Biotechnol. J. 2006, 1, 1085–1092
DOI 10.1002/biot.200600126
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Research Article
Endoplasmic reticulum-retention C-terminal sequence enhances production of an 11S seed globulin from Amaranthus hypochondriacus in Pichia pastoris Sergio Medina-Godoy1,2, Angel Valdez-Ortiz1,3, María Elena Valverde1 and Octavio Paredes-López1 1Departamento
de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Irapuato, Guanajuato, México 2Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional, CIIDIR-IPN, Guasave, Sinaloa, México 3Programa Regional del Noroeste para el Doctorado en Biotecnología, Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, México
The methylotrophic yeast Pichia pastoris was used to express an 11S seed globulin from Amaranthus hypochondriacus. Three different plasmids were tested for expression of amarantin. One of them, which included the untranslated regions (UTR) of the full cDNA, failed to express the amarantin under tested conditions, whereas the other plasmids, one without UTR and the other similar but including the endoplasmic reticulum-retention signal KDEL, were able to express the proamarantin in P. pastoris. After 48 h of induction, KDEL-proamarantin had accumulated quite significantly compared to unmodified proamarantin. Different solubilization patterns were also obtained from both proamarantin versions; only soluble protein was obtained from the system that included the KDEL retrieval signal. Protein fractionation was carried out by differential precipitation with ammonium sulfate, and proamarantin purification was performed using an HPLC ion exchange column. The endoplasmic reticulum-retention C-terminal sequence (KDEL retrieval signal), not commonly employed in this heterologous expression system, can therefore be used to enhance accumulation of recalcitrant protein in P. pastoris. The results obtained here also suggest that this expression system is suitable for expression and evaluation of engineered seed globulin proteins.
Received 12 July 2006 Revised 16 August 2006 Accepted 21 August 2006
Keywords: Amarantin · Heterologous expression · KDEL retrieval signal · Recombinant amarantin · Storage protein
1
Introduction
Globulin seed storage proteins have been proposed as an excellent candidate to be incorporated into major crops to impact both functional and nutraceutical properties [1]. Amarantin, an 11S seed globulin, contains a good balance of essential amino acids, remarkable heat stability and
Correspondence: Dr. Octavio Paredes-López, Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Apdo. Postal 629, Irapuato, Gto 36500, México E-mail:
[email protected] Fax: +52-462-624-5996 Abbreviation: UTR, untranslated region
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emulsifying properties [2]; these features make this protein a suitable model to generate transgenic crops [3]. Moreover, this type of proteins is susceptible to protein engineering to further improve some characteristics, such as those cited above. Amarantin is synthesized as a single polypeptide, named preproamarantin, consisting of acidic and basic polypeptides with a single transit sequence; the signal sequence is removed cotranslationally. The resultant proamarantin subunits (of ca. 50 kDa) self-assemble into trimers of ca. 150 kDa in the endoplasmic reticulum (ER) [4–6]. As a trimer, proamarantin migrates to protein storage vacuoles via the endomembrane system, where it is processed to its mature form by cleaving at a highly conserved sequence by a specific vacuolar processing en-
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zyme, an asparaginyl endopeptidase, while the acidic and basic polypeptides generated remain joined by a disulfide bond [7–9]. Research at our laboratory has been focused on the recombinant expression of amarantin in different systems. A His-tagged version was expressed and accumulated in E. coli as a trimer, and proamarantin was purified by immobilized metal ion affinity chromatography (IMAC) [6]. This system does not generate post-translational modifications required to obtain the proper mature form. Expression in maize plants was also performed, resulting in important increases of seed protein contents [3]. Moreover, Sinagawa-García et al. [10] concluded that no allergenic reactions were generated with transgenic maize. A modified version of amarantin was expressed in seeds of transgenic tobacco, resulting in a proper accumulation pattern [11]. Using protein engineering, further characteristics could be incorporated to this interesting protein, such as biopeptides or modified amino acid sequence, to enhance functional and nutraceutical properties; for this reason, protein expression system should be carefully selected. A single-cell eukaryotic system may be a good option to carry out protein engineering on proamarantin, since it shares both easy manipulation and short production times and has high quality protein controls. In this sense, the methylotrophic yeast Pichia pastoris is a highly successful system for expression of heterologous proteins [12–15]. It is also well suited for protein production that requires post-transcriptional modifications, such as disulfide bond formation [13]. For these reasons, we expected to be able to express proamarantin at a high level in P. pastoris, and that, once this goal is reached, engineered proamarantins could be evaluated before introduction into a plant model. Even when excellent results have been reported in this expression system previously [16], alternative strategies to improve accumulation of recombinant protein should be assayed. The tetrapeptide KDEL is commonly found in ER soluble proteins; it contributes to their localization and long life in plants [17–20]. Callewaert et al. [21] reported the N-glycan engineering in P. pastoris by expression of a HDEL-tagged mannosidase to properly express a therapeutic glycoprotein. This retrieval signal could be assayed to produce proteins, like 11S seed globulins, in P. pastoris, which due to their nature are transported to vacuoles, and may be susceptible to endogenous vacuolar proteases. We report here the accumulation of both native and modified amarantin (containing a KDEL retrieval signal at its C terminus) in P. pastoris. Differences in both levels and accumulation patterns are described.
