A final refolding
`yield was 16.8% which is very similar to that obtained
`zymogen has been shown to promote correct folding
`
`TABLE 1
`Purification Summary of Recombinant CPY from E. coli Cell Lysatea
`
`Purification step
`
`Total protein
`(mg)
`
`CPY
`(mg)b
`
`Purity
`(%)
`
`Recovery yield
`(%)
`
`34
`85
`250
`Total cell lysate
`69
`75
`109
`Preparation of inclusion bodies after washing with 3 M urea
`24
`16
`66
`Soluble fraction after refolding
`92
`11
`12
`p-aminobenzylsuccinic acid affinity chromatography
`a The total cell lysate containing 250 mg protein was obtained from about 450 mL of cell culture (,4.7 g of cells, wet weight).
`b The amount of CPY was quantified by scanning the CPY band on SDS–polyacrylamide gel with a densitometer (Bio-Rad Model GS-700
`imaging densitometer, Bio-Rad Laboratories, Hercules, CA).
`
`100
`88
`19
`13
`
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`REFOLDING AND PURIFICATION OF YEAST CPY
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`107
`
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`428–434.
`14. Winther, J. R., and Sorensen, P. (1991) Propeptide of carboxypep-
`tidase Y provides a chaperone-like function as well as inhibition
`of the enzyme activity. Proc. Natl. Acad. Sci. USA 88, 9330–9334.
`15. Winther, J. R., Sorensen, P., and Kielland-Brandt, M. C. (1994)
`Refolding of carboxypeptidase Y folding intermediate in vitro
`by low-affinity binding of the proregion. J. Biol. Chem. 269,
`22007–22013.
`16. Johansen, J. T., Breddam, K., and Ottesen, M. (1976) Isolation
`of carboxypeptidase Y by affinity chromatography. Carlsberg Res.
`Commun. 41, 169–182.
`17. Winther, J. R., Stevens, T. H., and Kielland-Brandt, M. C. (1991)
`Yeast carboxypeptidase Y requires glycosylation for efficient in-
`tracellular transport, but not for vacuolar sorting, in vivo stabil-
`ity, or activity. Eur. J. Biochem. 197, 681–689.
`
`FIG. 8. Time course of native CPY refolding in the presence (m)
`and absence (m) of 10 molar eq of CPYPR-His6 after complete denatur-
`ation.
`
`of the protein by providing a chaperone-like activity.
`Hence, the method described in this study might be
`used for the production of a variety of proteins that are
`initially synthesized as zymogens, in cases in which E.
`coli is preferred as an expression host.
`
`ACKNOWLEDGMENTS
`
`This work was supported by grant from Korea Science and Engi-
`neering Foundation through the Center for Advanced Bioseparation
`Technology (BSEP).
`
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