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Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris
Biochemical and Biophysical Research Communications 363 (2007) 708–714 www.elsevier.com/locate/ybbrc
Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris q Nobuyuki Ide, Tetsuya Masuda, Naofumi Kitabatake
*
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan Received 3 September 2007 Available online 18 September 2007
Abstract Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the a-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNAencoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis. 2007 Elsevier Inc. All rights reserved. Keywords: Thaumatin; Sweet-tasting protein; Recombinant protein; Secretion signal; Pichia pastoris
The methylotropic yeast Pichia pastoris is widely used for production of various proteins [1–3]. This system allows proper folding and considerable posttranslational modification of the expressed protein molecule, and culture of cells at high density. Protein production can be maximized by controlling oxygen supply and some proteins can be secreted at high levels [1–3]. P. pastoris can produce heterologous proteins either intracellularly or extracellularly. The most commonly used secretion signal in the P. pastoris expression system is the a-factor prepro-leader sequence of Saccharomyces cerevisiae, which consists of a pre-region of 19 amino acids signal peptide and a pro-region of 60 hydrophilic amino acids [1,4]. The signal sequence can be removed by signal peptidase during the translocation process and the pro-region can be cleaved by the yeast Kex2
q Nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the Accession No. AB265690. * Corresponding author. Fax: +81 774 38 3743. E-mail address: [email protected] (N. Kitabatake).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.09.021
protease, resulting in the release of the mature and processed protein. Many other secretion signals are used in yeast expression systems, such as acid phosphatase signal peptide, yeast invertase signal sequence [1,2], and others [5,6]. Furthermore, some proteins have been expressed and secreted by P. pastoris using the native signal sequence of the protein concerned. For instance, the native signal sequence of phytohemagglutinin (PHA) permitted secretion of recombinant PHA, correct folding, and correct processing of the amino terminus [7]. When the a-factor sequence was used instead of the native signal sequence, the a-factor sequence at the amino terminus was retained in the secreted PHA molecule [7]. Thaumatin is a sweet-tasting plant protein extracted from the aril of the fruit of Thaumatococcus daniellii Benth [8]. The predominant thaumatins are thaumatin I and II, which consist of 207 amino acid residues [9] and contain eight disulfide bonds [10]. A comparison of the deduced amino acid sequence from cDNA with the protein sequence derived from amino acid sequencing suggests that the pre-
N. Ide et al. / Biochemical and Biophysical Research Communications 363 (2007) 708–714
cursor form contains both a 22-amino acid N-terminal presequence and 6-amino acid C-terminal pro-sequence [11] (Fig. 1). The function of the N-terminal pre-sequence is presumed to be the compartmentalization of thaumatin [11]. Although several reports show that C-terminal peptides are cleaved during protein maturation in plants, little is known about their precise function. The biological function of the acidic C-terminal extension of thaumatin also remains obscure. In our previous study [12], P. pastoris was transformed with the thaumatin cDNA fused with both the a-factor signal sequence and the Kex2 protease cleavage site, and secretion of thaumatin by the transformant was achieved using 3 L jar fermenter. However, N-terminal sequence analysis of the recombinant thaumatin revealed that two or three unexpected amino acid residues were still attached. This may be from deficiency of Kex2 protease, which cleaves the peptide bond besides the dibasic spacer sequence of Lys–Arg. Although these additional amino acid residues at N-terminal did not influence the threshold value of sweetness of thaumatin [12], they could affect its expression, secretion, and structure. In this study, we clarified the effects of the prepro-sequence of thaumatin on its secretion and production efficiency. Materials and methods Materials and culture media. Escherichia coli strain XL1-Blue supercompetent cells (Stratagene, La Jolla, CA, USA) were used as the host for
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DNA manipulations. P. pastoris wild type X-33 (Invitrogen, Carlsbad, CA, USA) was used for production of the recombinant thaumatin. P. pastoris was grown in YPD (1% yeast extract, 2% peptone, and 2% dextrose) or Buffered Minimal Glycerol (BMG) medium (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 · 105% biotin, and 1% glycerol) for preparation of inoculum. Buffered Minimal Methanol (BMM) medium (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 · 105% biotin, 0.5% methanol, and 1% casamino acid) was used for induction of heterologous protein synthesis. Blasticidin hydrochloride was from Kaken Pharmaceutical Co. Ltd. (Tokyo, Japan). Thaumatin I was purified from crude thaumatin powder as described previously [13]. All other chemicals were biochemical reagent grade. Cloning of the prepro-thaumatin cDNA. The mRNA was purified from the fruit of T. daniellii Benth, and the first-strand cDNA was synthesized as previously described [14]. The primers including the pre- and the proregion of thaumatin were constructed on the basis of the sequence deposited as the prepro-thaumatin II cDNA (GenBank Accession No. J01209) [11]. The 5 0 sense primer, ThPre-SfuI, was: 5 0 -GAATTC GAAATGGCCGCCACCACTTGCTTC-3 0 and the 3 0 antisense primer, ThPro-XbaI, was: 5 0 -GCTCTAGATTACTCGTCTTCAAGTTCAAGC3 0 . ThPre-SfuI primer includes both EcoRI (GAATTC) and SfuI (TTCGAA) restriction sites, and ThPro-XbaI includes an XbaI (TCTAGA) site. The PCR was performed using Pfu Turbo Hotstart DNA polymerase (Stratagene) with the following thermal cycling parameters: 1 cycle of 2 min at 94 C, followed by 30 cycles of 30 s at 94 C, 30 s at 55 C, 1 min at 72 C, with a final elongation step of 5 min at 72 C. The DNA sequence of the cloned thaumatin cDNA was confirmed using an ABI 310 DNA sequencer (Applied Biosystems, Warrington, UK). The resulting plasmid containing a prepro-thaumatin cDNA was named ‘‘pCR2.1-PPTh’’. Construction of the expression vector of thaumatin. To investigate the effects of introducing the prepro-sequence of thaumatin into the mature thaumatin cDNA on both production level and processing efficiency in P. pastoris, four different expression plasmids were constructed (Fig. 2).
