Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo 0600810, 1 Department of Physics, School of Science, University of Tokyo, Bunkyo-ku, Tokyo 1130033 and 2 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Aoba-ku, Sendai 9808579, Japan
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Abstract |
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Keywords: calcium-binding lysozyme/molten globule state/refolding/synthetic gene
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Introduction |
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Canine milk lysozyme belongs to the family of calcium-binding lysozymes (Grobler et al., 1994; Kikuchi et al., 1998
) and its physico-chemical properties are known to be very different from those of non-calcium-binding c-type lysozyme (conventional lysozyme). A notable difference has been observed in their unfolding profiles (Kikuchi et al., 1998
). A stable equilibrium intermediate has been shown to accumulate during the guanidine hydrochloride-induced unfolding of canine milk lysozyme, although conventional lysozymes usually unfold in a cooperative two-state manner without the accumulation of an intermediate. Thus, the unfolding behavior of this protein is more similar to that of
-lactalbumin, which has been shown to exhibit a well characterized molten globule state in its equilibrium unfolding (Kuwajima et al., 1976
; Kuwajima, 1989
, 1996
; Uchiyama et al., 1995
) and the unfolding intermediate of canine milk lysozyme may also be in the molten globule state. Among the known examples of calcium-binding lysozymes, only canine and equine milk lysozymes are known to show a stable equilibrium molten globule state (Nitta et al., 1993
; Van Dael et al., 1993
; Griko et al., 1995
). In
-lactalbumin and equine milk lysozyme, the molten globule state has been shown to be identical with an early transient intermediate in kinetic folding from the fully unfolded state (Kuwajima, 1977
, 1992
, 1995
, 1996
; Kuwajima et al., 1985
; Ikeguchi et al., 1986
; Mizuguchi et al., 1998
). Interestingly, the molten globule state of equine milk lysozyme is stabilized, at least in part, by native-like specific interactions and is more stable than that of
-lactalbumin (Mizuguchi et al., 1998
).
A high-expression plasmid of the canine milk lysozyme gene has been constructed in order to study the physico-chemical properties of canine milk lysozyme in relation to lysozyme--lactalbumin evolution and its folding mechanisms and the results are reported in this paper. Because the cDNA sequence of canine milk lysozyme has not yet been determined, chemically synthesized deoxyoligonucleotides, the sequences of which are based on the known amino acid sequences of the protein (Grobler et al., 1994
; K.Nitta, unpublished data), were used for constructing the plasmid. We show that the protein over-expressed as inclusion bodies in Escherichia coli is efficiently refolded to the native state through the use of an E.coli thioredoxin refolding system (Koshiba et al., 1998
) and that the refolded protein exhibits full enzymatic activity as an authentic lysozyme. Furthermore, the thermal unfolding transitions of the protein have shown that the molten globule state of canine milk lysozyme is extraordinarily stable, even more stable than that of equine milk lysozyme (Van Dael et al., 1993
; Griko et al., 1995
). Therefore, canine milk lysozyme is a useful model protein for elucidating the molecular mechanisms by which the molten globule state is stabilized and for determining the role of this state in protein folding.
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Materials and methods |
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All enzymes used for the genetic experiments were purchased from Takara Shuzo (Kyoto, Japan). The 21 deoxyoligonucleotides that were used for construction of the canine milk lysozyme gene (Figure 1) were synthesized at Rikaken (Tokyo, Japan). SP-Sepharose FF and Sephacryl S-200 were obtained from Pharmacia Biotech (Uppsala, Sweden). Micrococcus lysodeikticus for measuring the enzymatic activity of lysozymes was obtained from Sigma Chemical (St Louis, MO). Hen egg white lysozyme and human lysozyme were also supplied by Sigma Chemical. Authentic equine milk lysozyme and canine milk lysozyme have been prepared previously (Kikuchi et al., 1998
; Mizuguchi et al., 1998
). All other reagents were of biochemical research grade.
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The canine milk lysozyme gene was assembled from three gene segments, PS1, PS2 and PS3, and each segment was constructed from seven oligonucleotides (Figure 1).
