Departamento de Bioloxía Celular e Molecular, Facultade de Ciencias, Campus da Zapateira s/n, 15071-A Coruña, Spain
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Abstract |
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Keywords: ß-galactosidase/Kluyveromyces lactis/protein secretion
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Introduction |
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The secretion of the intracellular protein into the culture medium, achieved by genetic modification, could facilitate its purification. The fusion of a secretion signal sequence 5' to the gene has proved to be useful in the case of small peptides, but it is especially problematic when high molecular weight or oligomeric proteins such as ß-galactosidase are involved. Over the last decade, several authors have tried to achieve the heterologous secretion of bacterial and fungal ß-galactosidases by Saccharomyces cerevisiae. In Table I we observe that the protein from Escherichia coli remained mainly periplasmic, as if the high molecular weight prevented ß-galactosidase, which is a tetramer of 116 kDa sub-unit (Jacobson et al, 1994
), from passing through the cell wall. Rossini et al. obtained a maximum of 33% secretion in the best of several culture conditions tested: high temperature and rich media supplemented with reducing agents (Rossini et al., 1993
). The 40% secretion obtained by Kumar et al. (Kumar et al., 1992
) may be explained by the extracellular nature of the Aspergillus niger ß-galactosidase. Based on these data, we conclude that adding a signal sequence is not sufficient to lead ß-galactosidase out of the cell: culture conditions play an important role, the wall acts as a molecular sieve but, moreover, structural determinants of the protein, at present under study, may influence secretion efficiency (Boyd and Beckwith, 1990
; Kowalski et al., 1998
; Zhu et al., 1998
; Katakura et al., 1999
).
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Materials and methods |
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The following strains were used: Kluyveromyces lactis MW 190-9B (MATa, lac4-8, uraA, Rag+), Kluyveromyces lactis NRRL-Y1140 (wild type), Saccharomyces cerevisiae BJ3505 (pep4::HIS3, prb-1.6R HIS3, lys2-208, trp1-
101, ura3-52, gal2, can1).
The BJ3505 strain was purchased from Eastman Kodak.
Liquid cultures of transformed strains were performed (a) in Erlenmeyer flasks filled with a 2:5 volume of culture medium at 30°C and 250 r.p.m. As inocula, a suitable volume of a stationary phase pre-culture in a complete medium (CM) without the corresponding auxotrophic amino acid was added to obtain an initial absorbance at 600 nm of 0.2. Samples were taken at regular time intervals to measure growth (absorbance at 600 nm), intra- and extracellular ß-galactosidase activity and sugar consumption; (b) in a Biostat-MD (Braun-Biotech) 2 l vessel chemostat. The working volume of the culture was 2 l and the temperature was maintained at 30°C. The air flow was 2 l/min sparged through the culture with an agitation speed of 250 r.p.m. Dissolved oxygen was measured with a polarographic electrode and growth was monitored with a turbidity probe, previously calibrated to absorbance at 600 nm values. The pH was maintained at about 7 by the addition of sodium hydroxide. A 100 ml volume of a liquid preculture on CM-Ura was used as inocula. Samples and measurements were as described above.
YPD (1% yeast extract, 0.5% bactopeptone, 0.5% dextrose), YPL (1% yeast extract, 0.5% bactopeptone, 4% lactose), YPHSM (1% yeast extract, 8% bactopeptone, 1% dextrose, 3% glycerol, 20 mM CaCl2) and cheese-whey (ultrafiltration permeate supplemented with 1% yeast extract) were used as culture media. The ultrafiltration permeate of cheese-whey (about 5% lactose) was obtained from the local dairy plant Quegalsa (Ferrol, A Coruña, Spain). If a protein precipitate was observed after autoclave sterilization at 121°C for 15 min, then it was removed by centrifugation (15 min at 10 000 r.p.m.) under sterile conditions. A complete medium (CM) (Lowry et al., 1983) without the corresponding auxotrophic amino acid was used for the inocula.
