Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system

Nozomi Koganesawa1, Tomoyasu Aizawa3, Kazuo Masaki1, Atsushi Matsuura1, Taisuke Nimori1,2, Hisanori Bando2, Keiichi Kawano3,4 and Katsutoshi Nitta1

1 Division of Biological Sciences, Graduate School of Science and 2 Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Sapporo 060-0810 and 3 Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of {alpha}-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals. When the {alpha}-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme. Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal. However, its level of expression was not increased. This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather {alpha}-factor leader inhibits the secretion. Silkworm lysozyme with the {alpha}-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.

Keywords: level of expression/lysozyme/Pichia pastoris/protein stability/signal sequence


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The construction of an over-expression system using molecular biology strategies is essential for the investigation of protein engineering and enzymology. This is especially the case when the protein of interest is rare and it is difficult to obtain a sufficient amount for physicochemical research or when a sufficient amount of the protein of interest must be arranged by site-directed mutagenesis. In order to acquire the protein of interest in as large an amount as possible, the optimum condition for efficient expression must be elucidated.

Escherichia coli, yeast, insect and mammalian cells have been widely used for recombinant protein expression. E.coli is the most frequently used expression host, although the polypeptide expressed from it remains in the host cell and is often yielded as insoluble inclusion bodies in the case of high level expression. In order to gain the active form protein, the inclusion body must be solubilized and refolded. Further, when protein is expressed in E.coli, a methionine residue is sometimes left at the N-terminus of the expressed protein. It has been reported that this extra methionine affects the conformational stability of the protein (Chaudhuri et al., 1999Go; Takano et al., 1999Go)

On the other hand, the polypeptide chain can be secreted to an extracellular medium by selecting the appropriate signal sequence from eucaryotic cells like yeast. This feature is greatly advantageous to the expression system using E.coli. Moreover, the expression level in yeast is generally much higher than that in animal cells (Ueda et al., 2000Go). Many kinds of secretion signals are used in yeast expression systems, such as PHO1 secretion signal, yeast invertase signal sequence (Chang et al., 1986Go; Payne et al., 1995Go), {alpha}-factor secretion signal, etc. Each signal has its particular advantage, and there is no common rule by which to determine the most effective sequence. Among these signals, {alpha}-factor, a yeast peptide pheromone consisting of 13 amino acid residues, plays an important role in the process of {alpha}-mating initiation (Thorner, 1981Go). It is known that {alpha}-factor is synthesized initially as a larger precursor, prepro-{alpha}-factor (Brake et al., 1983Go). Prepro-{alpha}-factor consists of a signal sequence, prosegment and the repeats of spacer peptides followed by mature {alpha}-factor sequences. This leader sequence is popular for its high level of expression in the yeast system (Brake et al., 1984Go; Hashimoto et al., 1998aGo,bGo).

In this investigation, expression systems of human and silkworm Bombyx mori lysozymes were constructed using the methylotrophic yeast Pichia pastoris, developed as an efficient host for the production of heterologous proteins (Sreekrishna et al., 1988Go; Cregg et al., 1989Go, 1993Go; Wegner, 1990Go; Buckholz and Gleeson, 1991Go; Mine et al., 1999Go; Oka et al., 1999Go). Among the many signal sequences developed for yeast expression, the precursor of human and silkworm lysozymes and the leader sequence of {alpha}-factor were selected.

Lysozyme has been well studied as a model protein (Canfield, 1963Go; Prager and Jollès, 1996Go) and many kinds of lysozymes have been expressed in the Pichia expression system (Digan et al., 1989Go; Brierley et al., 1990Go).