Fig. 1. Nucleotide sequence of the precursor of thaumatin I. The nucleotide sequence of the precursor of thaumatin I was deposited in the DDBJ/EMBL/ GenBank databases under Accession No. AB265690. The amino acid sequence deduced from its nucleotide sequence is given in single-letter abbreviations under the DNA sequence. The N-terminal pre-sequence and C-terminal pro-sequence are boxed.
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(i) pPIC6a-TH: This plasmid was constructed as previously described [12]. Briefly, the mature thaumatin cDNA fragment was prepared by XhoI and XbaI digestion of pCR2.1-Th [12], and subsequently ligated between the XhoI and XbaI sites of pPIC6a vector which contained a-factor signal, 5 0 AOX promoter, and 3 0 AOX transcription termination sequence flanking the multicloning site. (ii) pPIC6a-proTH: The pro-thaumatin cDNA was amplified by PCR using ThXhoI and ThPro-XbaI primer set with pCR2.1-PPTh plasmid as a template. ThXhoI primer encodes the mature thaumatin region without its pre-sequence [12]. After TA cloning using pCR2.1-TOPO (Invitrogen), the pro-thaumatin cDNA fragment was prepared by XhoI and XbaI digestion. The resulting fragment was ligated between the XhoI and XbaI site of pPIC6a vector. (iii) pPIC6-preTH: The pre-thaumatin cDNA was amplified by PCR using the ThPre-SfuI and ThstopXbaI primer set with pCR2.1PPTh plasmid as a template. ThstopXbaI primer contains a stop codon without the coding region of pro-sequence of thaumatin [12]. After the TA cloning, the pre-thaumatin cDNA fragment was prepared by SfuI and XbaI digestion. To remove the a-factor sequence from pPIC6a vector, the vector was digested by SfuI and XbaI, and the approximately 3.3-kb fragment was gel-puri-
fied. We confirmed that the a-factor signal sequence had been removed from the resulting vector, pPIC6. The pre-thaumatin cDNA was then inserted into the SfuI and XbaI site of the pPIC6 vector. (iv) pPIC6-preproTH: The prepro-thaumatin cDNA fragment was prepared by SfuI and XbaI digestion of pCR2.1-PPTh plasmid. The prepared prepro-thaumatin cDNA was inserted into the SfuI and XbaI sites of pPIC6 vector.
Transformation of P. pastoris by electroporation. The constructed expression plasmids were digested with PmeI and the linearized plasmid was transformed into P. pastoris X-33 by electroporation as described previously [12]. The transformants were selected by plating on YPD plates containing 300 lg mL1 blasticidin. Expression of the recombinant thaumatin in a shaking-flask. Pichia pastoris transformants were inoculated into 50 mL of BMG medium in 500-mL baffled flasks. The cells were pre-cultured at 28 C to an OD600 of 2–6. The cells were harvested by centrifugation at 1500g for 5 min at 4 C, and resuspended in 50 mL of BMM medium and diluted up to an OD600 of 1.0. The cells were grown at 28 C with continuous shaking (250 rpm)
Fig. 2. Construction of the expression vectors. (A) The mature thaumatin gene (i) and the pro-thaumatin gene (ii) were ligated into the pPIC6a vector with a-factor signal sequence. The pre-thaumatin gene (iii) and the prepro-thaumatin gene (iv) were ligated into the pPIC6 vector without a-factor signal sequence. (B) The schematic representation of the expression cassettes and potential cleavage site are shown.