Molecular biology procedures
E.coli DH5 strain (supE44,
lacU169,
80lacZ
M15, hsdR17, recA1, endA1, gyrA96, thi-1, relA1) was used for the construction of the plasmids and propagation by means of the usual DNA recombinant techniques (Sambrook et al., 1989
; Ausubel et al., 1995
).
Yeast strains were transformed using the lithium acetate procedure of Ito et al. (Ito et al., 1983). Plasmid uptake and ß-galactosidase production by the transformed strains were identified in plates with the chromogenic substrate X-gal in the corresponding auxotrophic medium.
Plasmid stability was determined by taking samples at different culture times and spreading 100 µl of a suitable dilution on YPD plates. After growing at 30°C, they were replica plated on CM without the corresponding auxotrophic amino acid plus X-gal and the ratio of viable and ß-galactosidase-expressing cells (containing the plasmid) was estimated.
PCR conditions
A 20 ng amount of template DNA was incubated with 30 pmol of primer-1 and 30 pmol of primer-2 in the presence of 0.25 mM dNTPs, Taq or Pwo polymerase buffer 1X and 2 U of the corresponding polymerase. Initial denaturation was done at 94°C for 2 min, followed by 30 cycles of 1 min at 95°C, 2 min at 5057°C and 1.52.5 min at 72°C. There was a final incubation at 72°C for 10 min to fill in ends.
Vectors and DNA constructions
The plasmids used as vectors, the constructed ß-galactosidase secretion cassettes and the corresponding transformed strains are shown in Figure 1.
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Plasmid pSPGK1-LAC4 was constructed by cloning the LAC4 gene, which codes for K.lactis ß-galactosidase, in the single cloning site of pSPGK1; this cloning site was previously modified by inserting an XbaI linker in the EcoRI site; the LAC4 gene was amplified from pLX8 plasmid (Das and Hollenberg, 1982) with the following oligonucleotides that create an XbaI site on the ends of the PCR product: GCTCTAGATTATTCAAAAGCGAGATC, GCTCTAGATGTCTTGCCTTATTCCT. The K.lactis strain MW190-9B was transformed with plasmid pSPGK1-LAC4.
Plasmid pSPGK1-Ser-LAC4 was constructed as follows: plasmid pSPGK1 was digested with EcoRI, treated with S1-nuclease and ligated to the following complementary oligonucleotides containing an XhoI site (ATCCCTCGAG, CTCGAGGGAT). The LAC4 gene was PCR-amplified using oligonucleotides A and B in Table II and ligated in the XhoI site of the modified pSPGK1. The K.lactis strain MW190-9B was transformed with plasmid pSPGK1-Ser-LAC4.
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Four constructs were performed, PCR-amplifying from pLX8 plasmid the complete LAC4 gene or deleted mutants, using different pairs of the oligonucleotides shown in Table II as primers and cloning them in the XhoI site of the multiple cloning site of the YEpFLAG1 vector. Oligonucleotides A and B allowed us to obtain the entire LAC4 gene with XhoI sites at both ends (the resulting plasmid was called YEpFLAG1-LAC4). Oligonucleotides B and C allowed us to obtain the LAC4 gene but removing the 10 N-terminal amino acids (the resulting plasmid was called YEpFLAG1-BC). Oligonucleotides B and D allowed us to obtain the LAC4 gene but removing the 25 N-terminal amino acids (the resulting plasmid was called YEpFLAG1-BD). Oligonucleotides E and F allowed us to obtain the region of the LAC4 gene corresponding to the active site of the protein (the resulting plasmid was called YEpFLAG1-EF). The Saccharomyces cerevisiae BJ3505 strain was transformed with these four plasmids (YEpFLAG1-LAC4, YEpFLAG1-BC, YEpFLAG1-BD, YEpFLAG1-EF).