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2 2.1
Materials and methods Bacterial and yeast strains
E. coli TOP10F´ (Invitrogen, Carlsbad, CA) was used for routine plasmid transformation and propagation. P. pastoris strains X-33 and KM71H (Invitrogen) were used for expression of proamarantin. 2.2 Plasmid
The plasmid pSPORT1-AMAR, which contains the full preproamarantin cDNA (GenBank accession no. X82121), was used for further constructions and as a source of DNA template for PCR. The full preproamarantin cDNA includes the 5´-untranslated region (UTR), a 5´ signal peptide sequence, the amarantin coding region, and the 3´UTR [22]. The expression plasmid pPICZB (Invitrogen) was used for proamarantin expression in P. pastoris. This plasmid contains the Sh ble gene, which confers zeocin resistance to both E. coli and P. pastoris. Expression of proamarantin was under the control of the aox1 promoter induced with methanol. 2.3 Construction of proamarantin expression plasmids
The expression plasmid pPICZFULL-AMAR (Fig. 1a) was constructed by insertion of the full amarantin cDNA, excised from pSPORT1-AMAR, into pPICZB. The 5´ region of amarantin gene was located downstream of the aox1 promoter. Expression plasmid pPICZPPAMAR was constructed to evaluate the effect of the UTRs over proamarantin synthesis. Plasmid pPICZPPAMAR (Fig. 1b) is the same as pPICZFULL-AMAR except for the absence of the UTRs; it only contains the signal peptide sequence and the amarantin coding region. On the other hand, pPICZPPAMARKDEL (Fig. 1c) is the same as pPICZPPAMAR, except for the addition of the ER retention signal KDEL at its C terminus, which has been documented as a positive factor for heterologous protein expression in plants [18, 20, 23]. To generate pPICZPPAMAR, specific oligonucleotide primers were designed to amplify only the amarantin coding sequence by PCR, eliminating the UTRs from full amarantin cDNA. In addition, a 5´ EcoRI site and a 3´ XbaI site were incorporated into amarantin of 1.44-kb length. The sequences of these primers were as follows: forward 5´CCAGAATTCAACAAAAATGG-3´ (EcoRI restriction site, italics; start codon, underlined); and reverse 5´-TTTATACTCTAGAACTTAGGCAATGC-3´ (XbaI restriction site, italics; stop codon, underlined). For construction of pPICZPPAMAR-KDEL, the same forward oligonucleotide used with pPICZPPAMAR was employed, using the reverse oligonucleotide to introduce KDEL sequence. The sequence of this primer was as follows: Reverse 5´-TCTA-
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Figure 1. Proamarantin expression cassettes for P. pastoris. (A) pPICZFULL-AMAR, (B) pPICZPPAMAR, (C) pPICPPAMAR-KDEL. Paox1: aox1 promoter; Taox1: aox1 terminator; black box: UTR regions; Amar ORF: amarantin open reading frame; KDEL: ER-retention C-terminal sequence.