To prepare the PS1 segment, each of the seven oligonucleotides from PS1-1 to PS1-7 was phosphorylated in 20 µl of a reaction mixture that contained 50 pmol of the purified oligonucleotide, 50 mM TrisHCl (pH 8.0), 10 mM MgCl2, 10 mM ß-mercaptoethanol, 5 µM ATP and 40 units of T4 polynucleotide kinase. After being inclubated for 30 min at 37°C, the kinase was inactivated by heating at 65°C for 15 min. The phosphorylated oligonucleotides were then mixed and annealed in 10 µl of a reaction mixture that contained 1 µl of the above phosphorylation reaction mixture of each oligonucleotide and 3 µl of water. The oligonucleotide mixture was heated at 100°C for 3 min and then cooled slowly to room temperature over a period of 1 h to allow the complementary strands to anneal. The annealed segment, PS1, was ligated into a cloning vector (pMT7); pMT7 was double-digested with NcoI and BamHI, then mixed with the annealed segment with a molar ratio of 1:10 (vector:insert) and ligated. The ligation was carried out using a Takara Version 2 ligation kit and this generated a plasmid pMT7PS1 (Figure 1B). E.coli strain MV1184 was transformed by the ligation mixture and the pMT7PS1 DNA was purified from the transformed cells. The inserted segment was confirmed by DNA sequencing with an ALF express autosequencer (Pharmacia).
The other two segments, PS2 and PS3, were prepared in the same manner as above. The PS2 segment was inserted into pMT7PS1 to generate pMT7PS1/2 and the PS3 segment was inserted into pMT7PS1/2 to generate pMT7PS1/2/3, which contained the entire canine milk lysozyme gene (Figure 1B).
Construction of a versatile expression plasmid (pHK7CML)
To construct a versatile expression plasmid of canine milk lysozyme, the entire gene segment excised from pMT7PS1/2/3 was cloned into a plasmid pSCREEN 1-b(+) (Novagen, Madison, WI). The pMT7PS1/2/3 was digested with PvuII and XbaI and the canine milk lysozyme gene segment obtained was ligated into the EcoRVXbaI sites of pSCREEN 1-b(+). The resulting plasmid was called pHK7CML. The pHK7CML contains T7 transcription and translation signals, both of which result in the high expression of canine milk lysozyme and the f1 origin of replication, which allowed us to prepare the single-stranded plasmid DNA.
Expression and partial purification of recombinant canine milk lysozyme
Recombinant canine milk lysozyme was overexpressed in E.coli (BL21(DE3)/pLysS) that had been transformed by pHK7CML. The protein was expressed as inclusion bodies and was solubilized in 6 M guanidine hydrochloride containing 50 mM TrisHCl (pH 8.0), 1 mM EDTA and 650 mM ß-mercaptoethanol. The solubilized protein was partially purified by gel filtration on a column of Sephacryl S-200 as described previously (Koshiba et al., 1998).
Refolding and isolation of recombinant canine milk lysozyme
The refolding of recombinant canine milk lysozyme that had been partially purified by gel filtration (see above) was carried out by the method of Koshiba et al. (1998) with slight modifications. Thioredoxin (50 µM; 20 ml) was first preincubated with 10 µl of DTT (10 mM) for 30 min and the resultant reduced thioredoxin solution was mixed with 20 ml of oxygenated thioredoxin (50 µM) to make a refolding buffer solution. After mixing the refolding buffer solution, 4.1 ml of denatured recombinant canine milk lysozyme (25 µM) and 90 µl of CaCl2 (0.5 M) were immediately added to this refolding buffer solution. The reaction mixture was incubated for 4 days at 25°C, then dialyzed against 20 mM TrisHCl buffer (pH 8.0) containing 1 mM CaCl2 and applied to an SP-Sepharose FF column (8x50 mm, Pharmacia). Refolded recombinant canine milk lysozyme was eluted from the column by a linear NaCl gradient from 0 to 0.3 M. The eluate containing lysozyme was collected, dialyzed against 0.01 M HCl and lyophilized.
Measurements of lysozyme activity
The bacteriolytic activity of lysozymes was assayed by the method of Kumagai et al. (1992).
