Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein

Takumi Koshiba, Tomohiro Hayashi, Ishido Miwako1,3, Izumi Kumagai2, Teikichi Ikura1, Keiichi Kawano4, Katsutoshi Nitta and Kunihiro Kuwajima1,5

Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo 060–0810, 1 Department of Physics, School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113–0033 and 2 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Aoba-ku, Sendai 980–8579, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A high-expression plasmid of the canine milk lysozyme, which belongs to the family of calcium-binding lysozymes, was constructed in order to study its physico-chemical properties. Because the cDNA sequence of the protein has not yet been determined, a 400 base-pair gene encoding canine milk lysozyme was first designed on the basis of the known amino acid sequence. The gene was constructed by an enzymatic assembly of 21 chemically synthesized oligonucleotides and inserted into an Escherichia coli expression vector by stepwise ligation. The expression plasmid thus constructed was transformed into BL21(DE3)/pLysS cells. The gene product accumulated as inclusion bodies in an insoluble fraction. Recombinant canine milk lysozyme was obtained by purification and refolding of the product and showed the same characteristics in terms of bacteriolytic activity and far- and near-UV circular dichroism spectra as the authentic protein. The NMR spectra of refolded lysozyme were also characteristic of a native globular protein. It was concluded that recombinant canine milk lysozyme was folded into the correct native structure. Moreover, the thermal unfolding profiles of the refolded recombinant lysozyme showed a stable equilibrium intermediate, indicating that the molten globule state of this protein was extraordinarily stable. This expression system of canine milk lysozyme will enable biophysical and structural studies of this protein to be extended.

Keywords: calcium-binding lysozyme/molten globule state/refolding/synthetic gene


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lysozyme and {alpha}-lactalbumin are homologous with each other and have undoubtedly diverged from a common ancestral protein (Brew et al., 1970Go). Lysozyme is widely distributed in animal and plant tissues and occurs in especially large amounts in avian egg white. It catalyzes the hydrolysis of glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues of peptidoglycans in bacterial cell walls. In contrast, {alpha}-lactalbumin is found in the milk of lactating mammals and its gene is expressed only in the mammary gland. The role of {alpha}-lactalbumin is to modify the enzymatic specificity of galactosyl transferase; it forms a complex with the enzyme and converts the complex into lactose synthase (Ebner et al., 1966Go; Richardson and Brew, 1980Go; McKenzie and White, 1991Go). Thus, the functions of these two proteins are completely different in spite of similarities in their sequences (Brew et al., 1970Go) and three-dimensional structures (Smith et al., 1987Go; Acharya et al., 1989Go). The calcium-binding properties of the two proteins are also known to be different. All the findings to date show that {alpha}-lactalbumin strongly binds with a calcium ion (Hiraoka et al., 1980Go; Sugai and Ikeguchi, 1994Go), whereas chicken type (c-type) lysozyme does not specifically bind to calcium ions in most cases. Our previous studies have also revealed, however, that there is a group of calcium-binding lysozymes, although only several such examples have been identified at present (Nitta et al., 1987Go; Nitta and Sugai, 1989Go). These calcium-binding lysozymes are especially important in understanding the evolutional relationship between lysozyme and {alpha}-lactalbumin, because they seem to provide an evolutionary link between the two proteins (Nitta and Sugai, 1989Go).

Canine milk lysozyme belongs to the family of calcium-binding lysozymes (Grobler et al., 1994Go; Kikuchi et al., 1998Go) 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., 1998Go). 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 {alpha}-lactalbumin, which has been shown to exhibit a well characterized molten globule state in its equilibrium unfolding (Kuwajima et al., 1976Go; Kuwajima, 1989Go, 1996Go; Uchiyama et al., 1995Go) 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., 1993Go; Van Dael et al., 1993Go; Griko et al., 1995Go). In {alpha}-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, 1977Go, 1992Go, 1995Go, 1996Go; Kuwajima et al., 1985Go; Ikeguchi et al., 1986Go; Mizuguchi et al., 1998Go). 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 {alpha}-lactalbumin (Mizuguchi et al., 1998Go).

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-{alpha}-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., 1994Go; 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., 1998Go) 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., 1993Go; Griko et al., 1995Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

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 1Go) 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., 1998Go; Mizuguchi et al., 1998Go). All other reagents were of biochemical research grade.




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Fig. 1. (A) The DNA sequence of the synthetic canine milk lysozyme gene. Each box indicates each of 21 oligonucleotides used to assemble the lysozyme gene. The thick lines indicate the ends of three gene segments (PS1, PS2 and PS3) (see text). The amino acid sequence is shown above the DNA sequence. (B) Assembly of the PS gene segments and construction of the expression plasmid (pHK7CML) of recombinant canine milk lysozyme. A total of 21 oligonucleotides were used to assemble the three gene segments (PS1–3) that varied from 115 to 152 bp in length. Each DNA fragment corresponding to each gene segment was isolated and used to create the functional PS1/2/3 gene that encodes canine milk lysozyme via stepwise ligation into the cloning vector, pMT7.

