Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris

Shuichiro Goda1, Kazufumi Takano1, Yuriko Yamagata2, Yoshio Katakura3 and Katsuhide Yutani1,4

1 Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871, 2 Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita, Osaka 565-0871 and 3 Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
A human lysozyme expression system by Pichia pastoris was constructed with the expression vector of pPIC9, which contains the {alpha}-factor signal peptide known for high secretion efficiency. P.pastoris expressed the human lysozyme at about 300 mg/l broth, but four extra residues (Glu–4–Ala–3–Glu–2–Ala–1–) were added at the N-terminus of the expressed protein (EAEA–lysozyme). To determine the effect of the four extra residues on the stability, structures and folding of the protein, calorimetry, X-ray crystal analysis and GuHCl denaturation experiments were performed. The calorimetric studies showed that the EAEA–lysozyme was destabilized by 9.6 kJ/mol at pH 2.7 compared with the wild-type protein, mainly caused by the substantial decrease in the enthalpy change ({Delta}H). On the basis of structural information on the EAEA–lysozyme, thermodynamic analyses show that (1) the addition of the four residues slightly affected the conformation in other parts far from the N-terminus, (2) the large decrease in the enthalpy change due to the conformational changes would be almost compensated by the decrease in the entropy change and (3) the decrease in the Gibbs energy change between the EAEA and wild-type human lysozymes could be explained by the summation of each Gibbs energy change contributing to the stabilizing factors concerning the extra residues.

Keywords: crystal structure/extra N-terminal residues/human lysozyme/Pichia pastoris/protein stability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human lysozyme (EC 3.2.1.17) is widely distributed in several human tissues and secretions, including milk, tears and saliva (Peters et al., 1989Go). It is composed of 130 amino acid residues and the molecular weight is 14700. Its physico-chemical properties have been extensively studied (Artymiuk and Blake, 1981; Redfield and Dobson, 1990; Takano et al., 1999a; and references cited in these articles). Furthermore, two types of human lysozyme variants (I56T and D67H) are known to be amyloidogenic (Pepys et al., 1993Go). Therefore, human lysozyme is expected to be a model protein in order to solve the mechanism of the amyloid disease caused by protein misfolding (Carell and Lomas, 1997). The expression systems have been reported using Saccharomyces cerevisiae, but the expression yield is about 2.4 mg/l broth (Muraki et al., 1987Go; Taniyama et al., 1988Go). Therefore, we planned to construct the expression systems of the human lysozyme by Pichia pastorisGS115(His) to obtain more human lysozyme variants in order to determine the characteristics of amyloid formation.

The methylotropic yeast P.pastoris has recently attracted attention for overexpressing proteins and has been used to express many kinds of proteins. Some proteins are expressed by several grams per liter of broth (Tschopp et al., 1987Go; Paifer et al., 1994Go). However, the expression yields depend on the signal peptide and protein sequence in the yeast expression system (Hashimoto et al., 1998Go). In some cases, a few residues are left at the N-terminal residue of the expressed protein derived from the signal peptide sequence problem (Steinlein et al., 1995Go).

When a protein is expressed in Escherichia coli, a methionine residue is sometimes left at the N-terminal residue of an expressed protein when the penultimate residue is relatively bulky and/or charged (Flinta et al., 1986Go; Moerschell et al., 1990Go). For lysozymes and {alpha}-lactoalbumins, this is also the case, because their N-terminal residues are lysine. It has been reported that the extra methionine residues at the N-terminus considerably affect the conformational stability (Ishikawa et al., 1998Go; Chaudhuri et al., 1999Go; Takano et al., 1999bGo). On the other hand, polyhistidine tags in the N/C-terminal regions of proteins are widely used to isolate proteins easily (Kuliopulos and Walsh, 1994Go). However, the effect of several elongation residues to the N/C-terminus on structure, stability and folding have not yet been well examined. Recently, Matsuura et al. (1999) have shown that elongation to the N/C-terminus would lead to the construction of proteins with higher stability and activity, because it provides a new protein sequence variety. More information on the stability–structure relationships of proteins with extra residues at the N/C-termini is then required.