The function and structure of many vertebrate lysozymes have been well studied. Hen egg white and human lysozymes are two of the most frequently studied proteins. On the other hand, very little is known about the lysozyme of insects (Lee and Brey, 1995Go). For example, the invertebrate silkworm B.mori lysozyme is composed of 119 amino acids and has 11 amino acids fewer than human lysozyme, with which it shares only 40% amino acid homology. Therefore, vertebrate lysozymes (such as hen egg white or human lysozyme) and invertebrate lysozymes (such as B.mori lysozyme) are thought to be considerably different in terms of their structural and functional features. A comparison of the behavior of these proteins would be useful for the development of protein engineering and protein science. From this viewpoint, the correlation between their levels of expression and the stability of the protein with various signal sequences, especially the leader sequence of {alpha}-factor and the native signal sequences of some kinds of lysozymes, is discussed.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amplifying and subcloning of lysozyme cDNAs

Human lysozyme cDNA was amplified by PCR methodology using QUICK-Clone Human Placenta cDNA (CLONTECH) as a template. PCR methodology was performed using 5' and 3' primers as follows:

without native signal sequence (for expression using {alpha}-factor) plus EAEA:

5'-CTCGAGAAAAGAGAGGCTGAAGCTAAGGTCTTTGAAAGG-TGTAA-3',

5'-CCGAGCTCGAATTCGCGGCCGCTTACACTCCACAACCTT-GAAC-3'

without native signal sequence (for expression using {alpha}-factor):

5'-CCTTAGGGCCCCTCGAGAAAAGAAAGGTCTTTGAAAGGT-GTGAG-3',

5'-CCGAGCTCGAATTCGCGGCCGCTTACACTCCACAACCTT-GAAC-3';

and with native signal sequence:

5'-CCTTAGGGCCCGGATCCAAACCATGAAGGCTCTCATTGT-TCTG-3',

5'-CCGAGCTCGAATTCGCGGCCGCTTACACTCCACAACCTT-GAAC-3'.

Silkworm lysozyme cDNA was reverse transcribed from mRNA extracted from the body fat of B.mori N124 and was amplified by PCR methodology. The primers for this reaction were as follows:

without native signal sequence (for expression using {alpha}-factor) plus EAEA:

5'-CTCGAGAAAAGAGAGGCTGAAGCTAAAACGTTCACGAGA-TGCGG-3'

5'-CCGTCGACGAATTCGCGGCCGCTTAGCAGCTGCTAATAT-CAGGTAAGGAGCCCTG-3'

without native signal sequence (for expression using {alpha}-factor):

55'-CCTTAGGGCCCCTCGAGAAAAGAAAAACGTTCACGAGAT-GCGGT-3',

5'-CCGTCGACGAATTCGCGGCCGCTTAGCAGCTGCTAATAT-CAGGTAAGGAGCCCTG-3';

and with native signal sequence:

5'-CCTTAGGGCCCGGATCCAAACCATGCAGAAGTTAATTAT-TTTC-3',

5'-CCGTCGACGAATTCGCGGCCGCTTAGCAGCTGCTAATAT-CAGGTAAGGAGCCCTG-3'.

cDNAs were then ligated to expression vectors pPIC9 and pPIC3 (Invitrogen). The diagrams of constructed vectors are shown in Figure 1Go. Amino acid sequences of human lysozyme and silkworm lysozyme are shown in Figure 2Go ( Masaki et al., 2001Go; Matsuura,A., Yao,M., Aizawa,T., Koganesawa,N., Masaki,K., Demura,M., Tanaka,I., Kawano,K. and Nitta,K. in preparation). The human lysozyme mutant having the silkworm lysozyme signal sequence was also constructed by using PCR methodology using pfu polymerase (Stratagene) and ligated to pPIC3. Ligation samples were linearized by SacI and were transformed into P.pastoris GS115 (his4) by electroporation.



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Fig. 1. Diagram of the expression vector for the P.pastoris system. pPIC9 is the vector constructed for the expression using the {alpha}-factor leader sequence as the secretion signal and this vector contains the {alpha}-factor leader sequence. PIC3 is the vector that has no signal sequence.

 


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Fig. 2. Amino acid sequences of the complete {alpha}-factor prepro sequence, human lysozyme, silkworm lysozyme and `silkworm lysozyme with EAEA'. The arrows in {alpha}-factor prepro indicate the positions of incorrect cleavage sites in the case of expression from the {alpha}-factor signal. The underlined sequence is the spacer sequence, which is excluded in the case of expression using the {alpha}-factor signal with no EAEA.