 
Construction of a synthetic canine milk lysozyme gene

The canine milk lysozyme gene was assembled from three gene segments, PS1, PS2 and PS3, and each segment was constructed from seven oligonucleotides (Figure 1Go).

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 Tris–HCl (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 1BGo). 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 1BGo).

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 EcoRV–XbaI 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 Tris–HCl (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., 1998Go).

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 Tris–HCl 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., 1998Go).

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 ({Delta}Hcal) was evaluated using Origin computer software (MicroCal).

One-dimensional 1H NMR spectra

One-dimensional 1H NMR spectra were obtained on a JEOL {alpha}-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, 1978Go).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE)

The expression level of recombinant canine milk lysozyme in E.coli was analyzed by SDS–PAGE 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., 1998Go) and E1%1 cm = 11.4 for thioredoxin (Pigiet and Conley, 1978Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Design of the synthetic canine milk lysozyme gene

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., 1994Go; 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, 1987Go). 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 1Go). 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., 1997Go), to create the plasmid pMT7PS1/2/3 (Figure 1BGo). 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 1BGo). The map of the expression plasmid thus obtained, pHK7CML, is shown in Figure 2Go.



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Fig. 2. Schematic representation of the E.coli expression plasmid, pHK7CML, for canine milk lysozyme. The plasmid contains a T7 transcription expression region to regulate the expression level of the recombinant protein.

 
Expression and purification of recombinant canine milk lysozyme

The expression plasmid pHK7CML was transformed into competent BL21(DE3)/pLysS cells and the protein was expressed as inclusion bodies (Figure 3Go). The expression level of the protein was 40–50 mg/l of culture as estimated by SDS–PAGE. 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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Fig. 3. SDS–PAGE pattern of recombinant canine milk lysozyme expressed in E.coli BL21(DE3)/pLysS cells. Lanes: (a) native human lysozyme; (b) whole cells grown in LB media, after 3 h of IPTG induction; (c) supernatant of the ultrasonicated cells; (d) precipitate of the ultrasonicated cells that includes inclusion bodies. The concentration of polyacrylamide gel was 18.8%.

 
Refolding of recombinant canine milk lysozyme

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., 1998Go). 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 4Go 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., 1998Go).



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Fig. 4. Kinetics of the refolding of denatured recombinant canine milk lysozyme in the thioredoxin refolding system containing 1 mM CaCl2. A 20 ml volume of 50 µM thioredoxin was preincubated with 10 µl of 10 mM DTT for 30 min and added to 20 ml of 50 µM thioredoxin that was bubbled with oxygen immediately prior to the addition of 4.1 ml of 25 µM denatured recombinant canine milk lysozyme. The refolding reaction was carried out in 20 mM Tris–HCl buffer (pH 8.0) containing 1 mM CaCl2 and 0.2 M NaCl at 25°C.

 
Characterization of refolded recombinant canine milk lysozyme

We measured the bacteriolytic lysozyme activity, far- (210–250 nm) and near-UV (250–310 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 5Go 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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Fig. 5. Bacteriolytic activities of various lysozymes and refolded recombinant canine milk lysozyme. The relative activities are expressed by taking the activity of hen egg white lysozyme as 100%. Aut. CML, rec. CML, HEL, EML and HL represent authentic canine milk lysozyme, recombinant canine milk lysozyme, hen egg white lysozyme, equine milk lysozyme and human lysozyme, respectively.

 
Figure 6Go shows the CD spectra of recombinant canine milk apo- and holo-lysozymes measured in 50 mM sodium acetate buffer (pH 4.5) containing 1 mM EDTA and 1 mM CaCl2, respectively. The spectra of both recombinant canine milk apo- and holo-lysozymes in the regions from 210 to 250 nm and from 250 to 310 nm are very similar to the spectra of the authentic canine milk lysozyme studied previously (Kikuchi et al., 1998Go), suggesting that recombinant canine milk lysozyme has folded into the same secondary and tertiary structures as the authentic lysozyme. The native structures of recombinant lysozyme in the apo- and holo-states were also characterized by 1H NMR spectra (Figure 8A and BGo). Both the apo- and holo-state spectra show characteristic chemical-shift dispersions, including upfield shifts of aliphatic protons between 0 and –1 p.p.m., for a globular protein having the folded native structure.