N. Ide et al. / Biochemical and Biophysical Research Communications 363 (2007) 708–714 for 96 h. Methanol was added to a final concentration of 1% (v/v) every 24 h during the culture period. The growths of transformants were monitored by measuring OD600 periodically. Aliquots of the culture supernatant were taken and protein production was checked by Western-blotting as described below. Separation of yeast proteins. The washed cell pellet (equivalent of 500 lL with an OD600 of 25) was resuspended in 500 lL of breaking buffer (50 mM sodium phosphate buffer, pH 7.0, 1 mM EDTA, 1 mM PMSF, and 5% glycerol) and mixed with an equal amount of glass beads. Cells were lysed by 10 repetitions of 30-s vortexing at maximum speed and 1-min chilling on ice. After centrifugation at 15,000g for 10 min at 4 C, the supernatants containing the soluble cytosolic proteins were collected. The pellet containing the membrane-associated protein fraction was further treated with breaking buffer containing 2% SDS, and the suspension was centrifuged at 2500g for 5 min at 4 C. The supernatants containing membrane-associated proteins were collected. The protein concentration was determined using bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA) and thaumatin I purified from the crude sample of commercially available thaumatin powder as a standard. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and Westernblotting. SDS–PAGE was carried out using 13.5% polyacrylamide gel and the gels were stained with Coomassie Brilliant Blue R-250. For Westernblotting, protein samples were electrophoresed on SDS–PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Clear Blot membrane-P, ATTO Co., Tokyo, Japan) with semidry blotting technique using a HorizeBlot apparatus (ATTO) at 2 mA cm2 for 1 h. The PVDF membrane was blocked with TBS-T (25 mM Tris–HCl, pH 7.4, 140 mM NaCl, and 0.1% Tween-20) containing 1% bovine serum albumin (Wako Pure Chemical Industries). The blocked membrane was incubated with mouse anti-thaumatin sera (1:2000) [14] as the primary antibody, washed with TBS-T, and incubated with alkaline phosphatase-conjugated antimouse IgG antibody (1:20,000; Promega, Madison, WI, USA). After being washed, staining was achieved with NBT/BCIP stock solution (Roche Diagnostics, Mannheim, Germany). The secretion efficiency of recombinant thaumatin was assessed by densitometric analysis of the protein bands on SDS–PAGE and Western-blotting by using the Gel-Pro analyzer software Version 3.1 (Media Cybernetics, Silver Spring, MD, USA). N-terminal sequence analysis. The concentrated culture supernatant was loaded on a 13.5% SDS gel. After electrophoresis the proteins on the gel were transferred to a PVDF membrane and the membrane strip containing the objective band was cut off. This piece was subjected to N-terminal sequence analysis, which was performed in a gas-phase sequencer (Procise 492; Applied Biosystems) using the Edman degradation method.
Results and discussion Cloning of prepro-thaumatin cDNA The open reading frame of thaumatin II consists of three parts of DNA portion: (i) an N-terminal pre-sequence, (ii) a mature form of thaumatin, and (iii) a C-terminal prosequence [11]. In our previous study, the cDNA of the mature form of thaumatin I was cloned and its sequence was confirmed [12]. For the cloning of thaumatin cDNA containing prepro-sequence, the design of the primers including the pre-sequence and pro-sequence was based on the prepro-thaumatin II cDNA sequence determined by Edens et al. [11]. The product of PCR using the firststrand cDNAs as a template and using the ThPre-SfuI/ ThPro-XbaI primer set gave a 720-bp band (the size expected for prepro-thaumatin). After TA cloning, the clones were treated with StyI to separate the putative thau-
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matin II cDNA from the putative thaumatin I cDNA, as the thaumatin II cDNA contains a StyI site (CCWWGG), whereas thaumatin I cDNA does not [12]. The thaumatin I cDNA was used for the present study. From the DNA sequencing, the cloned cDNA was a prepro-thaumatin I cDNA, and both pre-sequence and pro-sequence of thaumatin I were identical to those of the prepro-thaumatin II cDNA [11] (Fig. 1). The N-terminal pre-sequence was deduced from the nucleotide sequence and its function is assumed to be compartmentalization of thaumatin in the plant. The presequence for the putative signal peptide with putative cleavage sites was further analyzed using SignalP 3.0 software [15] set at default values. The 22-amino acid N-terminal peptide was predicted as the secretion signal. Effect of pre-sequence of thaumatin The pPIC6a-TH plasmid contains cDNA encoding mature thaumatin I, a-factor secretion signal, and a spacer comprised of dibasic amino acids (-KR-) cleavable by the endogenous Kex2 protease. The pPIC6-preTH plasmid was constructed by inserting the cDNA encoding pre-thaumatin at the downstream of AOX1 promoter without the cDNA encoding a-factor secretion signal. The constructed pPIC6-preTH contains the ATG initiation codon of thaumatin cDNA flanked by the 5 0 AOX promoter without the Kozak consensus sequence (Fig. 2). Since it was not certain whether addition of the plant signal peptide at the N-terminal of the mature thaumatin would affect the secretion of thaumatin from P. pastoris, the efficiency of thaumatin secretion in transformants containing thaumatin mature gene with native signal peptide (preTH gene) was compared with that in transformants containing mature gene with a-factor secretion signal (aTH gene). Three colonies of each transformants were randomly chosen and cultured in 50 mL of medium in shaking 500-mL baffled flasks. The preTH and a-TH transformants did not differ in the growth rates significantly during 96-h induction phase (data not shown). Western-blotting analysis revealed the immunoreactive bands in the supernatant of the culture medium of preTH transformant, whereas the immunoreactive band was not detectable in a-TH transformant cultures (Fig. 3). The N-terminal amino acid sequence of the recombinant thaumatin secreted from preTH transformant was NH2-A-T-F-E-I-V-N-, which is identical to that of plant thaumatin I [9]. Thus, P. pastoris recognized and processed the pre-sequence of thaumatin through the secretion pathway. Since the pre-sequence of thaumatin does not contain a Kex2-like processing site, there should be another system in P. pastoris to process thaumatin. The mechanism for pre-sequence processing of thaumatin is similar in both T. daniellii Benth and P. pastoris. The threshold value (mean values ± SD) of sweetness of the recombinant thaumatin secreted from preTH transformant was 44.6 ± 12.1 nM, which is close to that of plant thaumatin I, i.e. 46.0 ± 23.0 nM.