The human lysozyme could be expressed by 300 mg/l of broth using the system of P.pastoris GS115(His). However, four extra residues (glutamic acid–4–alanine–3–glutamic acid–2– alanine–1–; EAEA–) derived from the signal peptide remained at the N-terminal residue of the expressed human lysozyme. It is called the EAEA–lysozyme. The EAEA–lysozyme also shows better amyloidgenic behavior than the wild-type protein (unpublished data). It is important to know the physico-chemical properties of proteins which have good amyloidgenic behavior. In this work, the effects of the four extra residues of the EAEA–lysozyme on stability, structure and folding were examined using circular dichroism (CD), differential scanning calorimetry (DSC) and X-ray crystal diffraction methods. We discuss the mechanism of the changes in the conformational stability on the basis of the EAEA–lysozyme structure.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Construction of expression vector and introduction into P.pastoris

The human lysozyme gene was amplified from pGEL125 (Taniyama et al., 1988Go) using 5'-TTTGGCTCGAGAAAAG-AGAGGCTGAAGCTAAGGTTTTCGAGAGATGCGAATT-AGCC and 5'-AAGGATCCCGAATTCAGCTATTAAACACCAC as primers (underlining indicates XhoI and EcoRI sites, respectively). The PCR product was digested with XhoI and EcoRI and the resulting fragment was cloned into pPIC9 (Invitrogen, CA, USA) digested with XhoI and EcoRI. In the resulting plasmid, designated human lysozyme/pPIC9 (Figure 1Go), the human lysozyme gene was fused with the pre–pro sequence that originated from the {alpha}-factor of S.cerevisiae. The expression of the fused gene was controlled by the methanol-inducible AOX1 promoter that originated from the alcohol oxidase gene of P.pastoris. The plasmid was digested with BglII and introduced into P.pastoris GS115 (his4) by electropolation according to the manufacturer's instructions (Invitrogen). A transformant slowly consuming methanol (Muts) was selected and used throughout this study.



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Fig. 1. Restriction map of human lysozyme/pPIC9. Human lysozyme genes were inserted between the XhoI site and the EcoRI site of pPIC9.

 
Protein expression and purification

The protein expression was carried out using a methanol control system as described by Katakura et al. (1998), except that 1% casamino acid was added to the basal salt medium and the methanol concentration in the medium was controlled at 0.3% during the production phase.

For purification, the supernatant of the culture was diluted 5-fold with water and loaded on an SP-Sepharose Fast Flow column (26x50 mm) (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 20 mM sodium phosphate buffer (pH 6.5). The bound protein was eluted with 0.5 M sodium chloride in 20 mM sodium phosphate buffer (pH 6.5). The eluate was diluted 4-fold with water and loaded on an SP-Sepharose High Performance column (26x100 mm), then eluted with a linear gradient of 0.06 to 0.36 M Na2SO4 in 50 mM sodium phosphate buffer (pH 6.5). The concentration of the protein solution was determined spectrophotometrically using A1%1 cm = 25.65 at 280 nm for the human lysozyme (Parry et al., 1969Go). The molar absorptivity of the EAEA protein is assumed to be the same as that of the wild-type protein because the X-ray structure of the EAEA, except for the N-terminal region newly introduced, and the CD spectrum in the near-UV region were the same as those of the wild-type protein. The N-terminal amino acid sequence was analyzed using the HP G1005A Protein Sequencing System.

The purified human lysozyme was exhaustively dialyzed against distilled water and was lyophilized for storage. The wild-type human lysozyme was obtained as described by Takano et al. (1995).

Enzymatic activities of human lysozyme

Bacteriolytic activity of lysozyme was assayed by the method of Loquet et al. (1968) with slight modifications. A solution (25 µl) containing the lysozyme was added to 1 ml of a suspension of Micrococcus lysodeikticus cells (0.2 mg/ml) in 0.1 M potassium phosphate buffer (pH 6.2) at 25°C. The change in absorbance at 450 nm was measured for 3 min.

