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Heterologous expression of a hydrophobin HFB1 and evaluation of its contribution to producing stable foam
Accepted Manuscript Heterologous expression of a hydrophobin HFB1 and evaluation of its contribution to producing stable foam Azadeh Lohrasbi-Nejad, Masoud Torkzadeh-Mahani, Saman Hosseinkhani PII:
S1046-5928(15)30075-9
DOI:
10.1016/j.pep.2015.09.025
Reference:
YPREP 4800
To appear in:
Protein Expression and Purification
Received Date: 27 April 2015 Revised Date:
22 September 2015
Accepted Date: 25 September 2015
Please cite this article as: A. Lohrasbi-Nejad, M. Torkzadeh-Mahani, S. Hosseinkhani, Heterologous expression of a hydrophobin HFB1 and evaluation of its contribution to producing stable foam, Protein Expression and Purification (2015), doi: 10.1016/j.pep.2015.09.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Heterologous expression of a hydrophobin HFB1 and evaluation of its contribution to producing stable foam
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Azadeh Lohrasbi-Nejada, Masoud Torkzadeh-Mahanib, Saman Hosseinkhania* a
Department of Biochemistry, Faculty of Biological Sciences, TarbiatModares University, Tehran, Iran Department of Biotechnology, Institute of Science, High Technology and Environmental Science, Graduate University of Advanced Technology, Kerman, Iran. * Corresponding author. Tel.: +98 21 82884407; fax: +98 21 82884484. E-mail address: [email protected] (S. Hosseinkhani).
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Hydrophobins are small secreted proteins belong to filamentous fungi. These proteins possess a unique ability to self-assemble at air/water interfaces. Hydrophobins have a broad range of biotechnological applications such as stabilizing emulsions and foams, immobilizing proteins on a surface, designing biosensors, affinity tag for protein purification, and drug delivery. We have successfully expressed HFB1 from Trichoderma reesei belonged to class II of hydrophobins in Pichia pastoris. The recombinant gene was under the control of the methanol-inducible AOX1 promoter (alcohol oxidase 1) in the pPICZAα vector. The amount of secreted HFB1 was increased in 90-hours using methanol induction. The recombinant HFB1 was purified based on the presence of His-tag and foam formation. Furthermore, HFB1 was able to produce macro and micro stable air bubbles in the liquid due to the presence of hydrophobic patches on its surface. The liquid medium containing HFB1 becomes turbid after shaking, and then the stable bubbles are formed and remained for three weeks.
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Keywords: Hydrophobin; HFB1; heterologous expression; Pichia pastoris
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Introduction Hydrophobins are small secreted proteins with a surface active, which are produced by filamentous fungi [1]. They consist of about 100 amino acids including 8 cysteine residues in conserved position, which can form 4 intracellular disulfide bonds [2]. Self-assembly in hydrophobic/hydrophilic (such as air/water) interfaces is one of the most important qualities of these proteins [3]. Their role in growth and development of fungi is of utmost importance. Hydrophobins help fungal hyphae to emerge from water by reducing the surface tension of water when secreted into the environment [4-5]. Moreover, they participate in coverage of cell wall fungi and subsequently lead to reverse hydrophobicity. These proteins facilitate appropriate gas exchange, aerial dispersal of spores and connection of hyphae to hydrophobic surfaces by aggregation on the surface of some structures such as fruiting bodies, spores, and hyphae, respectively [6-7]. Based on hydropathy profile, biophysical characteristics, and amino acid sequence similarity, hydrophobins are divided into two groups: class I and class II [8]. All hydrophobins can form amphipathic membranes. Membranes constructed by class I hydrophobins are resistant to dissolution and dissolved in formic acid or trifluoroacetic acid. But membranes created by class II hydrophobins are dissolved in 2% hot SDS or 60% ethanol [9]. Hydrophobins are useful in some branches of biotechnology such as stabilizing enzyme [10-11], biosensor and electrode construction [12], tissue engineering, drug delivery [13], foam stabilizer [14-15], coating techniques [16], and fusion tag for the purification of recombinant proteins [17]. There are many potential practical applications for this protein in technology; however, mass production with low cost is a major obstacle in this regard. Using a proper host such as Pichia pastoris could help surmount this problem. Protein production in Pichia pastoris is preferred among other hosts due to its wide range of post-translational modifications and production of secretory protein [18]. In this study, we have focused on the recombinant production of HFB1 from Trichoderma reesei belong to the class II of hydrophobins. The native form of HFB1 consists of 97 amino acids including 16 amino acids belong to the secretory signal sequence, 6 amino acids belong to the prepropeptide, and 75 amino acids belong to the main structural sequence. The final 7.5 KDa protein is delivered after elimination of the first two parts from N-terminal through intracellular processing. Moreover, we transformed hfb1 gene without a signal and prepropeptide sequences in Pichia pastoris and investigated the secretory expression of HFB1. We have also compared the efficiency of different methods on the yield of purification and finally presented a high-throughput method for purification of HFB1 with high purity and high concentration.