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In our previous study, recombinant thaumatin was obtained from P. pastoris transformed with pPIC6a-TH expression construct using 3 L jar fermenter under the control of temperature, pH, and dissolved oxygen, but this thaumatin was incompletely processed at the N terminus [12]. In the expression system of Aspergillus awamori, the secretion signal sequence was also incompletely processed, and the recombinant thaumatin contained extra Lys and Arg residues [16]. On the other hand, recombinant thaumatin was secreted when N-terminal deleted thaumatin was fused to signal sequence derived from the host strain Bacillus subtilis [17] and Streptomyces lividans [18]. Therefore, N-terminal processing of thaumatin is important for thaumatin secretion and high yield of secreted thaumatin was achieved by addition of the pre-sequence of thaumatin at the N-terminal end.
Fig. 3. Comparison of secretion yields of recombinant thaumatin. (A) Twenty microliters of culture supernatant and 40 ng of thaumatin I from plant were loaded onto each lane of SDS–PAGE gels, and Westernblotting was performed using anti-thaumatin sera. All numbers shown above the picture are clone ID numbers. (B) Levels of extracellular thaumatin measured by densitometric analysis of the protein bands on Western-blotting. The error bars indicate standard deviations for three measurements. N.D. indicates not detected.
Effect of the C-terminal pro-sequence of thaumatin The pro-sequences are known to play a role in the transport of precursor protein [19,20], and some pro-sequences function as an intramolecular chaperone in protein folding [21]. The function of the thaumatin acidic pro-peptide is obscure, although a role in the folding of the basic thaumatin molecule has been suggested [18]. Here, pPIC6a-proTH and the pPIC6-preproTH plasmids were used to examine the effect of the pro-sequence of thaumatin on its production and secretion efficiency of the recombinant thaumatin. The pPIC6-preproTH contains the full-length ORF of the thaumatin I, which is located downstream of the AOX1 promoter, and the pPIC6a-proTH contains the a-factor sequence instead of the pre-sequence of thaumatin as a secretion signal (Fig. 2). Although the growths of P. pastoris transformants bearing pPIC6a-proTH (a-proTH) and the transformants bearing pPIC6-preproTH (preproTH) were similar to those of the a-TH and preTH transformants (data not shown), the a-proTH transformants secreted little amount of recombinant thaumatin and the preproTH transformants secreted abundant recombinant thaumatin (Fig. 3), suggesting that secretion was controlled by the signal peptide of thaumatin. The molecular weight of the recombinant thaumatin secreted from preproTH transformants was slightly higher than that secreted from preTH transformants (Fig. 3). This indicated that C-terminal pro-sequence would not be processed correctly and some additional amino acids still remain. The yield from preproTH transformants estimated by band density on Western-blotting was comparable with that from preTH transformants. According to Edens et al. deletion of C-terminal pro-
Fig. 4. Western blot analysis of intracellular samples. Exactly 20 lg of total protein from cell lysate (C) and of membrane fraction (M) were loaded for analysis. The concentration of the total protein was determined by BCA assay.
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sequence reduced the expression of thaumatin to 1/20 of that without deletion in S. cerevisiae [22]. However, our results show that the C-terminal pro-sequence does not affect secretion yield (Figs. 3 and 4). This difference might be due to the host strain and/or detection method.
useful on an industrial level. This expression system is easily scaled-up and provides sufficient samples for various purposes.
Some thaumatin molecules were retained intracellularly
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Secretion levels from a-TH and a-proTH transformants were extremely low. To investigate whether this low secretion is due to inefficient cleavage of a-factor sequence by Kex2 protease, the amount of intracellular thaumatin recovered from P. pastris was measured by Western-blotting. Immunoreactive bands were detected in the cell extracts of preTH and preproTH, whereas bands were not observed in those of a-TH and a-proTH transformants (Fig. 4). This indicates that a-TH and a-proTH could not produce recombinant thaumatin even in yeast cells and that the thaumatin pre-sequence improved the total production yield of thaumatin. The recombinant thaumatin molecule fused with a-factor sequence may be rapidly digested before secretion from yeast cells. Although the promoter strength, codon bias, and gene copy number may also influence the expression of thaumatin, the use of the same expression vector and the same thaumatin gene in all constructs rules these factors out as major causes of the very low expression of thaumatin by transformants expressing the protein with a-factor. Differing from a-TH and a-proTH, both preTH and preproTH produced detectable recombinant thaumatin in the membrane fraction (Fig. 4). The band mobility of thaumatin extracted from preTH transformants was the same as that secreted into the culture medium, which showed the same mobility of plant thaumatin I, whereas that of preproTH was slightly slower, meaning that the pre-sequence had been removed and the pro-sequence did not. These findings indicate that thaumatin were stored in some secretory apparatus of the cell even after the removal of pre-sequence from preTH and preproTH in the endoplasmic reticulum (ER). One of the reasons for retention of processed pre-thaumatin molecules in P. pastoris may be incorrect folding of molecule as this involves the formation of eight disulfide bonds. Overexpression of protein disulfide isomerase (PDI) and molecular chaperone (BiPA) increased thaumatin secretion in A. awamori [23,24], suggesting the importance of folding in the ER for effective secretion of thaumatin. In the P. pastoris expression system, the a-factor secretion signal did not promote recombinant thaumatin secretion, whereas mature thaumatin with the native signal of thaumatin was recognized and secreted by the P. pastoris translocation machinery. These findings demonstrate that the pre-sequence (secretion signal) of thaumatin must be present before secretion of the recombinant protein, and the pro-sequence of thaumatin is not apparently necessary for secretion. The method of producing recombinant thaumatin efficiently and processing it correctly in a microorganism is
