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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: crystal structure/extra N-terminal residues/human lysozyme/Pichia pastoris/protein stability
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1987; Paifer et al., 1994
). However, the expression yields depend on the signal peptide and protein sequence in the yeast expression system (Hashimoto et al., 1998
). 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., 1995
).
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., 1986; Moerschell et al., 1990
). For lysozymes and
-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., 1998
; Chaudhuri et al., 1999
; Takano et al., 1999b
). On the other hand, polyhistidine tags in the N/C-terminal regions of proteins are widely used to isolate proteins easily (Kuliopulos and Walsh, 1994
). 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 stabilitystructure 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 acid4alanine3glutamic acid2 alanine1; EAEA) derived from the signal peptide remained at the N-terminal residue of the expressed human lysozyme. It is called the EAEAlysozyme. The EAEAlysozyme 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 EAEAlysozyme 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 EAEAlysozyme structure.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human lysozyme gene was amplified from pGEL125 (Taniyama et al., 1988) 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 1
), the human lysozyme gene was fused with the prepro sequence that originated from the
-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.
|
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., 1969). 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., 1991). The sample solution for the DSC measurements was prepared by dissolving the human lysozyme in 50 mM glycineHCl buffer between pH 2.6 and 3.1. The concentration of the human lysozyme was 0.91.5 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., 1992; Funahashi et al., 1996
). 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 glycineHCl 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., 1992). Kinetic experiments were performed in 40 mM glycineHCl 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 EAEAlysozyme 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, 1991) 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, 1991
). The data were processed with the program DENZO (Otwinowski, 1990
). The structure of the EAEAlysozyme was solved by the molecular replacement technique using the program AMoRe (Navaza, 1994
) with the wild-type structure (Takano et al., 1995
) as a search model. The structure was refined with the program X-PLOR (Brunger, 1992
) as already described (Takano et al., 1995
; Yamagata et al., 1998
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2 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 2
). 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.
|
DSC measurements
In order to determine the effect of the four extra residues of the EAEAlysozyme 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 3 shows typical DSC curves of the wild-type human lysozyme and EAEAlysozyme at pH 2.7. Table I
shows the thermodynamic parameters of the EAEAlysozyme obtained from the DSC curves at different pHs. As shown in Figure 4
, the Td values of the EAEAlysozyme were lower than those of the wild-type protein in the measured pH region and the calorimetric enthalpies (
Hcal) of the EAEAlysozyme were considerably decreased compared with those of the wild-type protein. The
Cp value of the EAEAlysozyme obtained from the slope of Td versus
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 II
:
![]() |
![]() |
![]() |
|
|
|
|
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 5). The transition of the EAEAlysozyme was highly cooperative, as shown in Figure 5
, 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:
![]() |
![]() |
![]() |
![]() |
|
|
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 denaturationrefolding of the EAEAlysozyme 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 EAEAlysozyme 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:
![]() |
|
|
Crystal structure
The data collection and refinement statistics for the EAEAlysozyme are summarized in Table IV. The EAEAlysozyme had a different crystal form from the wild-type and other mutant ones already reported (Takano et al., 1995
, 1997a
,Takano et al., b
, 1999b
). The crystal of the EAEAlysozyme belongs to the space group P6122 and has the highest solvent content (Matthews, 1968
) of 67% in the six crystal forms found in the mutant human lysozyme crystals (Takano et al., 1995
, 1997a
,Takano et al., b
, 1999b
). The overall structure of the EAEAlysozyme was essentially identical with that of the wild-type protein, with an r.m.s. deviation of 0.47 Å for the C
atoms. The structural position of the first residue (Glu4) 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 Glu4 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 EAEAlysozyme) and of residues 4749 and 6769 (1.5 Å in r.m.s. deviations) which are far from the N-terminal region. The regions of residues 4749 and 6769 are on the surface area of the molecule and located in the loop structure, respectively. This suggests that changes in the regions 4749 and 6769 reflect the conformational flexibility of these regions in addition to the difference in the crystal packings.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human lysozyme gene was cloned into the XhoI site of pPIC9 and the -factor prepro sequence for the extracellular production of human lysozyme was regenerated for good processing efficiency. The processing of the prepro sequence occurs in three steps (Julius et al., 1983
; Bussey, 1988
; Anna-Arriola and Herskowitz, 1994
): (1) the pre-sequence is cleaved by a signal peptidase in the endoplasmic reticulum; (2) the pro-sequence is cleaved between Arg5 and Glu4 by an endo-protease (Kex2p) in the Golgi apparatus; (3) finally, a repeat of spacer sequence GluAla is cleaved by a dipeptidyl aminopeptidase (Ste13p). There is a case where the Ste13p cleavage of the GluAla 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., 1995
). 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 Glu2 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 EAEAlysozyme was destabilized by 9.6 kJ/mol compared with the wild-type protein owing to the substantial decrease in the enthalpy change (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
Sconf) due to three extra residues (Ala3Glu2Ala1) was estimated to be 31 kJ/mol at 64.9°C. Among the additional EAEA residues, the first Glu4 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
S) of 21 kJ/mol at 64.9°C (Table II
). 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 1993
; Makhatadze and Privalov 1995). Using the parameters of Oobatake and Ooi (1993), the values of the hydration enthalpy (
Hhu) and hydration entropic energy (T
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
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
S) (Takano et al., 1999b
). In the case of the EAEAlysozyme (Figure 8
), the entropic effect due to the release of two water molecules is 15 kJ/mol (T
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
S at 64.9°C. This value did not agree with the experimental results (T
S = 21 kJ/mol at 64.9°C). Calorimetric studies of the EAEAlysozyme showed a significant decrease in the enthalpy and entropy (
H = 31 kJ/mol and T
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 enthalpyentropy 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
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 4749 and 6769, 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 (GHP), conformational energy (
Gconf), hydrogen bonding (
GHB) and introduction of a water molecule (
GH2O). The value of
GHP between the EAEAlysozyme and the wild-type protein was calculated to be 7.7 kJ/mol.