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Fig. 6. CD spectra in the near- and far-UV regions of refolded recombinant canine milk apo-lysozyme in the presence of 1 mM EDTA and holo-lysozyme in the presence of 1 mM CaCl2, at 25°C and pH 4.5. The solid line and dashed line represent recombinant canine milk lysozyme in the apo and holo states, respectively.

 


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Fig. 8. 1H NMR spectra of recombinant canine milk lysozyme at 25°C in D2O: (A) holo-lysozyme with 1 mM CaCl2 at pH 4.5; (B) apo-lysozyme at pH 4.5; (C) apo-lysozyme at pH 2.0; and (D) apo-lysozyme in 6 M guanidine hydrochloride at pH 4.5.

 
DSC measurements of the thermal unfolding of recombinant canine milk lysozyme were carried out in 50 mM sodium acetate buffer (pH 4.5) containing 10 mM CaCl2. Under these conditions, {alpha}-lactalbumin and equine milk lysozyme are known to show a single heat absorption peak caused by a cooperative transition from the native to the thermally unfolded state (Yutani et al., 1992Go; Van Dael et al., 1993Go; Griko et al., 1994Go, 1995Go). Figure 7AGo shows a DSC curve of the thermal transition of recombinant canine milk lysozyme. The results indicate that the unfolding transition of recombinant canine milk lysozyme occurs around 68°C in a highly cooperative manner, which is characteristic of a folded globular protein. However, unlike {alpha}-lactalbumin and equine milk lysozyme, canine milk lysozyme shows the second heat absorption peak centered at 90°C, indicating that there is a stable unfolding intermediate in the thermal transition (see below).



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Fig. 7. Thermal unfolding transitions of refolded recombinant canine milk apo- and holo-lysozyme at pH 4.5. The unfolding transition is expressed by excess heat capacity as a function of temperature. (A) Holo-lysozyme in the presence of 10 mM CaCl2 and (B) apo-lysozyme in the absence of CaCl2.

 
The molten globule state of canine milk lysozyme

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 8CGo 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 BGo) and the spectrum in the fully unfolded state at 6 M guanidine hydrochloride (Figure 8DGo). As previously observed in {alpha}-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., 1986Go; Nitta et al., 1993Go; Van Dael et al., 1993Go). 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 7BGo). 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., 1993Go; Griko et al., 1995Go). This behavior has also been observed in the DSC curve of canine milk lysozyme (Figure 7BGo), 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., 1995Go); the two peaks of canine milk lysozyme are located at 45 and 90°C (Figure 7BGo). 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 7AGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The purpose of the present study was to build up an expression system of canine milk lysozyme in E.coli and to characterize the expressed protein. Because the cDNA sequence of the protein has not yet been determined, we designed and constructed a synthetic gene based on the reported amino acid sequence (Grobler et al., 1994Go; K.Nitta, unpublished data). The protein, which was overexpressed from the synthetic gene in E.coli, formed insoluble inclusion bodies, but it was efficiently refolded through the use of the E.coli thioredoxin refolding system developed by Koshiba et al. (1998). The refolded recombinant protein showed the same enzymatic activity in the lysis of Micrococcus lysodeikticus cells as the authentic lysozyme (Figure 5Go), suggesting that the protein correctly folded into the native and active conformation.

The results of the CD, 1H NMR and DSC measurements (Figures 6–8GoGoGo) 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 7Go). The cooperative thermal transition is characteristic of a globular protein having a specific tertiary structure in the native state (Khechinashvili et al., 1973Go; Privalov and Khechinashvili, 1974Go; Pfeil and Privalov, 1976Go; Yutani et al., 1992Go).

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 {alpha}-lactalbumin and equine milk lysozyme (Figure 8Go). 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., 1995Go). 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., 1990Go). 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., 1987Go). Recently, Ishikawa et al. (1998) and Chaudhuri et al. (1998) have also shown that the extraneous N-terminal methionine residue in recombinant {alpha}-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.


    Acknowledgments
 
We gratefully acknowledge Professor Yue Ke of the Normal University of Inner Mongolia, China, and Mr T. Tatsuki of Morinaga Milk Industry Co., Ltd, Japan, for the generous gift of equine and canine milk, respectively. Mr Yoshihiro Kobashigawa and Mr Tetsuya Suetake of Hokkaido University are acknowledged for measurements of the 1H NMR spectra. This study was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.


    Notes
 
3 Present address: Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606–8501 Back

4 Present address: Faculty of Pharmaceutical Science, Toyama Medical and Pharmaceutical University, Toyama, Toyama 930–0194, Japan Back

5 To whom correspondence should be addressed. E-mail: kuwajima{at}phys.s.u-tokyo.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received November 2, 1998; revised January 5, 1999; accepted January 8, 1999.