GACTTACAGTTCATCTTTGCTGATTTTCCTTCGGTACT-3´ (XbaI restriction site, italics; stop codon, underlined and bold; KDEL codons, italics and underlined). PCR products were quantified and digested overnight at 37°C with EcoRI and XbaI restriction enzymes. The digested cDNA was ligated into the pPICZB plasmid (previously digested with EcoRI and XbaI). E. coli TOP10F´ was transformed by the calcium chloride method [24] and transformants were selected on low salt LB plates containing zeocin at 25 µg/mL. Zeocin-resistant E. coli clones were analyzed by PCR with specific oligonucleotide primers designed for the amplification of the aox1 gene in pPICZB. Positive clones were further analyzed by restriction endonuclease analyses. 2.4 Transformation of yeast cells
P. pastoris strains X-33 and KM71H were transformed in the Cell Porator System (Gibco-BRL, Life Technologies Inc., Gaithersburg, MD, USA) according to the instruction manual using the following parameters: 400 V, low resistance and 10 µF. 2.5 Expression and detection of proamarantin in P. pastoris
Single colonies of P. pastoris harboring amarantin cDNA were used to inoculate 10 mL of MGY medium [1.34% yeast nitrogen base (YNB) with ammonium sulfate, amino acid free, 1% glycerol and 4 µg/ml biotin] in 125 mL Erlenmeyer flasks. Cultures were shaken at 250 rpm in an orbital incubator shaker at 30°C. After 16 h, cells were harvested by centrifugation at 3000 × g, for 10 min at room temperature. Cell pellets were resuspended in minimal medium (MM) (1.34% YNB, 4 µg/ml biotin) containing 0.5% methanol to a final OD600 nm of 1.0; then, cultures were placed back in the incubator and shaken again.
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Methanol was added every 24 h to a final concentration of 0.5%; after this point, 1 mL of each expression culture was transferred to a 1.5 mL centrifuge tube at 0, 24, 48, and 72 h, to determine the optimal time for proamarantin expression after induction. Sample cells were harvested by centrifugation at 12 000 × g in a tabletop microcentrifuge for 3 min at room temperature. Supernatants were discarded and cell pellets were stored at –80°C for protein assays. Total protein extractions were carried out according to Yaffe and Schatz [25]. This method consists of an alkaline cell disruption followed by protein precipitation with TCA. SDS-PAGE and Western blot analyses were carried out to assess protein expression. Rabbit polyclonal antibodies were raised against the whole amarantin subunit purified from amaranth seeds. As negative controls, both pPICZB-transformed strains X-33 and KM71H were cultured with induction and sampled as described for those transformed with the expression plasmids. In addition, P. pastoris strains transformed with pPICZPPAMAR or pPICZPPAMAR-KDEL were employed for a preparative expression assay. One-hundred-milliliter aliquots of MGY medium were inoculated with a single colony. Cultures were incubated at 30°C with agitation at 250 rpm for 24 h. Cells were harvested by centrifugation at 3000 × g for 10 min at room temperature. Cells were resuspended in MM expression medium to an OD600 nm of 1.0; then, cells were harvested after 48 h of induction by centrifugation as described above. The supernatants were decanted and the pellets were kept for cell disruption. Protein extraction was performed using the Yeast Protein Extraction Reagent (Y-PER®, Pierce, Rockford, IL, USA), which is designed to extract soluble protein from yeast. Typically, 1 g wet cell pellet was resuspended in 2.5 mL extraction reagent; the mixture was agitated at 40°C for 20 min at 200 rpm. Cell debris were pelleted by centrifugation at 14 000 × g for 20 min and supernatants containing soluble proteins were stored until assayed. Soluble protein fractions were subjected to SDS-PAGE according to the procedure of Laemmli [26]. The electrophoresed proteins were visualized with CBB R-250 and proamarantin was detected by Western blot analysis after transferring proteins to a PVDF membrane (Bio-Rad, Hercules, CA, USA) and exposing it first to rabbit polyclonal anti-amarantin IgGs and then to goat anti-rabbit IgG (H+L) conjugated to alkaline phosphatase (Bio-Rad) [27]. Image documentation (gels and membranes analyses of two independent experiments, n=2) was obtained using a Gel-Doc system (Bio-Rad), and densitometric determinations were performed with the Quantity One Software (Bio-Rad).