 
Selection by MD plate and MM-lysoplate analysis

Genotypic selection and phenotypic screening were performed on MD plates [1.34% (w/v) YNB, 4x10–5% (w/v) biotin, 1% (w/v) dextrose, 1.5% (w/v) agar] and MM-lysoplates [1.34% (w/v) YNB, 4x10–4% (w/v) biotin, 3% (w/v) methanol, 0.061% (w/v) Micrococcus lysodeikticus and 3% (w/v) agar in 1/15 M potassium phosphate buffer, pH 6.0], respectively. Colonies were inoculated on to MM-lysoplates, fed 200 µl of 100% methanol in the plate cover and incubated at 30°C for about 1–2 days. The radius of translucent plaque around the colony was measured as an estimate of its level of expression of lysozyme.

Expression of recombinant lysozyme by batch cultivation

Cells were grown in BMGY medium [1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4x10–5 biotin, 1% glycerol] at 30°C in a shaking incubator until culture reached log-phase growth. The cells were harvested by centrifugation and resuspended into BMMY medium (same components as BMGY, except that 2% methanol was used instead of glycerol) and incubated with shaking at 30°C for 4–5 days. Methanol was fed every 24 h to sustain a concentration of 2%.

Purification of lysozymes

The culture was centrifuged for 20 min at 5000 g at 4°C. The supernatant was filtered through a nitrocellulose membrane (pore size 0.45 nm). After 4-fold dilution, the supernatant was applied to a 5 ml HiTrap SP column (Pharmacia) equilibrated with 50 mM phosphate buffer (pH 6.0), then the lysozyme was eluted with 50 mM phosphate buffer (pH 6.0)–1 M NaCl and the main peak was determined. The sample was then dialyzed (MWCO 8000) to H2O and 10 mM ammonium bicarbonate and freeze-dried.

Ion-exchange high-performance liquid chromatography

After freeze-drying, lysozymes were dissolved in 20 ml of deionized water and 1 ml of the solution was loaded on to a cation-exchange HPLC column [CM Toyopearl 650S (Tosoh), 7.5x100 mm]. The column was equilibrated with 50 mM phosphate buffer (pH 6.0) and the sample was eluted with 50 mM phosphate buffer (pH 6.0)–1 M NaCl. The HPLC elution pattern of silkworm lysozyme is shown in Figure 3Go. Concentrations of the lysozymes were calculated using the values of integration of peaks and the absorption coefficient, the latter of which was calculated as described elsewhere (Yin et al., 1988Go).



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Fig. 3. Cation-exchange HPLC elution patterns of silkworm lysozyme expressed from its native signal sequence. The peak of lysozyme is indicated by an arrow. Equilibration buffer and elution buffer are described in Materials and methods.

 
Differential scanning calorimetric measurements

Differential scanning calorimetric (DSC) measurements were performed on an MC-2 instrument (Micro Cal) at a heating rate of 60 K/h.

All samples were purified as descibed under Purification of lysozymes and dissolved to 1–2 mg/ml with 50 mM sodium acetate buffer (pH 3.0). At the Hi Trap SP column purification step, human lysozyme with EAEA and silkworm lysozyme with EAEA were sufficiently pure and DSC measurements could be used. Thermodynamic parameters were determined from the first scan.


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 Materials and methods
 Results
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Construction of two lysozyme expression systems using {alpha}-factor signal with EAEA as the secretion signal sequence

It is reported that {alpha}-factor with its prepro sequence (Glu–Ala repeats) is cleaved by two kinds of peptidase, KEX1 and STE13 (Emter, et al., 1983Go; Julius, et al., 1984Go). First, lysozymes with this spacer sequence were constructed and expressed.

Amino acid sequences of these samples are shown in Figure 2Go. However, it was revealed that the spacer sequence (Glu–Ala–Glu–Ala) remained in the N-terminus of lysozymes expressed from these systems (defined here as `lysozyme with EAEA'). From the analysis of amino acid sequences, each lysozyme with EAEA constituted a single molecular species (data not shown) and these results suggested that the digestion occurred not after but before the spacer sequence and that the cleavage was achieved with high efficiency. Thus, the two lysozymes were sufficiently purified to be measured by DSC with only Hi Trap SP column purification. In order to remove the EAEA spacer sequence, we also constructed the expression system without the spacer sequence.