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Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris q Nobuyuki Ide, Tetsuya Masuda, Naofumi Kitabatake
*
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan Received 3 September 2007 Available online 18 September 2007
Abstract Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the a-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNAencoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis. 2007 Elsevier Inc. All rights reserved. Keywords: Thaumatin; Sweet-tasting protein; Recombinant protein; Secretion signal; Pichia pastoris
The methylotropic yeast Pichia pastoris is widely used for production of various proteins [1–3]. This system allows proper folding and considerable posttranslational modification of the expressed protein molecule, and culture of cells at high density. Protein production can be maximized by controlling oxygen supply and some proteins can be secreted at high levels [1–3]. P. pastoris can produce heterologous proteins either intracellularly or extracellularly. The most commonly used secretion signal in the P. pastoris expression system is the a-factor prepro-leader sequence of Saccharomyces cerevisiae, which consists of a pre-region of 19 amino acids signal peptide and a pro-region of 60 hydrophilic amino acids [1,4]. The signal sequence can be removed by signal peptidase during the translocation process and the pro-region can be cleaved by the yeast Kex2
q Nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the Accession No. AB265690. * Corresponding author. Fax: +81 774 38 3743. E-mail address: [email protected] (N. Kitabatake).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.09.021
protease, resulting in the release of the mature and processed protein. Many other secretion signals are used in yeast expression systems, such as acid phosphatase signal peptide, yeast invertase signal sequence [1,2], and others [5,6]. Furthermore, some proteins have been expressed and secreted by P. pastoris using the native signal sequence of the protein concerned. For instance, the native signal sequence of phytohemagglutinin (PHA) permitted secretion of recombinant PHA, correct folding, and correct processing of the amino terminus [7]. When the a-factor sequence was used instead of the native signal sequence, the a-factor sequence at the amino terminus was retained in the secreted PHA molecule [7]. Thaumatin is a sweet-tasting plant protein extracted from the aril of the fruit of Thaumatococcus daniellii Benth [8]. The predominant thaumatins are thaumatin I and II, which consist of 207 amino acid residues [9] and contain eight disulfide bonds [10]. A comparison of the deduced amino acid sequence from cDNA with the protein sequence derived from amino acid sequencing suggests that the pre-
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cursor form contains both a 22-amino acid N-terminal presequence and 6-amino acid C-terminal pro-sequence [11] (Fig. 1). The function of the N-terminal pre-sequence is presumed to be the compartmentalization of thaumatin [11]. Although several reports show that C-terminal peptides are cleaved during protein maturation in plants, little is known about their precise function. The biological function of the acidic C-terminal extension of thaumatin also remains obscure. In our previous study [12], P. pastoris was transformed with the thaumatin cDNA fused with both the a-factor signal sequence and the Kex2 protease cleavage site, and secretion of thaumatin by the transformant was achieved using 3 L jar fermenter. However, N-terminal sequence analysis of the recombinant thaumatin revealed that two or three unexpected amino acid residues were still attached. This may be from deficiency of Kex2 protease, which cleaves the peptide bond besides the dibasic spacer sequence of Lys–Arg. Although these additional amino acid residues at N-terminal did not influence the threshold value of sweetness of thaumatin [12], they could affect its expression, secretion, and structure. In this study, we clarified the effects of the prepro-sequence of thaumatin on its secretion and production efficiency. Materials and methods Materials and culture media. Escherichia coli strain XL1-Blue supercompetent cells (Stratagene, La Jolla, CA, USA) were used as the host for
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DNA manipulations. P. pastoris wild type X-33 (Invitrogen, Carlsbad, CA, USA) was used for production of the recombinant thaumatin. P. pastoris was grown in YPD (1% yeast extract, 2% peptone, and 2% dextrose) or Buffered Minimal Glycerol (BMG) medium (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 · 105% biotin, and 1% glycerol) for preparation of inoculum. Buffered Minimal Methanol (BMM) medium (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 · 105% biotin, 0.5% methanol, and 1% casamino acid) was used for induction of heterologous protein synthesis. Blasticidin hydrochloride was from Kaken Pharmaceutical Co. Ltd. (Tokyo, Japan). Thaumatin I was purified from crude thaumatin powder as described previously [13]. All other chemicals were biochemical reagent grade. Cloning of the prepro-thaumatin cDNA. The mRNA was purified from the fruit of T. daniellii Benth, and the first-strand cDNA was synthesized as previously described [14]. The primers including the pre- and the proregion of thaumatin were constructed on the basis of the sequence deposited as the prepro-thaumatin II cDNA (GenBank Accession No. J01209) [11]. The 5 0 sense primer, ThPre-SfuI, was: 5 0 -GAATTC GAAATGGCCGCCACCACTTGCTTC-3 0 and the 3 0 antisense primer, ThPro-XbaI, was: 5 0 -GCTCTAGATTACTCGTCTTCAAGTTCAAGC3 0 . ThPre-SfuI primer includes both EcoRI (GAATTC) and SfuI (TTCGAA) restriction sites, and ThPro-XbaI includes an XbaI (TCTAGA) site. The PCR was performed using Pfu Turbo Hotstart DNA polymerase (Stratagene) with the following thermal cycling parameters: 1 cycle of 2 min at 94 C, followed by 30 cycles of 30 s at 94 C, 30 s at 55 C, 1 min at 72 C, with a final elongation step of 5 min at 72 C. The DNA sequence of the cloned thaumatin cDNA was confirmed using an ABI 310 DNA sequencer (Applied Biosystems, Warrington, UK). The resulting plasmid containing a prepro-thaumatin cDNA was named ‘‘pCR2.1-PPTh’’. Construction of the expression vector of thaumatin. To investigate the effects of introducing the prepro-sequence of thaumatin into the mature thaumatin cDNA on both production level and processing efficiency in P. pastoris, four different expression plasmids were constructed (Fig. 2).