DSC measurements

DSC was carried out with a DASM4 adiabatic microcalorimeter equipped with an NEC personal computer. The scan rate was 1.0 K/min. This system is the same as previously reported (Yutani et al., 1991Go). The sample solution for the DSC measurements was prepared by dissolving the human lysozyme in 50 mM glycine–HCl buffer between pH 2.6 and 3.1. The concentration of the human lysozyme was 0.9–1.5 mg/ml. The data were analyzed by Origin software (Micro Cal, MA, USA).

Equilibrium studies of guanidine hydrochloride-induced denaturation

The denaturation of proteins by guanidine hydrochloride (GuHCl) was monitored by measuring changes in the circular dichroism (CD) values at 222 nm. The CD measurements were performed on a Jasco J-720 recording spectropolarimeter using a cell of 10 mm pathlength (Taniyama et al., 1992Go; Funahashi et al., 1996Go). The protein solution (0.05 mg/ml) was incubated for at least 24 h in various concentrations of GuHCl at 25°C and at a pH of 3.0, 4.0 or 7.0 (50 mM glycine–HCl buffer for pH 3.0 and 4.0 and 50 mM sodium phosphate buffer for pH 7.0).

Kinetic studies of denaturation and refolding

The reactions of denaturation and refolding by GuHCl were monitored by measuring changes in the fluorescence intensity above 300 nm with excitation at 280 nm. Fluorescence stopped-flow experiments were carried out with a Photal RA-401 stopped-flow spectrophotometer equipped with a mixing device using 1:10 volumes of two solutions (Otsuka Electronics, Osaka, Japan). The system for fluorescence measurements is the same as previously described (Taniyama et al., 1992Go). Kinetic experiments were performed in 40 mM glycine–HCl buffer, pH 4.0, at 10°C.

Crystallization and X-ray crystallography

Purified human lysozyme was crystallized by the hanging drop vapor-diffusion method. As crystals of the EAEA–lysozyme were not obtained under the usual crystallization conditions with 2 M sodium chloride as the precipitant in 20 mM acetate buffer (pH 4.5), the sparse matrix approach (Jancarik and Kim, 1991Go) was used to search for the best crystallization conditions. A 3 µl drop of aqueous protein (15 mg/ml) was mixed with 3 µl of a reservoir solution containing 0.1 M cadmium chloride, 0.1 M sodium acetate (pH 4.6) and 30% (v/v) polyethylene glycol 400. Crystals grew in a few days at 10°C. The data were collected at 100K using synchrotron radiation on the beam line 18B of the Photon Factory (Tsukuba, Japan) with a Weissenberg camera (Sakabe, 1991Go). The data were processed with the program DENZO (Otwinowski, 1990Go). The structure of the EAEA–lysozyme was solved by the molecular replacement technique using the program AMoRe (Navaza, 1994Go) with the wild-type structure (Takano et al., 1995Go) as a search model. The structure was refined with the program X-PLOR (Brunger, 1992Go) as already described (Takano et al., 1995Go; Yamagata et al., 1998Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression and purification of lysozyme

The protein expression was induced by the addition of methanol to the broth and the methanol concentration was maintained at 0.3% using the semiconductor methanol control system. Figure 2Go shows the time course of the cell concentration of P.pastoris monitored by measuring its absorbance at 600 nm and the lysozyme concentration in the broth was evaluated from the enzymatic activity after induction. The cell concentration and lysozyme concentration in the culture increased exponentially (Figure 2Go). Although the production rate decreased after 70 h owing to the decrease in dissolved oxygen in the culture, the concentration of human lysozyme increased to 300 mg/l broth.



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Fig. 2. Time course of cell growth and expression of human lysozyme after induction. Circles and squares represent cell density and amount of lysozyme, respectively. Cell density is denoted by absorbance (optical density, OD) at 600 nm (left scale) and the amount of lysozyme in broth (right scale) is based on enzymatic activity.