Construction of two lysozyme expression systems using {alpha}-factor signal with no EAEA as the secretion signal sequence

Expression systems using the {alpha}-factor signal without the EAEA spacer sequence as the secretion signal sequence were constructed merely by removing the spacer sequence from the prepro-{alpha}-factor leader sequence.

Pilot expression on MM-lysoplate was performed to select the strains that expressed the lysozyme. MM-lysoplate analysis was developed in our laboratory to investigate the expression of lysozyme in recombinant P.pastoris directly and conveniently, using a lysoplate (Osserman and Lawlor, 1966Go) containing minimal medium for yeast. Production levels of recombinant lysozymes can also be simply estimated by MM-lysoplate analysis. The results of MM-lysoplate analysis are shown in Figure 4Go. When using the {alpha}-factor leader sequence without the spacer sequence as secretion signals, the radius of plaque on the MM-lysoplate showed that the level of expression of human lysozyme (Figure 4cGo) was greater than that of silkworm lysozyme (Figure 4dGo).



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Fig. 4. Results of MM-lysoplate analysis. The radius of plaque around the colony indicates the approximate level of expression of lysozyme. (a) The human lysozyme with its native signal; (b) the silkworm lysozyme with its native signal; (c) the human lysozyme with the {alpha}-factor signal; (d) the silkworm lysozyme with the {alpha}-factor signal; (e) the human lysozyme with the native signal of silkworm lysozyme.

 
The SDS–PAGE (Laemmli, 1970Go) results for human lysozyme purified using a cation-exchange Hi Trap SP column (Figure 5Go, lane A) revealed that products were a mixture of several molecular species in which the {alpha}-factor signal sequences were not digested completely or were digested at incorrect positions. From the analysis of N-terminal amino acid sequencing, these molecular species were lysozymes whose signal sequences had been digested incorrectly and those such as AKEEGVSLEKR- or EEGVSLEKR-, for example, were attached to the N-terminus (see Figure 2Go). A similar result was observed in the case of expression of silkworm lysozyme secreted by {alpha}-factor.



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Fig. 5. SDS–PAGE result{sigma} for recombinant lysozymes secreted from the P.pastoris system. (a) Human lysozyme expressed using the {alpha}-factor leader as the secretion signal, indicating that the secreted protein is a mixture of several molecular species. The upper band is a mixture of lysozyme molecules with prepro {alpha}-factors not cleaved completely and the lower band is the mature human lysozyme. (b) Silkworm lysozyme secreted by its native signal used as the secretion signal. All samples were purified by cation-exchange HPLC.

 
In Figure 5Go, it can be seen that the molecular weight of silkworm lysozyme (10 amino acids shorter than human lysozyme) appears equal to that of human lysozyme, about 14 700 Da. However, the actual molecular weight of silkworm lysozyme measured by mass spectrometry is smaller than that of human lysozyme.

The lysozymes yielded were purified by Hi Trap SP and then loaded on a cation-exchange HPLC column to be further purified. The levels of expression estimated from the results of HPLC are shown in Table IGo, indicating that the level of expression of human lysozyme with {alpha}-factor is more than 100 times higher than that of silkworm lysozyme with {alpha}-factor.


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Table I. Expression levels (mg/l) of five kinds of lysozymes measured by cation-exchange HPLC, the amount of protein being calculated from the value of OD280 and absorption coefficient of each enzyme
 
Construction of two lysozyme expression systems using their native signals as the secretion signal sequence

Expression systems using secretion signals of the two lysozymes were then constructed and levels of the expression systems were compared. We defined the secretion signal sequences of the two lysozymes used in this case as their respective `native signal' sequences. The amino acid sequences of the two precursors are indicated in Figure 2Go. In order to construct the expression system using the native signal, the pPIC3 vector, the plasmid of which has the same DNA sequence that of pPIC9 except that it has no {alpha}-factor signal sequence, was used (Figure 1Go).