Fig. 1. Nucleotide sequence of the precursor of thaumatin I. The nucleotide sequence of the precursor of thaumatin I was deposited in the DDBJ/EMBL/ GenBank databases under Accession No. AB265690. The amino acid sequence deduced from its nucleotide sequence is given in single-letter abbreviations under the DNA sequence. The N-terminal pre-sequence and C-terminal pro-sequence are boxed.
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(i) pPIC6a-TH: This plasmid was constructed as previously described [12]. Briefly, the mature thaumatin cDNA fragment was prepared by XhoI and XbaI digestion of pCR2.1-Th [12], and subsequently ligated between the XhoI and XbaI sites of pPIC6a vector which contained a-factor signal, 5 0 AOX promoter, and 3 0 AOX transcription termination sequence flanking the multicloning site. (ii) pPIC6a-proTH: The pro-thaumatin cDNA was amplified by PCR using ThXhoI and ThPro-XbaI primer set with pCR2.1-PPTh plasmid as a template. ThXhoI primer encodes the mature thaumatin region without its pre-sequence [12]. After TA cloning using pCR2.1-TOPO (Invitrogen), the pro-thaumatin cDNA fragment was prepared by XhoI and XbaI digestion. The resulting fragment was ligated between the XhoI and XbaI site of pPIC6a vector. (iii) pPIC6-preTH: The pre-thaumatin cDNA was amplified by PCR using the ThPre-SfuI and ThstopXbaI primer set with pCR2.1PPTh plasmid as a template. ThstopXbaI primer contains a stop codon without the coding region of pro-sequence of thaumatin [12]. After the TA cloning, the pre-thaumatin cDNA fragment was prepared by SfuI and XbaI digestion. To remove the a-factor sequence from pPIC6a vector, the vector was digested by SfuI and XbaI, and the approximately 3.3-kb fragment was gel-puri-
fied. We confirmed that the a-factor signal sequence had been removed from the resulting vector, pPIC6. The pre-thaumatin cDNA was then inserted into the SfuI and XbaI site of the pPIC6 vector. (iv) pPIC6-preproTH: The prepro-thaumatin cDNA fragment was prepared by SfuI and XbaI digestion of pCR2.1-PPTh plasmid. The prepared prepro-thaumatin cDNA was inserted into the SfuI and XbaI sites of pPIC6 vector.
Transformation of P. pastoris by electroporation. The constructed expression plasmids were digested with PmeI and the linearized plasmid was transformed into P. pastoris X-33 by electroporation as described previously [12]. The transformants were selected by plating on YPD plates containing 300 lg mL1 blasticidin. Expression of the recombinant thaumatin in a shaking-flask. Pichia pastoris transformants were inoculated into 50 mL of BMG medium in 500-mL baffled flasks. The cells were pre-cultured at 28 C to an OD600 of 2–6. The cells were harvested by centrifugation at 1500g for 5 min at 4 C, and resuspended in 50 mL of BMM medium and diluted up to an OD600 of 1.0. The cells were grown at 28 C with continuous shaking (250 rpm)
Fig. 2. Construction of the expression vectors. (A) The mature thaumatin gene (i) and the pro-thaumatin gene (ii) were ligated into the pPIC6a vector with a-factor signal sequence. The pre-thaumatin gene (iii) and the prepro-thaumatin gene (iv) were ligated into the pPIC6 vector without a-factor signal sequence. (B) The schematic representation of the expression cassettes and potential cleavage site are shown.