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).
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,
GHB was assumed to be 0. Two water molecules around the Lys1 were lost in the EAEAlysozyme (Figure 8
). The entropic effect due to the release of two water molecules is 15.0 kJ/mol (
GH2O), as described above. The summation of the four effects (
GHP,
Gconf,
GHB and
GH2O) is 7.1 kJ/mol. This value is similar to the experimental value (9.6 kJ/mol), suggesting that the contribution to
G is mainly due to the N-terminal region. The contribution of the conformational changes in other parts far from the N-terminus to
G could be neglected by the enthalpyentropy compensation.
It has been reported that extra methionine at the N-terminal residue of goat -lactoalbumin expressed by E.coli destabilizes the protein owing to a conformational entropy effect (Chaudhuri et al., 1999
). On the other hand, the extra methionine residue contributes to the large decrease in the enthalpy change (
H = 77 kJ/mol at 64.9°C) in the human lysozyme (Takano et al., 1999b
).
Effects on folding of human lysozyme
The denaturation rate constant of the EAEAlysozyme 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 EAEAlysozyme was significantly destabilized. These results indicate that the destabilization of the EAEAlysozyme was mainly caused by the increase in the denaturation rate constant. In studies of goat -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., 1999
). According to the HD 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., 1994
). 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 (Glu4Ala3Glu2Ala1) were added (EAEAlysozyme). Calorimetric studies showed that the EAEAlysozyme was destabilized by 9.6 kJ/mol compared with the wild-type protein, mainly caused by the substantial decrease in the enthalpy change (H). The thermodynamic parameters obtained were analyzed on the basis of the structural information for the EAEAlysozyme. 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 (
G) could be explained by the summation of the Gibbs energies contributing to each stabilizing factor concerning the extra residues.
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Artymiuk,P.J. and Blake,C.C.F. (1981) J. Mol. Biol., 152, 737762.[ISI][Medline]
Brunger,A.T. (1992) X-PLOR Manual, Version 3.1. Yale University, New Haven, CT.
Bussey,H. (1988) Yeast, 4, 1726.[ISI][Medline]
Carrell,R.W. and Lomas,D.A. (1997) Lancet, 350, 134138.[ISI][Medline]
Chaudhuri,T.K. et al. (1999) J. Mol. Biol., 285, 11791194.[ISI][Medline]
Flinta,C., Persson,B., Jornvall,H. and von Heijne,G. (1986) Eur. J. Biochem., 154, 193196.[Abstract]
Funahashi,J., Takano,K., Ogasahara,K., Yamagata,Y. and Yutani,K. (1996) J. Biochem., 120, 12161223.[Abstract]
Funahashi,J., Takano,K., Yamagata,Y. and Yutani,K. (1999) Protein Engng, 12, 841850.
Hashimoto,Y., Koyabu,N. and Imoto,T. (1998) Protein Engng, 11, 7577.
Hooke,S.D., Radford,S.E. and Dobson,C.M. (1994) Biochemistry, 33, 58675876.[ISI][Medline]
Ishikawa,N., Chiba,T., Chen,L.T., Shimizu,A., Ikeguchi,M. and Sugai,S. (1998) Protein Engng, 11, 333335.[Abstract]
Jancarik,J. and Kim,S.H. (1991) J. Appl. Crystallogr., 24, 409411.[ISI]
Julius,D., Blair,L., Brake,A., Sprague,G. and Thoner,J. (1983) Cell, 32, 839852.[ISI][Medline]
Katakura,Y., Zhang,W., Zhuang,G., Omasa,T., Kishimoto,M., Goto,Y. and Suga,K. (1998) J. Ferment. Bioengng, 86, 482487.