ß-Galactosidase activity assays
Intra- and extracellular ß-galactosidase activity was measured by the method of Guarente (Guarente, 1983). For extracellular activity, a suitable volume of the culture medium was used instead of the permeabilized cellular suspension. Liberated o-nitrophenol (ONP) was measured spectrophotometrically at 420 nm (extinction coefficient 4500 l/mol.cm). One enzyme unit (EU) is defined as the quantity of enzyme that catalyzes the liberation of 1 µmol of ONP from o-nitrophenyl-ß-D-galactopyranoside per minute under assay conditions. Unless stated otherwise, EU are expressed per milliliter of culture medium.
Polyacrylamide gel electrophoresis and Western blotting
These were performed as described (Becerra et al., 1997) but using anti-FLAG M1 monoclonal antibody (10 µg/ml).
Sugars
Total sugars in the culture medium were determined by the method of Dubois et al. (Dubois et al., 1956). Lactose was determined using a commercial test kit (Boehringer-Mannheim).
Cell fractionation
A modification of a published method (Jigami et al., 1986) was used. Cells grown in YPL were harvested by centrifugation at 5000 r.p.m. for 5 min. Pellet cells were suspended in 1.2 M sorbitol, 10 mM KH2PO4, pH 6.8 and 25 mM ß-mercaptoethanol, washed twice and resuspended in 1.2 M sorbitol, 10 mM KH2PO4, pH 6.8 and 0.6% lyticase (from Arthrobacter luteus; Sigma Chemical, St.Louis, MO, Ref. L-8012). The mixture was incubated at 30°C for 4560 min and centrifuged at 2500 r.p.m. at 4°C for 5 min. The supernatant constituted the periplasmic fraction. The pellet was suspended in distilled water and after lysis the suspension was centrifuged at 2500 r.p.m. for 5 min. The supernatant contains the cytoplasmic fraction and the pellet contains the cell debris fraction. ß-Galactosidase activity was measured in the extracellular culture medium, periplasmic, cytoplasmic and cell debris fractions.
Experimental design and statistical data analysis
Factorial experimental designs were created and data were analyzed with the aid of version 1 of the Statgraphics software for Windows (Manugistics and Statistical Graphics).
The statistical significance of differences between means was determined by Student's t-test performed with the same software; p values <0.05 were considered significant.
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Results and discussion |
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ß-Galactosidase secretion in K.lactis
Representative parameters of the chemostat batch culture of the ß-galactosidase-mutant K.lactis strain MW190-9B transformed with plasmid pSPGK1-LAC4 (Figure 1) on cheese-whey permeate supplemented with yeast extract, as described in Materials and methods, are shown in Figure 2
. Plasmid stability was 56% after 45 h of culture, when lactose was exhausted. The percentage of ß-galactosidase secretion into the culture medium was higher in early culture phases, varying from 54% after 1014 h of culture to <10% at incubation times of over 35 h. However, absolute values of extracellular activity increased with absorbance during the exponential growth phase, owing to the rise in intracellular enzyme levels.
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Does the N-terminal amino acid affect secretion?
The half-life of a mature protein is partially determined by its N-terminal amino acid (Varshavsky, 1996). Since the N-terminal amino acid of the wild-type K.lactis ß-galactosidase is destabilizing (Ile) and for the purpose of testing whether secretion was influenced by this fact, we constructed the plasmid pSPGK1-Ser-LAC4 (Figure 1
) and transformed the K.lactis strain MW190-9B. The engineered ß-galactosidase coded by this plasmid is three amino acids longer and, after the secretion signal is processed and cut by the Kex1 K.lactis endopeptidase, a serine (a stabilizing amino acid) results in its N-terminus.
In Figure 3 we compare the cultures in YPD of strain MW190-9B/pSPGK1-Ser-LAC4 versus the control strain MW190-9B/pSPGK1-LAC4. Growth (absorbance) was higher in the case of strain MW190-9B/pSPGK1-Ser-LAC4, but the intracellular ß-galactosidase/absorbance ratio was lower, reaching 12.3 for cells of strain MW190-9B/pSPGK1-LAC4 approaching the stationary phase, and did not surpass 7.5 for strain MW190-9B/pSPGK1-Ser-LAC4. The average percentage of extra- versus intracellular ß-galactosidase after 2 and 3 days of culture, was significantly higher for strain MW190-9B/pSPGK1-Ser-LAC4 than for strain MW190-9B/pSPGK1-LAC4 (p = 0.0003; mean ± SE was 4.99 ± 0.007 and 1.28 ± 0.018, respectively, n = 2). Regardless of the molecular mechanism responsible for this fact, these results indicate that the percentage of secretion increased when the N-terminal amino acid was changed to a more stabilizing one.