 
In the final step of purification, the solution that contained the human lysozyme was applied to an SP-Sepharose High Performance column and eluted as a single peak. The elution time was obviously faster than that of the wild-type protein. N-Terminal amino acid analysis showed that four extra residues (Glu–4–Ala–3–Glu–2–Ala–1–) were left at the N-terminus of the wild-type human lysozyme.

DSC measurements

In order to determine the effect of the four extra residues of the EAEA–lysozyme on the thermodynamic parameters of the denaturation, DSC measurements were performed at acidic pHs between 2.6 and 3.1, where the denaturation of the human lysozyme is reversible. Figure 3Go shows typical DSC curves of the wild-type human lysozyme and EAEA–lysozyme at pH 2.7. Table IGo shows the thermodynamic parameters of the EAEA–lysozyme obtained from the DSC curves at different pHs. As shown in Figure 4Go, the Td values of the EAEA–lysozyme were lower than those of the wild-type protein in the measured pH region and the calorimetric enthalpies ({Delta}Hcal) of the EAEA–lysozyme were considerably decreased compared with those of the wild-type protein. The {Delta}Cp value of the EAEA–lysozyme obtained from the slope of Td versus {Delta}Hcal was 6.7, similar to that of the wild-type protein. The thermodynamic parameters of denaturation at a constant temperature, 64.9°C (Td at pH 2.70 for the wild-type protein) can be calculated using the following equations as shown in Table IIGo:



where the {Delta}Cp values are assumed to be independent of temperature (Privalov and Khechinashvili, 1974Go). The differences in the thermodynamic parameters between the wild-type and EAEA–lysozymes were {Delta}{Delta}G = –9.6 kJ/mol, {Delta}{Delta}H = –31 kJ/mol and T{Delta}{Delta}S = –21 kJ/mol (Table IIGo), indicating that the EAEA–lysozyme was substantially destabilized owing to the enthalpic effect.



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Fig. 3. Typical excess heat capacity curves for the EAEA and wild-type human lysozymes. EAEA–lysozyme at pH 2.68 (1), wild-type lysozyme at pH 2.7 (2). The previously reported data for the wild-type protein (Takano et al., 1995Go) were used. The increments of excess heat capacity were 10 kJ/mol.K.

 

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Table I. Thermodynamic parameters for denaturation of the EAEA–lysozyme obtained from calorimetry at different pHs
 


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Fig. 4. pH dependence of denaturation temperature (A) and the denaturation temperature dependence of calorimetric enthalpy change (B). Filled circles and open squares represent the wild-type and EAEA–lysozymes, respectively.

 

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Table II. Thermodynamic parameters for denaturation of the EAEA–lysozyme obtained from calorimetry at the denaturation temperature (64.9°C) of the wild-type protein at pH 2.7
 
Equilibrium experiments of denaturation by GuHCl

DSC measurements were carried out in the acidic pH region because of its high reversibility. However, in the neutral pH region, it is difficult to analyze the DSC data because of its low reversibility. To examine the effect of the four extra residues in a wide pH range on conformational stability, equilibrium denaturation experiments were performed by monitoring the CD values at 222 nm as a function of the GuHCl concentration at pH 3.0, 4.0 and 7.0 and at 25°C (Figure 5Go). The transition of the EAEA–lysozyme was highly cooperative, as shown in Figure 5Go, and the denaturation by GuHCl at the three pHs was completely reversible. The transition curve was analyzed in order to evaluate the denaturation thermodynamic parameters using the following equations and assuming a two-state transition:




where K, {Delta}G and fu(C) are the equilibrium constant of the denaturation reaction, the Gibbs energy change upon denaturation and the fraction of denaturation as a function of concentration of GuHCl, respectively, and m is the slope of the linear correlation between {Delta}G and the concentration of GuHCl, [C]. {Delta}GH2O is the Gibbs energy change upon denaturation in the absence of GuHCl (in water). We used a computer program to produce a least-squares fit of the experimental data from the GuHCl denaturation curves to Equation 7 to obtain {Delta}GH2O. {Delta}GH2O of the EAEA–lysozyme was calculated to be 25.9, 34.5 and 35.6 kJ/mol at pH 3.0, 4.0 and 7.0, respectively (Table IIIGo). These were lower than those of the wild-type protein at the three pHs, indicating that the EAEA–lysozyme was considerably destabilized by the four extra residues, which was in agreement with the results of the heat denaturation in the acidic pH region (Table IIGo).