N. Ide et al. / Biochemical and Biophysical Research Communications 363 (2007) 708–714 for 96 h. Methanol was added to a final concentration of 1% (v/v) every 24 h during the culture period. The growths of transformants were monitored by measuring OD600 periodically. Aliquots of the culture supernatant were taken and protein production was checked by Western-blotting as described below. Separation of yeast proteins. The washed cell pellet (equivalent of 500 lL with an OD600 of 25) was resuspended in 500 lL of breaking buffer (50 mM sodium phosphate buffer, pH 7.0, 1 mM EDTA, 1 mM PMSF, and 5% glycerol) and mixed with an equal amount of glass beads. Cells were lysed by 10 repetitions of 30-s vortexing at maximum speed and 1-min chilling on ice. After centrifugation at 15,000g for 10 min at 4 C, the supernatants containing the soluble cytosolic proteins were collected. The pellet containing the membrane-associated protein fraction was further treated with breaking buffer containing 2% SDS, and the suspension was centrifuged at 2500g for 5 min at 4 C. The supernatants containing membrane-associated proteins were collected. The protein concentration was determined using bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA) and thaumatin I purified from the crude sample of commercially available thaumatin powder as a standard. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and Westernblotting. SDS–PAGE was carried out using 13.5% polyacrylamide gel and the gels were stained with Coomassie Brilliant Blue R-250. For Westernblotting, protein samples were electrophoresed on SDS–PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Clear Blot membrane-P, ATTO Co., Tokyo, Japan) with semidry blotting technique using a HorizeBlot apparatus (ATTO) at 2 mA cm2 for 1 h. The PVDF membrane was blocked with TBS-T (25 mM Tris–HCl, pH 7.4, 140 mM NaCl, and 0.1% Tween-20) containing 1% bovine serum albumin (Wako Pure Chemical Industries). The blocked membrane was incubated with mouse anti-thaumatin sera (1:2000) [14] as the primary antibody, washed with TBS-T, and incubated with alkaline phosphatase-conjugated antimouse IgG antibody (1:20,000; Promega, Madison, WI, USA). After being washed, staining was achieved with NBT/BCIP stock solution (Roche Diagnostics, Mannheim, Germany). The secretion efficiency of recombinant thaumatin was assessed by densitometric analysis of the protein bands on SDS–PAGE and Western-blotting by using the Gel-Pro analyzer software Version 3.1 (Media Cybernetics, Silver Spring, MD, USA). N-terminal sequence analysis. The concentrated culture supernatant was loaded on a 13.5% SDS gel. After electrophoresis the proteins on the gel were transferred to a PVDF membrane and the membrane strip containing the objective band was cut off. This piece was subjected to N-terminal sequence analysis, which was performed in a gas-phase sequencer (Procise 492; Applied Biosystems) using the Edman degradation method.
Results and discussion Cloning of prepro-thaumatin cDNA The open reading frame of thaumatin II consists of three parts of DNA portion: (i) an N-terminal pre-sequence, (ii) a mature form of thaumatin, and (iii) a C-terminal prosequence [11]. In our previous study, the cDNA of the mature form of thaumatin I was cloned and its sequence was confirmed [12]. For the cloning of thaumatin cDNA containing prepro-sequence, the design of the primers including the pre-sequence and pro-sequence was based on the prepro-thaumatin II cDNA sequence determined by Edens et al. [11]. The product of PCR using the firststrand cDNAs as a template and using the ThPre-SfuI/ ThPro-XbaI primer set gave a 720-bp band (the size expected for prepro-thaumatin). After TA cloning, the clones were treated with StyI to separate the putative thau-
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matin II cDNA from the putative thaumatin I cDNA, as the thaumatin II cDNA contains a StyI site (CCWWGG), whereas thaumatin I cDNA does not [12]. The thaumatin I cDNA was used for the present study. From the DNA sequencing, the cloned cDNA was a prepro-thaumatin I cDNA, and both pre-sequence and pro-sequence of thaumatin I were identical to those of the prepro-thaumatin II cDNA [11] (Fig. 1). The N-terminal pre-sequence was deduced from the nucleotide sequence and its function is assumed to be compartmentalization of thaumatin in the plant. The presequence for the putative signal peptide with putative cleavage sites was further analyzed using SignalP 3.0 software [15] set at default values. The 22-amino acid N-terminal peptide was predicted as the secretion signal. Effect of pre-sequence of thaumatin The pPIC6a-TH plasmid contains cDNA encoding mature thaumatin I, a-factor secretion signal, and a spacer comprised of dibasic amino acids (-KR-) cleavable by the endogenous Kex2 protease. The pPIC6-preTH plasmid was constructed by inserting the cDNA encoding pre-thaumatin at the downstream of AOX1 promoter without the cDNA encoding a-factor secretion signal. The constructed pPIC6-preTH contains the ATG initiation codon of thaumatin cDNA flanked by the 5 0 AOX promoter without the Kozak consensus sequence (Fig. 2). Since it was not certain whether addition of the plant signal peptide at the N-terminal of the mature thaumatin would affect the secretion of thaumatin from P. pastoris, the efficiency of thaumatin secretion in transformants containing thaumatin mature gene with native signal peptide (preTH gene) was compared with that in transformants containing mature gene with a-factor secretion signal (aTH gene). Three colonies of each transformants were randomly chosen and cultured in 50 mL of medium in shaking 500-mL baffled flasks. The preTH and a-TH transformants did not differ in the growth rates significantly during 96-h induction phase (data not shown). Western-blotting analysis revealed the immunoreactive bands in the supernatant of the culture medium of preTH transformant, whereas the immunoreactive band was not detectable in a-TH transformant cultures (Fig. 3). The N-terminal amino acid sequence of the recombinant thaumatin secreted from preTH transformant was NH2-A-T-F-E-I-V-N-, which is identical to that of plant thaumatin I [9]. Thus, P. pastoris recognized and processed the pre-sequence of thaumatin through the secretion pathway. Since the pre-sequence of thaumatin does not contain a Kex2-like processing site, there should be another system in P. pastoris to process thaumatin. The mechanism for pre-sequence processing of thaumatin is similar in both T. daniellii Benth and P. pastoris. The threshold value (mean values ± SD) of sweetness of the recombinant thaumatin secreted from preTH transformant was 44.6 ± 12.1 nM, which is close to that of plant thaumatin I, i.e. 46.0 ± 23.0 nM.