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2.6 Purification of recombinant proamarantin
To exchange buffer, an aliquot of the soluble protein fraction was applied to a HiTrap™ desalting column (Amersham Biosciences Biotech, Uppsala, Sweden), previously equilibrated with buffer A (20 mM Tris-HCl, pH 8.0), and proamarantin was differentially precipitated by the addition of ammonium sulfate at four distinct saturation ranges: 0–20%, 20–30%, 30–40% and 40–60%; fractions harboring proamarantin were applied to a HiTrap™ desalting column previously equilibrated with buffer A. After buffer exchange, protein was loaded onto an HPLC ion exchange column (Bio-Gel TSK-DEAE-5-PW, 75 × 7.5 mm, Bio-Rad) equilibrated with buffer A. The column was eluted with buffer A until an OD280 nm below 0.01 was detected in the elute; proteins were then eluted with a linear gradient of 0–0.5 M NaCl in buffer A for 30 min at 1 mL/min; protein elution absorbance (280 nm) was registered. Different peaks were analyzed by Western blot to determine the presence of proamarantin. The peak containing proamarantin was applied to an HPLC gel filtration column (Bio-Sil TSK 250, 300 × 7.5 mm, Bio-Rad), equilibrated with buffer A supplemented with 250 mM NaCl. Gel filtration standards (Bio-Rad) and blue dextran 2000 (Amersham Biosciences Biotech) were employed to produce a calibration curve to assess the molecular weight. Proamarantin was identified analyzing the protein peak by SDS-PAGE. 2.7
Protein measurement
Protein concentrations were measured in the crude extracts and in fractionated samples using the Bradford reagent according to the Sigma protocol (Sigma Chemical Company, St. Louis, MO, USA), or the Pierce BCA Protein Assay, a bicinchoninic acid method (Pierce). In both procedures, BSA (Sigma) was used as a protein standard. 2.8 Proamarantin control
A His-tagged proamarantin expressed and purified from E. coli [6] was used as a proamarantin loading control in electrophoretic analysis.
3
Results and discussion
3.1 Expression of amarantin in P. pastoris
To determine if P. pastoris was able to express the 11S globulin from amaranth, different plasmid versions harboring full cDNA were used to transform the yeast strains X-33 and KM71H. We were not able to detect proamarantin by SDS-PAGE or Western blot analysis in PCR-positive clones of both yeast strains harboring the pPICZFULLAMAR; however, those harboring the pPICZPPAMAR
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plasmid successfully expressed the proamarantin. This result could be due to the presence of UTRs in the full amarantin cDNA, present in the pPICZFULL-AMAR plasmid, which might have an adverse influence on mRNA stability and/or translation, negatively influencing amarantin production by P. pastoris [28]. These transformed yeast clones were, therefore, not further analyzed. These findings are similar to those obtained for a soybean 11S globulin in S. cerevisiae [29, 30]. Total proteins were visualized by staining with CBB, and no differences were detected between strains (Fig. 2a); however, as shown in Fig. 2b, a protein of ~52 kDa was detected by anti-amarantin antibodies when a duplicate of the SDS-PAGE shown in Fig. 2a was transferred and assayed by Western blot. This protein was detected in X-33 and KM71H strains (Fig. 2b, lanes 3 and 4), both of which harbored the pPICZPPAMAR containing the full coding region of amarantin. Anti-amarantin antibodies did not show cross-reaction against P. pastoris proteins (Fig. 2b, lanes 1 and 2). To evaluate if proamarantin was secreted by P. pastoris, the medium culture was concentrated (100×) and Western blot performed to assess the presence of proamarantin; since no positive evidence was detected (data not shown), we assume that proamarantin was expressed only in an intracellular fashion. The optimal expression time of proamarantin was after 48 h of induction (ca. 25 mg/L), as shown in Fig. 2c; after this point protein accumulation decreased, maybe due to proteolytic activity. Although some researchers have reported that strains X-33 and KM71H have different capabilities to accumulate the same protein [16], the results of the present work showed that there was no significant difference in proamarantin accumulation between both Pichia strains. The accumulation level was comparable to that described previously for 11S storage protein expression in E. coli [31–33] and in S. cerevisiae [29, 30], but lower than that reported in a previous study by our team in E. coli [6], or of other proteins expressed with P. pastoris system ([16] and references therein). According to seed storage globulin biogenesis, these proteins are translocated to storage vacuole, where the storage globulins are processed and finally accumulated [34, 35]. The seed storage globulins expressed in yeast could have the same fate. A soybean 11S seed globulin has been shown to accumulate in yeast vacuoles as an insoluble form with a native conformation after an in vitro refolding process [30]. Proteolytic activity is present in yeast vacuoles, which could be detrimental to the level of recombinant proteins accumulated into this organelle; thus, to increase proamarantin accumulation, we decided to introduce the ERretention C-terminal sequence (KDEL) at the C terminus of the recombinant proamarantin. Two independent clones harboring pPICZPPAMAR-KDEL were evaluated. The accumulation profiles of the recombinant protein expressed in P. pastoris under the same condition of both