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Fig. 5. GuHCl denaturation curves of the EAEA and wild-type human lysozymes at various pHs and at 25°C. (A) pH 3.0 in 50 mM glycine–HCl buffer; (B) pH 4.0 in 50 mM glycine–HCl buffer; (C) pH 7.0 in 50 mM sodium phosphate buffer. Filled circles and open squares represent the wild-type and EAEA–lysozymes, respectively.

 

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Table III. Thermodynamic parameters of the EAEA–lysozyme obtained from curves for denaturation by GuHCl at 25°C
 
Kinetic experiments on denaturation and refolding

To examine the effect of the four extra residues on the kinetic stability and the folding of the human lysozyme, kinetic experiments of the reversible denaturation–refolding of the EAEA–lysozyme were performed. The denaturation and refolding reactions were monitored by measuring the aromatic fluorescence intensity. The denaturation of the EAEA– lysozyme was followed by the concentration jump from 0 M to various concentrations of GuHCl. The denaturation rate of the EAEA–lysozyme was too fast to be monitored precisely in a stopped-flow apparatus at pH 3.0 and 25°C. Therefore, all measurements were performed at pH 4.0 and 10°C. The data were analyzed using the following equation:

where A(t) is the fluorescence intensity at a given time, A({infty}) is the value when no further change is observed, Ai is the amplitude of the ith phase and ki is the apparent rate constant of the ith phase. The denaturation kinetics of the EAEA–lysozyme were described by a single exponential, as reported for the wild-type protein (Taniyama et al., 1992Go). The kinetic amplitudes of the denaturation reactions at various final concentrations of GuHCl were almost 100%, as shown in Figure 6Go(A). The logarithm of the apparent rate constant (kapp) of the EAEA–lysozyme increased linearly with increasing GuHCl concentration as shown in Figure 7Go. The kapp values of the EAEA–lysozyme at all examined concentrations of GuHCl were 1–2 orders of magnitude higher than those of the wild-type protein, indicating that the addition of the four extra residues accelerates the denaturation rate of the human lysozyme.



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Fig. 6. Typical GuHCl-induced denaturation and refolding kinetic progress curves of the wild-type and EAEA–lysozymes. (A) and (B) represent denaturation and refolding curves, respectively. Solid and dotted lines represent the wild-type and EAEA proteins, respectively. The denaturation was initiated by a concentration jump from 0 to 4.51 M and the refolding process was initiated by a concentration jump from 5.0 to 0.65 M at 10°C in 40 mM glycine–HCl buffer at pH 4.0.

 


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Fig. 7. Dependence of the apparent rate constants (in s–1) of refolding and denaturation on GuHCl concentration at pH 4.0 and 10°C. Filled and open symbols represent the wild-type and EAEA proteins, respectively. Circles and squares represent the denaturation reactions. The up and down triangles refer to the slow and fast phases, respectively, in the biphasic refolding reactions.

 
The refolding kinetics were studied with a concentration jump from 5.5 M GuHCl solution, in which the protein was completely denatured, to various GuHCl concentrations at 10°C and pH 4.0 (Figure 6BGo). The GuHCl concentration dependence of log kapp is also shown in Figure 7Go, indicating that the refolding of the EAEA–lysozyme has two phases below 2 M GuHCl, similar to that of the wild-type protein. The amplitude of the fast phase of the two phases observed in the refolding kinetics was greater than that of the slow phase, indicating that the fast phase is predominant during the refolding reaction. The kapp values of the major fast phase for the EAEA–lysozyme were slightly lower than those of the wild-type protein, indicating that the addition of the four extra residues slightly affects the refolding rate of the human lysozyme.