Using the native signal, both human and silkworm lysozymes were produced in the extracellular space and each of the lysozymes had the correct mature sequence according to amino acid sequencing. The elution pattern of cation-exchange HPLC (Table IGo) shows that the level of expression of silkworm lysozyme with the native signal was higher than that of human lysozyme with the native signal. Silkworm lysozyme was expressed about 25-fold more than in the case using the {alpha}-factor leader sequence. Human lysozyme, on the other hand, was expressed more efficiently with the {alpha}-factor leader sequence than with its own secretion signal sequence.

Construction of the expression system of a human lysozyme mutant with the native signal of silkworm

The results described above showed that silkworm lysozyme was efficiently secreted by its native signal, as compared with secretion by the {alpha}-factor leader sequence, in the P.pastoris system. From this result, it is likely that the native signal of silkworm lysozyme is capable of increasing the level of production. To investigate the effect of the native signal of silkworm lysozyme in the P.pastoris expression system, a mutant of human lysozyme with the native signal sequence of silkworm lysozyme was constructed. The levels of expressed lysozymes including the human lysozyme with the native signal of silkworm were estimated by cation-exchange HPLC. The results are shown in Table IGo. It can be seen that the level of expression of human lysozyme secreted by its native signal and that of mutant human lysozyme with the signal of silkworm lysozyme showed little difference in level of expression.


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{alpha}-Factor is synthesized initially as a larger precursor, prepro-{alpha}-factor (Brake et al., 1983Go; Emter et al., 1983Go; Julius et al., 1984Go). During the translocation through the pathway, the prepro-{alpha}-factor is digested by three kinds of peptidases: endopeptidase, dipeptidyl aminopeptidase and carboxypeptidase. In the expression system using the {alpha}-factor leader sequence, several kinds of molecular species were secreted and it was revealed that these peptides correspond to the lysozyme molecules incompletely processed at the C-terminus of the {alpha}-factor signal. The misprocessed sub-products are also observed when another lysozyme is expressed in the yeast expression system using {alpha}-factor as secretion signal (Hashimoto et al., 1998aGo,bGo). In fact, the levels of expression of the samples measured by the lysis of Micrococcus lysodeikticus before purification by HPLC were higher than those measured after purification by HPLC in the case of expression derived from {alpha}-factor (data not shown). This is probably because it is difficult to remove these misprocessed molecular species without using an ion-exchange column (HPLC). These molecular species are produced because some proteases cannot digest the recombinants entirely. Factors that may inhibit the recognition of peptidases include the bulky side chain of Lys1, which is conserved in each lysozyme. As evidence to support this hypothesis, lysozymes with EAEA consist of nearly a single molecule. These molecular species have no bulky side chain in the neighborhood of the cleavage site.

The result that silkworm lysozyme with its native signal is expressed at a higher level than with that of {alpha}-factor indicates some possibility that the native signal of silkworm lysozyme may play an important role in increasing the level of expression of silkworm lysozyme. To confirm this assumption, we constructed a human lysozyme with the native signal sequence of silkworm and it revealed that the addition of the native signal of silkworm did not cause an increase in the level of expression. Thus, the native signal of silkworm lysozyme was not found to increase the level of production of silkworm lysozyme.

Therefore, it seems more reasonable to consider that the level of expression of silkworm lysozyme is reduced by the addition of the {alpha}-factor signal sequence. As an explanation for this decrease, it is likely that the thermodynamic stability of recombinants and the secretion signal may be relevant to this phenomenon.

Table IIGo shows a comparison of Tm values for four kinds of lysozymes. From these data, it is clearly shown that mature silkworm lysozyme has a lower thermal stability than human lysozyme. In order to clarify the effect of the addition of an amino acid sequence to the N-terminus of lysozyme, the data for `silkworm lysozyme with EAEA' are included. This molecular species harbors two-times repeats of the (Glu–Ala) spacer peptide at the N-terminus of silkworm lysozyme (see Figure 1Go). Table IIGo indicates that silkworm lysozyme with EAEA is much less stable than that of the wild type. Notably, Met–1 lysozyme expressed from E.coli becomes less stable than mature lysozyme (Yin et al., 1988Go; Mine et al., 1997Go) and human lysozyme with the EAEA sequence added to its N-terminus becomes less stable than that of the wild type (Table IIGo; Goda et al., 2000Go). These results clearly show that silkworm lysozyme with some amino acids added to its N-terminus becomes less stable than that of the wild type. Considering these findings, it is thought that nascent silkworm lysozyme with the full-length {alpha}-factor leader sequence may be unstable compared with mature silkworm lysozyme with no signal sequence.