Preparation of apo- and holo-proteins
Apo- and holo-proteins were prepared as described previously (Koshiba et al., 1998).
Measurements of circular dichroism (CD) spectra
The CD spectrum of recombinant canine milk lysozyme (27.5 µM) was measured on a Jasco (Tokyo, Japan) J-500 spectropolarimeter. Far- and near-UV CD spectra were measured using a cell with 0.1 and 1 cm optical path lengths, respectively, at pH 4.5 and 25°C.
Differential scanning calorimetry (DSC)
DSC measurements of recombinant canine milk lysozyme were carried out with an MC-2 microcalorimeter (MicroCal, Northampton, MA) at a scanning rate of 1.0 K/min. The sample solution for DSC measurement was prepared by dissolving lyophilized lysozyme in 50 mM sodium acetate buffer (pH 4.5). The lysozyme concentration used was 1.5 mg/ml. The pH of the sample solution was checked before and after each measurement. The calorimetric enthalpy (Hcal) was evaluated using Origin computer software (MicroCal).
One-dimensional 1H NMR spectra
One-dimensional 1H NMR spectra were obtained on a JEOL -400 spectrometer operating at 400 MHz. The sample solution for 1H NMR measurement was prepared by dissolving lyophilized lysozyme in D2O and the pH was adjusted to 4.5 with small quantities of NaOD or DCl. In all experiments, the sweep width was 15.0 p.p.m. and the digital resolution 0.36 Hz/point. The water signal was suppressed by the DANTE pulse sequence (Morris and Freeman, 1978
).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE)
The expression level of recombinant canine milk lysozyme in E.coli was analyzed by SDSPAGE according to the method of Laemmli (1970).
Estimation of protein concentration
The protein concentrations were estimated spectrophotometrically using the following extinction coefficients at 280 nm: E1%1 cm = 23.2 for authentic and recombinant canine milk lysozyme (Kikuchi et al., 1998) and E1%1 cm = 11.4 for thioredoxin (Pigiet and Conley, 1978
).
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Results |
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The cDNA sequence of canine milk lysozyme has not yet been determined; we therefore designed a synthetic gene that encodes canine milk lysozyme on the basis of its known amino acid sequence (Grobler et al., 1994; K.Nitta, unpublished data). The design of the synthetic gene progressed as follows. First, the amino acid sequence of the lysozyme was reverse-translated into a DNA sequence through the use of the triplets that occur most frequently in E.coli (Sharp and Li, 1987
). Second, the sequence was checked by a computer program (GENETYX) to ensure that there were no excessively stable secondary structures in the encoded mRNA, especially in the upstream region of the gene. Third, for obvious convenience, unique restriction endonuclease recognition sites were introduced into the sequence without changing the encoded amino acid sequence.
Construction of the synthetic gene and the expression plasmid
The three segments (PS1, PS2 and PS3) that made up the designed lysozyme gene were constructed from 21 oligonucleotides ranging in size from 20 to 56 (Figure 1). The three segments were (i) the 152 bp NcoI/BamHI fragment composed of seven oligonucleotides (PS1), (ii) the 133 bp BamHI/SphI fragment composed of seven oligonucleotides (PS2) and (iii) the 115 bp SphI/PvuII fragment composed of seven oligonucleotides (PS3). In order to use the NcoI site of the cloning vector pMT7, the PS1 segment had an NcoI cohesive end and the initiation codon (ATG) was added at the 5'-end of the synthetic lysozyme gene. Thus, the recombinant protein expressed in E.coli had an extra methionine residue at the N-terminus.
The assembly of the synthetic gene was carried out by a stepwise ligation of the three gene segments into pMT7 that had been used for the overexpression of staphylococcal nuclease in E.coli (Ikura et al., 1997), to create the plasmid pMT7PS1/2/3 (Figure 1B
). The insert of the assembled gene into pMT7 was confirmed by DNA sequencing.
To construct an expression plasmid of canine milk lysozyme, pMT7PS1/2/3 was digested with PvuII and XbaI and the lysozyme gene fragment was ligated directly into an expression vector, pSCREEN-1b(+) (Novagen), which had been digested with EcoRV and XbaI (Figure 1B). The map of the expression plasmid thus obtained, pHK7CML, is shown in Figure 2
.