Crystal structure

The data collection and refinement statistics for the EAEA–lysozyme are summarized in Table IVGo. The EAEA–lysozyme had a different crystal form from the wild-type and other mutant ones already reported (Takano et al., 1995Go, 1997aGo,Takano et al., bGo, 1999bGo). The crystal of the EAEA–lysozyme belongs to the space group P6122 and has the highest solvent content (Matthews, 1968Go) of 67% in the six crystal forms found in the mutant human lysozyme crystals (Takano et al., 1995Go, 1997aGo,Takano et al., bGo, 1999bGo). The overall structure of the EAEA–lysozyme was essentially identical with that of the wild-type protein, with an r.m.s. deviation of 0.47 Å for the C{alpha} atoms. The structural position of the first residue (Glu–4) was not determined, because the electron density corresponding to the first residue was poor. As the space where the first residue would be located was open, the first Glu–4 residue should be flexible and disordered. There were large changes in the regions of the N-terminal residues (2.0 Å in r.m.s. deviations of main-chain atoms between the wild-type and the EAEA–lysozyme) and of residues 47–49 and 67–69 (1.5 Å in r.m.s. deviations) which are far from the N-terminal region. The regions of residues 47–49 and 67–69 are on the surface area of the molecule and located in the loop structure, respectively. This suggests that changes in the regions 47–49 and 67–69 reflect the conformational flexibility of these regions in addition to the difference in the crystal packings.


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Table IV. Data collection and refinement statistics for the EAEA–lysozyme
 
The structures in the vicinity of the N-terminal regions of the EAEA and the wild-type lysozymes are illustrated in Figure 8Go. Three major structural changes were observed: changes in salt bridge pairs, those in the N-terminal ß-sheet structure and release of two water molecules. A salt bridge is observed between Lys1/N{zeta} and Glu7/O{varepsilon} in the wild-type structure. However, in the EAEA–lysozyme, there were two salt bridges between Glu–2/O{varepsilon} (third residue of the four extra residues) and Lys1/N{zeta} and between Glu7/O{varepsilon} and Arg10/N{varepsilon}. As a result, the side-chain positions of these residues, especially the structural position of Lys1, were significantly changed. The N-terminal region of the wild-type human lysozyme forms a ß-sheet structure with hydrogen bonds between Lys1/N and Thr40/O{gamma}1, between Lys1/O and Thr 40/O{gamma}1 and between Lys1/N and Asp87/O{delta}1. In the EAEA–lysozyme structure, Lys1/N forms a hydrogen bond with Asn39/O{delta}1 and Lys1/O for Thr40/O{gamma}1. The number of hydrogen bonds in the N-terminal region decreased from three to two bonds. In the wild-type protein, two water molecules were near the Lys1 residue, but they were lost in the EAEA–lysozyme.



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Fig. 8. Stereo drawings of the wild-type (A) and the EAEA human lysozyme (B) structures in the N-terminal region. Solvent water molecules are drawn as filled circles. The thin line represents a hydrogen bond. There are two water molecules in the wild-type protein, but none in the EAEA–lysozyme.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
High level expression of human lysozyme

The human lysozyme gene was cloned into the XhoI site of pPIC9 and the {alpha}-factor pre–pro sequence for the extracellular production of human lysozyme was regenerated for good processing efficiency. The processing of the pre–pro sequence occurs in three steps (Julius et al., 1983Go; Bussey, 1988Go; Anna-Arriola and Herskowitz, 1994Go): (1) the pre-sequence is cleaved by a signal peptidase in the endoplasmic reticulum; (2) the pro-sequence is cleaved between Arg–5 and Glu–4 by an endo-protease (Kex2p) in the Golgi apparatus; (3) finally, a repeat of spacer sequence Glu–Ala is cleaved by a dipeptidyl aminopeptidase (Ste13p). There is a case where the Ste13p cleavage of the Glu–Ala repeat is not efficient and this repeat is left at the N-terminus of the expressed protein. This is generally dependent on the expressed protein (Steinlein et al., 1995Go). In the present study, four extra residues (EAEA) were left at the N-terminus of the expressed human lysozyme owing to an inefficient cleavage of the Ste13p. X-ray analysis showed that the Glu–2 forms a salt bridge with the Lys1. This salt bridge might interfere the approach of the Ste13p to the cleavage sites. The EAEA spacer sequence was successful for high expression, but was left at the N-terminal residue.