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In our previous study, recombinant thaumatin was obtained from P. pastoris transformed with pPIC6a-TH expression construct using 3 L jar fermenter under the control of temperature, pH, and dissolved oxygen, but this thaumatin was incompletely processed at the N terminus [12]. In the expression system of Aspergillus awamori, the secretion signal sequence was also incompletely processed, and the recombinant thaumatin contained extra Lys and Arg residues [16]. On the other hand, recombinant thaumatin was secreted when N-terminal deleted thaumatin was fused to signal sequence derived from the host strain Bacillus subtilis [17] and Streptomyces lividans [18]. Therefore, N-terminal processing of thaumatin is important for thaumatin secretion and high yield of secreted thaumatin was achieved by addition of the pre-sequence of thaumatin at the N-terminal end.
Fig. 3. Comparison of secretion yields of recombinant thaumatin. (A) Twenty microliters of culture supernatant and 40 ng of thaumatin I from plant were loaded onto each lane of SDS–PAGE gels, and Westernblotting was performed using anti-thaumatin sera. All numbers shown above the picture are clone ID numbers. (B) Levels of extracellular thaumatin measured by densitometric analysis of the protein bands on Western-blotting. The error bars indicate standard deviations for three measurements. N.D. indicates not detected.
Effect of the C-terminal pro-sequence of thaumatin The pro-sequences are known to play a role in the transport of precursor protein [19,20], and some pro-sequences function as an intramolecular chaperone in protein folding [21]. The function of the thaumatin acidic pro-peptide is obscure, although a role in the folding of the basic thaumatin molecule has been suggested [18]. Here, pPIC6a-proTH and the pPIC6-preproTH plasmids were used to examine the effect of the pro-sequence of thaumatin on its production and secretion efficiency of the recombinant thaumatin. The pPIC6-preproTH contains the full-length ORF of the thaumatin I, which is located downstream of the AOX1 promoter, and the pPIC6a-proTH contains the a-factor sequence instead of the pre-sequence of thaumatin as a secretion signal (Fig. 2). Although the growths of P. pastoris transformants bearing pPIC6a-proTH (a-proTH) and the transformants bearing pPIC6-preproTH (preproTH) were similar to those of the a-TH and preTH transformants (data not shown), the a-proTH transformants secreted little amount of recombinant thaumatin and the preproTH transformants secreted abundant recombinant thaumatin (Fig. 3), suggesting that secretion was controlled by the signal peptide of thaumatin. The molecular weight of the recombinant thaumatin secreted from preproTH transformants was slightly higher than that secreted from preTH transformants (Fig. 3). This indicated that C-terminal pro-sequence would not be processed correctly and some additional amino acids still remain. The yield from preproTH transformants estimated by band density on Western-blotting was comparable with that from preTH transformants. According to Edens et al. deletion of C-terminal pro-
Fig. 4. Western blot analysis of intracellular samples. Exactly 20 lg of total protein from cell lysate (C) and of membrane fraction (M) were loaded for analysis. The concentration of the total protein was determined by BCA assay.
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sequence reduced the expression of thaumatin to 1/20 of that without deletion in S. cerevisiae [22]. However, our results show that the C-terminal pro-sequence does not affect secretion yield (Figs. 3 and 4). This difference might be due to the host strain and/or detection method.
useful on an industrial level. This expression system is easily scaled-up and provides sufficient samples for various purposes.
Some thaumatin molecules were retained intracellularly
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Secretion levels from a-TH and a-proTH transformants were extremely low. To investigate whether this low secretion is due to inefficient cleavage of a-factor sequence by Kex2 protease, the amount of intracellular thaumatin recovered from P. pastris was measured by Western-blotting. Immunoreactive bands were detected in the cell extracts of preTH and preproTH, whereas bands were not observed in those of a-TH and a-proTH transformants (Fig. 4). This indicates that a-TH and a-proTH could not produce recombinant thaumatin even in yeast cells and that the thaumatin pre-sequence improved the total production yield of thaumatin. The recombinant thaumatin molecule fused with a-factor sequence may be rapidly digested before secretion from yeast cells. Although the promoter strength, codon bias, and gene copy number may also influence the expression of thaumatin, the use of the same expression vector and the same thaumatin gene in all constructs rules these factors out as major causes of the very low expression of thaumatin by transformants expressing the protein with a-factor. Differing from a-TH and a-proTH, both preTH and preproTH produced detectable recombinant thaumatin in the membrane fraction (Fig. 4). The band mobility of thaumatin extracted from preTH transformants was the same as that secreted into the culture medium, which showed the same mobility of plant thaumatin I, whereas that of preproTH was slightly slower, meaning that the pre-sequence had been removed and the pro-sequence did not. These findings indicate that thaumatin were stored in some secretory apparatus of the cell even after the removal of pre-sequence from preTH and preproTH in the endoplasmic reticulum (ER). One of the reasons for retention of processed pre-thaumatin molecules in P. pastoris may be incorrect folding of molecule as this involves the formation of eight disulfide bonds. Overexpression of protein disulfide isomerase (PDI) and molecular chaperone (BiPA) increased thaumatin secretion in A. awamori [23,24], suggesting the importance of folding in the ER for effective secretion of thaumatin. In the P. pastoris expression system, the a-factor secretion signal did not promote recombinant thaumatin secretion, whereas mature thaumatin with the native signal of thaumatin was recognized and secreted by the P. pastoris translocation machinery. These findings demonstrate that the pre-sequence (secretion signal) of thaumatin must be present before secretion of the recombinant protein, and the pro-sequence of thaumatin is not apparently necessary for secretion. The method of producing recombinant thaumatin efficiently and processing it correctly in a microorganism is
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