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As mentioned in the Introduction, the large size (124 kDa/monomer) and oligomeric nature of K.lactis ß-galactosidase (Becerra et al., 1998) may be a handicap when attempting to obtain efficient secretion. Therefore, we tried to construct a reduced-size but fully active protein to facilitate its secretion into the culture medium. We used the YEpFLAG1 vector and the Saccharomyces cerevisiae strain BJ3505 as a host. Knowledge of the three-dimensional structure of the ß-galactosidase from E.coli (Jacobson et al., 1994
) and existing studies on primary sequence homology between this protein and other ß-galactosidases including that of K.lactis (Poch et al., 1992
) served as a theoretical basis on which we planned the corresponding deletions, for the additional purpose of finding out whether the high homology shown between both sequences (Figure 4
) corresponded to a certain structuralfunctional analogy.
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The BJ3505 cells transformed with plasmids YEpFLAG1-LAC4, coding the entire ß-galactosidase and YEpFLAG1-BC (see Figure 1 and Materials and methods for plasmids) showed ß-galactosidase activity when cultured in a YPHSM medium, but no activity (intra- or extracellular) was found in cells producing shorter proteins (from plasmids YEpFLAG1-BD and YEpFLAG1-EF). These results point to the existence of a functional analogy between the N-terminal segment of the K.lactis ß-galactosidase and the E.coli ß-galactosidase This region has been described as participating in the contact inter-domains and inter-monomers and, therefore, in the correct assembly of the tetramer and integrity of the active site which is made up of elements from two different sub-units (Jacobson et al., 1994
). A similar structurefunction relationship may account for the K.lactis ß-galactosidase, but three-dimensional structure data are necessary to corroborate this hypothesis.
Kinetics of growth and ß-galactosidase production for the two S.cerevisiae transformed strains producing active proteins are shown in Figure 5. Synthesis of the enzyme is accelerated after 24 h of culture, when glucose is exhausted, since the ADH2 promoter is glucose-repressed. Average percentages of secretion were within a similar range in both cases (p = 0.73; mean ± SE, 0.10 ± 0.03 for BJ3505/YEpFLAG1-LAC4, n = 6, 0.13 ± 0.04 for BJ3505/YEpFLAG1-BC, n = 4). Although such percentages were lower than those obtained with the K.lactis system developed here in glucose media (p = 0.034; mean ± SE, 0.12 ± 0.01, n = 10, 12.95 ± 1.91, n = 12) owing to the higher levels of intracellular ß-galactosidase, the extracellular ß-galactosidase absolute values shown by these cultures were equally important (p = 0.74; mean ± SE, 1.23 ± 0.08 EU/ml, n = 11, 1.41 ± 0.14 EU/ml, n = 12).
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Thus, strains BJ3505/YEpFLAG1-LAC4 and BJ3505/YEpFLAG1-BC are good tools for achieving an efficient K.lactis ß-galactosidase secretion and yields may be improved after an optimization of culture conditions.
Influence of culture conditions on secretion
Previous authors have reported that by accurately choosing the growth conditions, secretion into the culture medium of heterologous proteins by yeast may be improved (Rossini et al., 1993). Therefore, we decided to study the influence of several culture conditions on K.lactis ß-galactosidase heterologous secretion.