Changes in the thermodynamic parameters of denaturation

The calorimetric studies showed that the EAEA–lysozyme was destabilized by 9.6 kJ/mol compared with the wild-type protein owing to the substantial decrease in the enthalpy change ({Delta}H), although the conformational entropy in the denatured state should be increased when one or a few amino acid residues are added (inserted). Oobatake and Ooi (1993) have estimated the conformational entropy of each amino acid residue. Using these values, the change in conformational entropy (T{Delta}{Delta}Sconf) due to three extra residues (–Ala–3–Glu–2–Ala–1–) was estimated to be 31 kJ/mol at 64.9°C. Among the additional EAEA residues, the first Glu–4 residue was excluded from the calculation, because the residue was estimated to be flexible in the native state based on the X-ray analysis. However, the experimental results showed an entropy change (T{Delta}{Delta}S) of –21 kJ/mol at 64.9°C (Table IIGo). The extra residues would affect not only the conformational entropy, but also the hydration effect. The hydration energy is proportional to changes in the accessible surface area (ASA) of each amino acid due to denaturation (Oobatake and Ooi 1993Go; Makhatadze and Privalov 1995). Using the parameters of Oobatake and Ooi (1993), the values of the hydration enthalpy ({Delta}{Delta}Hhu) and hydration entropic energy (T{Delta}{Delta}Shu) were estimated to be –23 and –16 kJ/mol at 64.9°C, respectively. Using the parameters reported by Makhatadze and Privalov (1995), they were –32 and –20 kJ/mol, respectively. The calculated values indicate that the entropic effect of the hydration energy (T{Delta}{Delta}Shu) due to the extra residues contributes to protein stabilization. On the other hand, it has been reported that the release of a water molecule from the protein inside stabilizes the protein due to the entropic effect by 7.5 kJ/mol at 64.9°C (T{Delta}S) (Takano et al., 1999bGo). In the case of the EAEA–lysozyme (Figure 8Go), the entropic effect due to the release of two water molecules is –15 kJ/mol (T{Delta}{Delta}SH2O). The sum of the three effects, conformational entropy (31 kJ/mol), hydration entropy (–16 to –20 kJ/mol) and release of two water molecules (–15 kJ/mol), was 0 to –4 kJ/mol for T{Delta}{Delta}S at 64.9°C. This value did not agree with the experimental results (T{Delta}{Delta}S = –21 kJ/mol at 64.9°C). Calorimetric studies of the EAEA–lysozyme showed a significant decrease in the enthalpy and entropy ({Delta}{Delta}H = –31 kJ/mol and T{Delta}{Delta}S = –21 kJ/mol at 64.9°C), indicating that the significant decrease in enthalpy would be largely compensated by the decrease in entropy. These thermodynamic parameters might include the enthalpy–entropy compensation in the region except for the N-terminus, which would be affected owing to the extra N-terminus. These results suggest that the contribution to T{Delta}{Delta}S (–21 kJ/mol at 64.9°C) cannot be attributed only to the extra N-terminal residues and the addition of four residues affects the conformation in other parts far from the N-terminus. This is supported by the X-ray crystal analysis results; the structural changes were observed in the regions of residues 47–49 and 67–69, which are far from the N-terminal region.