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Table II. Tm values of human and silkworm lysozymes measured by DSC at pH 3.0
 
Of course, there are many kinds of stable fusion proteins. However, the fact that lysozyme with an extra amino acid sequence in its N-terminus tends to become unstable is common in the case of lysozyme. Silkworm lysozyme is less stable than other lysozymes, so it seems reasonable to consider that the destabilization caused by fusion to its N-terminus would be critical to its conformation.

Supposing that silkworm lysozyme is unstable to the extent that it can be easily digested by various intracellular proteases, recombinants would be easily digested, which would cause a decrease in total expression level.

A diagram of the yeast secretion system is shown in Figure 6Go. Prepro peptide translated at the ribosome should be translocated to the endoplasmic reticulum and in this step its signal sequence is cleaved by many kinds of proteases. In the endoplasmic reticulum, hyperglycosylation to the polypeptide chain occurs, then peptide is translocated to the Golgi body, then to secretion vesicles and at last to the periplasm space by exocytosis (Mellman and Simons, 1992Go; Pelham, 1992Go; Bennett and Scheller, 1994Go; Ferro-Novick and Jahn, 1994Go; Hammond et al., 1994Go; Nuoffer and Balch, 1994Go). One possibility for the decrease in silkworm lysozyme expression level by use of the {alpha}-factor leader sequence as the secretion signal is that the majority of this product is digested through this long secretion pathway, despite the fact that silkworm lysozyme is produced more efficiently with the {alpha}-factor leader than with its native signal sequence. Thus, total level of expression of silkworm lysozyme becomes lower than that with its native signal sequence. The decrease in stability caused by the addition of the {alpha}-factor leader can occur in the case of human lysozyme (see Table IIGo). However, the thermodynamic stability of wild-type human lysozyme is much higher than that of silkworm. If human lysozyme were stable enough to withstand the destabilization caused by the addition of extra residues to its N-terminus, reduction of expression level would not occur.



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Fig. 6. Modeling of the secretion pathway of protein derived from yeast.

 
Of course, the production level of the yeast expression system is influenced not only by stability of its recombinants. The fact that the expression level of silkworm lysozyme using its native signal is higher than that of human lysozyme with its own native signal cannot be elucidated only from the viewpoint of thermal stability. Various factors, such as the frequency of transcription start, the `strength' of the promoter, the stability of mRNA, the toxicity of expressed protein and codon bias should be considered when assessing production level (Dani et al., 1985Go; Hoekema et al., 1987Go; Kane, 1995Go). However, none of these factors can reasonably explain the reduction in the amount of silkworm lysozyme under the condition that the same expression vector and the same kinds of protein were used.

Here, the possibility that there is some relationship between the stability of recombinant protein with its secretion signal and its level of expression is suggested.

To our knowledge, this research is the first in which construction of the high-level expression system of insect lysozyme has been achieved and the first to demonstrate a correlation between the structural stability of a peptide with its secretion signal sequence and its level of expression. It is hoped that the present results will contribute to the selection of the optimum conditions of heterologous protein expression using eukaryotic host cells.


    Notes
 
4 To whom correspondence should be addressed. E-mail: kawano{at}ms.toyama-mpu.ac.jp Back


    Acknowledgments
 
We thank T.Ueda and Y.Hashimoto for suggestions regarding the method of cultivation of P.pastoris and methods for the separation and purification of lysozyme. This work was supported by the Center for the Enhancement of Excellence, Special Coordination Fund for Promoting Science and Technology, Science and Technology Agency, Japan.


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 Abstract
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 Materials and methods
 Results
 Discussion
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Received April 18, 2001; revised May 25, 2001; accepted June 12, 2001.