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has been demonstrated that natural and artificial reticuloplasmins share the same distribution into ER [37]. Proamarantin-KDEL may have a reticuloplasmin-like behavior, so that the colocalization with ER chaperons may increase its stability and long life; similar behavior was observed for a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER in leaf tissue of rice [37, 38]. This KDEL signal retrieval has been also proposed to serve in maintaining a pool of inactive protein that can rapidly be deployed to its site of activity when needed [39]. Because one of the most desirable characteristics of a recombinant protein is its production as a soluble protein, the solubilization pattern of both proamarantin versions was assessed. Analysis of the soluble protein fractions by
Figure 2. (A) SDS-PAGE and (B) Western blot analysis of cell extracts from P. pastoris cells. In each lane, approximately equal amounts of total cell protein were loaded. M, Molecular weight marker; lane 1, X-33 wild type; lane 2, KM71H wild type; lane 3, X-33::pPICZPPAMAR; lane 4, KM71H::pPICZPPAMAR; c1 & c2, proamarantin purified as loading control. Proamarantin (arrow) was detected with anti-amarantin antibodies. Time (in hours) after inductions are annotated at the top of (A) and (B). (C) Densitometry analysis of proamarantin detected in both strains (mean of values ± SEM, n=2); white squares represent KM71H::pPICZPPAMAR and black squares represent X-33::pPICZPPAMAR.
modified and unmodified versions are shown in Fig. 3a and b. Densitometric determinations showed increases in accumulation level of proamarantin carrying the KDEL retrieval signal in its C terminus, which was detectable from the beginning of induction and kept increasing up to 72 h of induction (Fig. 3c). Significant differences were detected after 48 h of inductions between modified and unmodified proamarantins (p=0.05), but not between clones harboring the pPICZPPAMAR-KDEL vector. This phenomenon has also been documented for recombinant proteins in plants, such as the single-chain Fv fragment [18] and phytase [20]. Introduction of KDEL retrieval signal into the C terminus of proamarantin may result in the generation of artificial reticuloplasmins (the term referring to soluble proteins that perform their functions in the lumen of the ER, [36]). These proteins are characterized by very long life, and their compartment of turnover is not known. It
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Figure 3. (A) SDS-PAGE and (B) Western blot analysis of cell extracts from P. pastoris cells. In each lane, approximately equal amounts of total cell protein were loaded. M, Molecular weight marker; lane 1, X-33 wild type; lane 2, X-33::pPICZPPAMAR; lane 3, X-33::pPICZPPAMAR-KDEL (clone 1); lane 4, X-33::pPICZPPAMAR-KDEL (clone 2). Proamarantin (arrow) was detected with anti-amarantin antibodies. (C) Densitometry analysis of proamarantin detected by anti-amarantin antibodies (values ± SEM, n=2). Square filled box represents X-33::pPICZPPAMAR; black box represents clone 1 of X-33::pPICZPPAMAR-KDEL, and white box represents clone 2 of X-33::pPICZPPAMAR-KDEL.
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might prevent ER-associated degradation of unfolded immunotoxin, resulting in a higher secretion level. This protein expression system may become a better alternative for expression of globulins than previously reported methods [6, 11], because it combines easy manipulation and short microbial expression time with an eukaryotic machinery to carry out post-translation modification, i.e., model plants; thus, modified proteins with new functional properties can be produced. 3.2 Purification of proamarantin expressed in P. pastoris
Figure 4. (A) SDS-PAGE and (B) Western blot analyses of soluble protein fractions produced by induced P. pastoris cells harboring a proamarantin expression cassette. M, Molecular weight marker; lane 1, X-33 wild type; lane 2, X-33::pPICZPPAMAR; lane 3, X-33::pPICZPPAMAR-KDEL. Each lane contains 10 μg protein.