Takano et al. (1999a) and Funahashi et al. (1999) have proposed parameters to estimate the difference in the denaturation Gibbs energy change on the basis of the structural information of a protein due to substitution. The contribution to the denaturation Gibbs energy change was divided into several stabilizing factors such as hydrophobic effect ({Delta}GHP), conformational energy ({Delta}Gconf), hydrogen bonding ({Delta}GHB) and introduction of a water molecule ({Delta}GH2O). The value of {Delta}{Delta}GHP between the EAEA–lysozyme and the wild-type protein was calculated to be 7.7 kJ/mol. {Delta}{Delta}Gconf was calculated to be –29.8 kJ/mol at 64.9°C using the values reported by Schellman (1955) and Pickett and Sternberg (1993). {Delta}{Delta}GHB can be calculated from the change in hydrogen bonds. However, the present results showed that the number of hydrogen bonds decreased from three to two, but the salt bridges increased from two to three. Therefore, in this case, {Delta}{Delta}GHB was assumed to be 0. Two water molecules around the Lys1 were lost in the EAEA–lysozyme (Figure 8Go). The entropic effect due to the release of two water molecules is 15.0 kJ/mol ({Delta}{Delta}GH2O), as described above. The summation of the four effects ({Delta}{Delta}GHP, {Delta}{Delta}Gconf, {Delta}{Delta}GHB and {Delta}{Delta}GH2O) is –7.1 kJ/mol. This value is similar to the experimental value (–9.6 kJ/mol), suggesting that the contribution to {Delta}{Delta}G is mainly due to the N-terminal region. The contribution of the conformational changes in other parts far from the N-terminus to {Delta}{Delta}G could be neglected by the enthalpy–entropy compensation.

It has been reported that extra methionine at the N-terminal residue of goat {alpha}-lactoalbumin expressed by E.coli destabilizes the protein owing to a conformational entropy effect (Chaudhuri et al., 1999Go). On the other hand, the extra methionine residue contributes to the large decrease in the enthalpy change ({Delta}{Delta}H = –77 kJ/mol at 64.9°C) in the human lysozyme (Takano et al., 1999bGo).

Effects on folding of human lysozyme

The denaturation rate constant of the EAEA–lysozyme was significantly higher than that of the wild-type protein, but the refolding rate constant was slightly lower. Calorimetric studies and equilibrium studies indicate that the EAEA–lysozyme was significantly destabilized. These results indicate that the destabilization of the EAEA–lysozyme was mainly caused by the increase in the denaturation rate constant. In studies of goat {alpha}-lactoalbumin, the addition of methionine at the N-terminal residue accelerates the denaturation rate constant and does not affect the refolding rate (Chaudhuri et al., 1999Go). According to the H–D exchange experiments in the NMR studies, two helices and the 310 helix near the C-terminus of the human lysozyme fold faster than the other sites. On the other hand, the N-terminal region does not play an important role in folding (Hooke et al., 1994Go). In the present study, the refolding rate constant was slightly affected by the four extra residues.

Conclusion

P.pastoris expressed the human lysozyme at about 300 mg/l broth. However, at the N-terminus of the expressed protein, four extra residues (Glu–4–Ala–3–Glu–2–Ala–1) were added (EAEA–lysozyme). Calorimetric studies showed that the EAEA–lysozyme was destabilized by 9.6 kJ/mol compared with the wild-type protein, mainly caused by the substantial decrease in the enthalpy change ({Delta}{Delta}H). The thermodynamic parameters obtained were analyzed on the basis of the structural information for the EAEA–lysozyme. The four extra residues also affected the thermodynamic properties in a region far from the N-terminus. However, the large change in enthalpy might almost be compensated by the changes in entropy. Therefore, changes in the Gibbs energy ({Delta}{Delta}G) could be explained by the summation of the Gibbs energies contributing to each stabilizing factor concerning the extra residues.


    Notes
 
4 To whom correspondence should be addressed E-mail: yutani{at}protein.osaka-u.ac.jp Back


    Acknowledgments
 
This work was supported in part by a Grant-in-Aid for special project research from the Ministry of Education, Science and Culture of Japan (K.Y. and Y.Y.), by a Grant-in-Aid for `REIMEI research' from the Japan Atomic Energy Research Institute (S.G. and K.Y.), by a Fellowship from the Japan Society for the Promotion of Science for Young Scientists (K.T.) and by the Sakabe project of TARA, University of Tsukuba (K.Y. and Y.Y.).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received December 17, 1999; revised February 29, 2000; accepted March 1, 2000.