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The expression plasmid pHK7CML was transformed into competent BL21(DE3)/pLysS cells and the protein was expressed as inclusion bodies (Figure 3). The expression level of the protein was 4050 mg/l of culture as estimated by SDSPAGE. The protein expressed as inclusion bodies was solubilized in 6 M guanidine hydrochloride solution and purified by gel filtration using Sephacryl S-200. Denatured recombinant canine milk lysozyme of >90% purity was obtained and this sample was used for refolding (see below).
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Our previous study has shown that the use of a refolding system that utilizes E.coli thioredoxin is very effective in completing the refolding of denatured and disulfide-reduced human lysozyme (Koshiba et al., 1998). This refolding system was therefore also used in the present study in the refolding of the recombinant canine milk lysozyme, with the modification that 1 mM CaCl2 was added to the refolding reaction mixture. Because canine milk lysozyme is a calcium-binding protein, calcium ions are expected to promote the refolding. In fact, when CaCl2 was present, a significant decrease in the turbidity of the refolding mixture of canine milk lysozyme was observed compared with the refolding mixture prepared in the absence of calcium ions.
Figure 4 shows the refolding process of canine milk lysozyme as monitored by the lysozyme activity as a function of the refolding time. Recombinant canine milk lysozyme fully refolded at 100 h after the refolding started. The refolding of recombinant canine milk lysozyme was found to be faster than that of the human lysozyme studied previously (Koshiba et al., 1998
).
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We measured the bacteriolytic lysozyme activity, far- (210250 nm) and near-UV (250310 nm) CD spectra and 400 MHz 1H NMR spectra of refolded recombinant canine milk lysozyme. The thermal transition of the protein measured by DSC was also investigated.
Figure 5 shows the bacteriolytic activities of various lysozymes including refolded canine milk lysozyme assayed in 50 mM sodium phosphate buffer (pH 6.2). The bacteriolytic activity of recombinant canine milk lysozyme, which was found to be the same as the activity of authentic canine milk lysozyme, was half that of hen egg white lysozyme.
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The molten globule state of canine milk lysozyme is stably populated at pH 2.0 in the absence of denaturants and at an intermediate concentration (~2 M) of a denaturant, guanidine hydrochloride, at neutral pH. Figure 8C shows a 1H NMR spectrum of recombinant canine milk lysozyme in the molten globule state at pH 2.0 and it is compared with the native spectra (Figure 8A and B
) and the spectrum in the fully unfolded state at 6 M guanidine hydrochloride (Figure 8D
). As previously observed in
-lactalbumin and equine milk lysozyme, the spectrum in the molten globule state is more similar to the spectrum in the unfolded state and the characteristic chemical shift dispersion has mostly disappeared. The spectral width is broader than those in the native and the fully unfolded states and this is also a characteristic of the molten globule state (Ikeguchi et al., 1986
; Nitta et al., 1993
; Van Dael et al., 1993
). However, it should also be noted that small resonance peaks are still present in the upfield region from 0 to 1 p.p.m., which arise from ring-current shifts of aliphatic protons and are due to specific packing interactions of the aliphatic side chains around an aromatic ring. Whether these ring current shifts are due to the specific interactions in the molten globule state or the incomplete unfolding of the native state has not yet been determined.
In order to investigate the stability of the molten globule state, the thermal transition of recombinant canine milk lysozyme in the apo state at pH 4.5 was investigated by DSC measurements (Figure 7B). Under these conditions, equine milk lysozyme is known to show two heat absorption peaks, one caused by unfolding from the native to the molten globule state and the other by unfolding from the molten globule to the thermally unfolded state (Van Dael et al., 1993
; Griko et al., 1995
). This behavior has also been observed in the DSC curve of canine milk lysozyme (Figure 7B
), but the second transition of the protein occurs at a much higher temperature than the corresponding transition of equine lysozyme (70°C) (Griko et al., 1995
); the two peaks of canine milk lysozyme are located at 45 and 90°C (Figure 7B
). The results indicate that the molten globule state of canine milk lysozyme is much more stable. Comparison of the DSC curves in the holo and apo states of canine milk lysozyme also indicates that the extraordinary stability of the molten globule state of this protein has led to the appearance of the second heat absorption peak in the holo-protein observed above (Figure 7A
).