SDS-PAGE and Western blot (Fig. 4a, b) showed that only proamarantin-KDEL proteins were detected in a soluble form (Fig. 4b, lane 3), which agrees with previous reports on recombinant proglycinin, an 11S seed globulin expressed in yeast [30]. These authors found ca. 95% of proglycinin as insoluble protein; as can be seen in Fig. 4a and b, line 2, wild-type proamarantin was not detected in this fraction. These findings of soluble protein combined with its accumulation enhances our proposal that the KDEL retrieval signal in P. pastoris is a good strategy for improving protein production. It is possible that even if proamarantin is expressed in P. pastoris, the unmodified version could be transported to lytic vacuoles, like proglycinin [30], where vacuolar environment could induce protein unfolding, which the proteolytic susceptibility of proamarantin may result in reducing its accumulation. On the other hand, proamarantinKDEL may be confined and accumulated in the ER, thus avoiding proteolytic attack. This strategy therefore has become an excellent strategy to express recombinant proteins in plants [18, 20], and has been applied to overexpress an 11S seed globulin in tobacco leaves, resulting in a higher accumulation [23]. The retrieval signal has been employed in P. pastoris to express enzymes or chaperon proteins to enhance their expression; expression of an 1,2α-D-mannosidase, including HDEL signal retrieval sequences, allowed the production of a recombinant therapeutic glycoprotein with human-compatible N-glycans added [21]. Expression of the major ER protein folding chaperone BiP/Kar2p resulted in higher levels of secretion of a bivalent anti-CD3 immunotoxin [39]. These latter authors concluded that overexpression of the chaperone
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Only the soluble fraction from yeast transformed with pPICZPPAMAR-KDEL was used for purification. After buffer exchange, proteins were differentially precipitated with ammonium sulfate; recombinant proamarantin was precipitated in two different saturation ranges: 0–20% and 30–40% as can be seen in the gel shown in Fig. 5a and by immunological detection in Fig. 5b. These precipitation profiles could be due to different association states. Proamarantin was one of the major proteins detected in the 0–20% fraction (Fig. 5a), for that reason it was used for further purifications steps. Figure. 6a shows a typical HPLC ion exchange chromatogram. Recombinant proamarantin was eluted as a single peak with ca. 250 mM NaCl (22.044 min); this result agrees with that previously reported [22], where amarantin was purified from seed. The protein peak was analyzed by HPLC gel filtration and the chromatogram is shown in Fig. 6b. Proamarantin eluted at two different times: one species eluted at 6.166 min
Figure 5. (A) SDS-PAGE and (B) Western blot analyses differential of precipitation fractions obtained by the addition of ammonium sulfate. In each lane 1/10 of each sample was loaded. M, Molecular weight marker; lane 1, X-33 wild type; lane 2, X-33::pPICZPPAMAR-KDEL. The protein pellet obtained from each saturation range was resuspended in half of the original volume of buffer A. Ammonium sulfate saturation ranges (%) are annotated at the top of each panel.
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Figure 6. Recombinant proamarantin purification. (A) HPLC ion exchange chromatogram (A280 nm) of protein precipitated at 0–20% saturation with ammonium sulfate, proamarantin was detected in peak at 22.04 min. (B) HPLC gel filtration chromatogram (A280 nm) of 22.04-min peak, obtained by HPLC ion exchange chromatogram. (C) SDS-PAGE of the two different peaks obtained by HPLC gel filtration. M, Molecular weight marker; lane 1, protein eluted at 6.166 min; lane 2, protein eluted at 7.194 min. Gel stained with CBB.
which may correspond to a trimer of proamarantin (ca. 150 kDa), and the other at 7.194 min, which may correspond to a monomer of ca. 52 kDa; however, further structural characterization needs to be performed. As can be seen in Fig. 6c, most of the proamarantin have a molecular mass of ca. 150 kDa, while the non-associated form is less abundant. This phenomenon may be due to low salt concentration used during HPLC gel filtration (0.2 M NaCl). This type of protein undergoes a reversible association-disassociation process as a function of salt concentration. 4
Concluding remarks
necessary for further characterization, using methods such as circular dichroism and fluorescence spectroscopy. The level of accumulation and conformational state of the protein are important considerations in protein engineering studies aimed at improving the functional and nutraceutical properties of mature amarantin.
S.M.G. thanks CONACYT-México for a scholarship to carry out this study. We also thank Yolanda Rodríguez from CINVESTAV-IPN by her help in performing HPLC chromatography.
In the present work, we were able to: (i) express the major seed storage protein of amaranth, the 11S globulin termed amarantin, in P. pastoris, and (ii) evaluate the effect of an ER-retention C-terminal sequence (KDEL) at its C-terminal amino acid sequence. The KDEL retrieval signal was able to significantly increase the accumulation of proamarantin, and these findings may thus help in evaluating the use of this retrieval signal when using P. pastoris as an expression system. Proamarantin purification is also
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