Kuliopulos,A. and Walsh,C.T. (1994) J. Am. Chem. Soc., 116, 45994607.[ISI]
Loquet,L.P., Saint-Blancard,J. and Jolles P. (1968) Biochim. Biophys. Acta, 167, 150153.[ISI][Medline]
Makhataze,G. and Privalov,P. (1995) Adv. Protein Chem., 47, 307425.[ISI][Medline]
Matsuura,T., Miya,i K., Trakulnaleamsai,S., Yomo,T., Shima,Y., Miki,S., Yamamoto,K. and Urabe,I. (1999) Nature Biotechnol., 17, 5861.[ISI][Medline]
Matthews,B.W. (1968) J. Mol. Biol., 33, 491497.[ISI][Medline]
Moerschell,R.P., Hosokawa,Y., Tsunasawa,S. and Sherman,F. (1990) J. Biol. Chem., 265, 1963819643.
Muraki,M., Jigami,Y., Morikawa,M. and Tanaka,H. (1987) Biochim. Biophys. Acta, 911, 376380.[ISI][Medline]
Navaza,J. (1994) Acta Crystallogr., A50, 157163.[ISI]
Oobatake,M. and Ooi,T. (1993) Prog. Biophys. Mol. Biol., 59, 237284.[ISI][Medline]
Otwinowski,Z. (1990). DENZO Data Processing Package. Yale University, New Haven, CT.
Paifer,E., Margolles,E., Cremata,J., Montesino,R., Herrera,L. and Delgado,J. (1994) Yeast, 10, 14151419.[ISI][Medline]
Parry,R.M.,Jr, Chandon,R.C. and Shahani,K.M. (1969) Arch. Biochem. Biophys., 130, 5965.[ISI][Medline]
Pepys,M.B. et al. (1993) Nature, 362, 553557.[ISI][Medline]
Peters,C.W., Kruse,U., Pollwein,R., Grzeschik,K.H. and Sippel,A.E. (1989) Eur. J. Biochem., 182, 507516.[Abstract]
Pickett,S.D. and Sternberg,M.J.E. (1993) J. Mol. Biol., 231, 825839.[ISI][Medline]
Privalov,P.L. and Khechinashvili,N.N. (1974) J. Mol. Biol., 86, 665684.[ISI][Medline]
Redfield,C. and Dobson,C.M. (1990) Biochemistry, 29, 72017214.[ISI][Medline]
Sakabe,N. (1991) Nucl. Instrum. Methods Phys. Res., A303, 448463.[ISI]
Schellman,J.A. (1955) C. R. Trav. Lab. Carlsberg, Ser. Chim., 29, 230259.
Steinlein,L.M., Graf,T.N. and Ikeda R.A. (1995) Protein Express. Purif., 6, 619624.[ISI][Medline]
Takano,K., Ogasahara,K., Kaneda,H., Ymagata,Y., Fujii,S., Kanaya,E., Kikuchi,M., Oobatake,M. and Yutani,K. (1995) J. Mol. Biol., 254, 6276.[ISI][Medline]
Takano,K., Yamagata,Y., Fujii,S. and Yutani,K. (1997a) Biochemistry, 36, 688698.[ISI][Medline]
Takano,K., Funahashi,J., Yamagata,Y., Fujii,S. and Yutani,K. (1997b) J. Mol. Biol., 274, 132142.[ISI][Medline]
Takano,K., Yamagata,Y., Funahashi,J., Hioki,Y., Kuramitsu,S. and Yutani,K. (1999a) Biochemistry, 38, 1269812708.[ISI][Medline]
Takano,K., Tsuchimori,K., Yamagata,Y. and Yutani,K. (1999b) Eur. J. Biochem., 266, 675682.
Taniyama,Y., Yamamoto,Y., Nakano,M., Kikuchi,M. and Ikehara,M. (1988) Biochem. Biophys. Res. Commun., 152, 962967.[ISI][Medline]
Taniyama,Y., Ogasahara,K., Yutani,K. and Kikuchi,M. (1992) J. Biol. Chem., 267, 46194624.
Tschopp,J.F., Sverlow,G., Kosson,R., Craig,W. and Grinna, l. (1987) Bio/Technology, 5, 13051308.[ISI]
Yamagata,Y., Kubota,M., Sumikawa,Y., Funahashi,J., Takano,K., Fujii,S. and Yutani,K. (1998) Biochemistry, 37, 93559362.
Yutani,K., Hayashi,S., Sugisaki,Y. and Ogasahara,K. (1991) Proteins: Struct. Funct. Genet., 9, 9098.[ISI][Medline]
Received December 17, 1999; revised February 29, 2000; accepted March 1, 2000.