We performed a full-factorial 24 experimental design focused on the study of the individual and combined influence of several culture parameters (temperature, aeration, NaCl concentration and incubation time) selected on a bibliographical basis (Rossini et al., 1993; Miksch et al., 1997
) on ß-galactosidase secretion by the strain BJ3505/YEpFLAG1-LAC4 growing on YPHSM. The experimental domain defined by the range of factors whose influence was studied and the corresponding coded values are presented in Table III
. The experiments performed and the results of the extracellular ß-galactosidase activity measured are shown in Table IV
.
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where the values of R (extracellular ß-galactosidase activity) are specified in their original units and are coded in the case of the factors, T is the temperature of the culture, A is the aeration rate, C is the NaCl concentration and t is the incubation time. The interactions AxC, Axt, Cxt, TxAxC, TxAxt, TxCxt, AxCxt and TxAxCxt failed to influence the response and were therefore excluded from the model.
Since the p-value for the lack of fit in the ANOVA is >0.05 (p = 0.069), the first-order model appears to be appropriate for the data observed. The R2 statistic indicates that the model as fitted explains 84.28% of the variability in R. The accuracy of the first-order model is also proven by the estimation of the curvature (5.85 ± 4.54, confidence interval) which is calculated as the difference between the mean ± SD of the results obtained in the four experiments at the central point (12.2 ± 1.61) and the mean ± SD of the results obtained in experiments 116 (6.35 ± 8.02). This parameter represents the sum of the coefficients of the four factors squared and, in the case at hand, is negligible, since there is no statistically significant difference between the means of the two samples (p = 0.17).
Based on the coefficients of the factors and the representation of the response surfaces (Figure 6) of the above equation, we conclude that, in the experimental domain studied, extracellular ß-galactosidase activity increases with a rise in aeration and incubation time and as temperature and NaCl concentration decrease. The temperature is the most influential factor. A 20-fold range of variation of the extracellular ß-galactosidase activity values predicted by the model was observed in the experimental plan used and there was an 8-fold increase with respect to previously obtained values (Figure 5A
).
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Although it has been reported that the secretion levels of heterologous proteins may be modified by the addition of reducing agents (De Nobel et al., 1990a, b
, De Nobel and Barnett, 1991
, Rossini et al., 1993
) or detergents (Becerra and González Siso, 1996
) to the culture medium, in the case of strain BJ3505/YEpFLAG1-LAC4, growing on YPHSM under the best conditions as previously determined, the addition to the medium of the reducing agents 2-mercaptoethanol (1020 mM) or dithiothreitol (12.525 mM) caused an inhibition of the growth of the recombinant yeast and a concomitant decrease in extracellular ß-galactosidase activity. The detergent Tween-80 (0.20.4%) did not affect growth but no increase in extracellular ß-galactosidase activity was observed (data not shown).
Conclusions
On fusing a signal sequence to the gene, although the secretion of the K.lactis ß-galactosidase is hindered by the high molecular weight and oligomeric nature of the enzyme, we were able to develop efficient molecular tools and to achieve appreciable levels of extra-cellular activity in the culture medium. Partial secretion of the enzyme was obtained in both K.lactis, by fusion to the killer toxin signal peptide, and in S.cerevisiae, by fusion to the -factor signal peptide. Observed percentages of secretion but not absolute values of extracellular ß-galactosidase activity in glucose media were higher with the system developed for K.lactis. We have improved secretion percentages by engineering the N-terminus of the protein introducing a stabilizing amino acid (Ser) in the first position of the mature protein. We propose that there is a structural and functional analogy between the K.lactis and E.coli ß-galactosidases based on the activity of the deletion mutants constructed, but we have not found a large segment of the K.lactis ß-galactosidase that can be removed without affecting activity but increasing secretion. Moreover, we have developed a model which describes the influence of several culture conditions on heterologous K.lactis ß-galactosidase secretion. Under the optimum conditions determined from this model, absolute values of extracellular ß-galactosidase produced by the S.cerevisiae recombinant strain BJ3505/YEpFLAG1-LAC4 were the highest of this work in glucose media.
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Notes |
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Acknowledgments |
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References |
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Received September 7, 2000; revised January 29, 2001; accepted February 15, 2001.