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Discussion |
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The results of the CD, 1H NMR and DSC measurements (Figures 68) showed that the recombinant canine milk lysozyme refolded from the inclusion bodies has the correct native structure. It also has secondary and tertiary structures, as indicated by the CD spectra, very similar to the spectra of the authentic protein in both the far- and near-UV regions. The typical excess heat capacity curve of the thermal unfolding of recombinant canine milk lysozyme suggests that the unfolding transition of this recombinant lysozyme occurs highly cooperatively, although it shows the two different thermal transitions (Figure 7
). The cooperative thermal transition is characteristic of a globular protein having a specific tertiary structure in the native state (Khechinashvili et al., 1973
; Privalov and Khechinashvili, 1974
; Pfeil and Privalov, 1976
; Yutani et al., 1992
).
The 1H NMR spectra at pH 2.0 show that the spectral features of the molten globule state are very similar to those found in -lactalbumin and equine milk lysozyme (Figure 8
). The thermal unfolding curves measured by DSC, however, show that the unfolding of the molten globule state occurs at ~90°C, so that it is remarkably more stable than the molten globule state of equine milk lysozyme, which shows unfolding at ~70°C (Griko et al., 1995
). The cooperative heat absorption from the molten globule also suggests that the molten globule state of this protein is stabilized, at least in part, by native-like specific interactions as suggested in equine milk lysozyme. Therefore, recombinant canine milk lysozyme is a useful model protein for elucidating the molecular mechanisms by which the molten globule state is stabilized and determining its role in protein folding.
Our preliminary study has also shown, however, that the thermal stability of the recombinant protein is significantly lower than that of authentic canine milk lysozyme; the melting temperatures in 3.0 M guanidine hydrochloride in the presence of 10 mM CaCl2 (pH 4.5) are 30.2 and 32.4°C for the recombinant and authentic proteins, respectively (Koshiba,T., Matsuki,N. and Nitta,K., unpublished data). Automated N-terminal amino acid (five residues) sequencing showed that the recombinant canine milk lysozyme expressed in E.coli possesses an extra methionine residue at the N-terminus (data not shown). This was made clear because the methionine aminopeptidase does not remove the N-terminal methionine residue when the mature sequence begins with lysine, which does occur with the sequence of the canine milk lysozyme (Moerschell et al., 1990). It is very likely that the decrease in the thermal stability of the recombinant protein compared with the authentic protein arises from the presence of the extra methionine residue at the protein N-terminus. Mine et al. (1997) reported that the presence of the extra methionine residue at the N-terminus in recombinant hen egg-white lysozyme brings about a decreased refolding yield and a decreased solubility in comparison with the authentic protein (Imoto et al., 1987
). Recently, Ishikawa et al. (1998) and Chaudhuri et al. (1998) have also shown that the extraneous N-terminal methionine residue in recombinant
-lactalbumin destabilizes the protein through an entropic effect and that the entropic destabilization arises from an increase in the conformational entropy of the recombinant protein in the unfolded state.
In conclusion, the expression system of the synthetic gene that encodes canine milk lysozyme should enable us to extend the biophysical and structural studies of this protein and its molten globule state. The high level of recombinant canine milk lysozyme expression from the expression plasmid (pHK7CML) will facilitate the purification of large amounts of mutant proteins required for such studies. As mentioned above, however, the recombinant protein possesses an extra N-terminal methionine residue that affects the protein stability. Therefore, it may still be necessary to modify this expression system if complete removal of the extra methionine residue is required.
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Acknowledgments |
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Notes |
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4 Present address: Faculty of Pharmaceutical Science, Toyama Medical and Pharmaceutical University, Toyama, Toyama 9300194, Japan
5 To whom correspondence should be addressed. E-mail: kuwajima{at}phys.s.u-tokyo.ac.jp
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Received November 2, 1998; revised January 5, 1999; accepted January 8, 1999.