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2. Materials and methods
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2.1. Strains, vectors, and reagents
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Pichia pastoris strain X-33, Escherichia coli strain DH5α was obtained from our laboratory. All restriction enzymes, T4 DNA ligase, and alkaline phosphatase were procured from Thermo Fisher Scientific, Inc. DNA extraction and plasmid purification kits were also supplied by Bioneer, Inc. The entire coding region of hfb1 gene was directly synthesized and cloned into the pUC57 vector by BioBasic, Inc. It was subsequently subcloned into the pPICZAα vector that was purchased from Iranian Gene Bank (Pasteur Insti-
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tute of Iran), and Ni-NTA-sepharose resin was purchased from Invitrogen, Inc. Other chemicals were of analytical grade and obtained from Merck & Co., Inc.
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2.2. Vector construction
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The entire coding region of the hfb1 gene (278 bp) was directly synthesized and cloned into the pUC57 vector. Its coding sequence was synthesized according to Genbank sequence (accession no. Z68124) without sequences related to secretory signal and prepropeptide (Fig. 1). Two nucleotide fragments were inserted into the 5’ and 3’ ends of the hfb1 gene. These fragments were possessed restriction enzyme cleavage sites and added to both ends of the gene. The upstream segment contains the kex2 protease recognition sequence which was located after the Xho1 cleavage site (5’ end of the gene) and followed by sequencing related to alanine and glutamate. The downstream fragment includes a nucleotide sequence encoding the 6 His-tag previous the Not1 recognition site (3’ end of the gene). The pPICZAα vector which contains α-mating factor signal sequence was selected to produce secretory HFB1 protein. The gene of interest, after digestion with Xho1 and Not1 endonuclease, was ligated into Xho1/Not1 linearized pPICZAα plasmid. So that, hfb1 gene placed in a frame with α-factor secretion signal to ensure the efficient secretion of HFB1.
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2.3. Pichia transformation
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The pPICZAα-hfb1 plasmid was linearized with sac1 restriction enzyme and transformed into the X-33 strain of the P. pastoris by electroporation. Transformed plasmids were cultured on a YPDS agar medium containing Zeocin (100 mg/ml) for 5 days. 11 Zeocin-resistant clones were selected, and then integration of the hfb1 gene into the host’s genome was approved by PCR.
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2.4. Selection and Expression of hfb1
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6 clones of transformed cells containing the recombinant gene and a single colony of untransformed cells (X-33 strain) were grown in 5 ml of YPG medium at 30 ℃ with agitation at 250 rpm until the cell density reached an OD600 of 2 to 6. The cells were harvested by centrifugation at 3000 g for 5 minutes at room temperature. After removing the supernatant, the cell pellet was resuspended in 50 ml of YPM medium and cultured at 28 ℃, 250 rpm. In order to maintain induction, methanol was added to the medium every 24 hours at a final concentration of 0.5% (V/V). To determine the optimum time interval for the high-level production of protein, 1 ml of the expression medium was removed and transferred into a microtube at various time intervals. After centrifugation of microtubes at 21000 g, the primary supernatant and cell pellets were separated from each other. Afterward, the pellets were resuspended in 1x PBS and lysed by bead beating (0.25–0.5 mm diameter). All lysate samples were centrifuged to separate the secondary supernatant from the cell debris, and finally 20 µl of these samples were assessed by SDSPAGE. In order to precipitate of the protein content of the primary supernatant, 23 µl of 100% trichloroacetic acid (TCA) was added to 200 µl of each sample and then stored at -20 ℃. Thereafter, the samples were centrifuged at 21000 g for 1 hour at 4 ℃. The supernatant was removed and the protein pellet was rinsed twice with 70% acetone. Then these pellets were dried on a heat block at 95 ℃ for 5 minutes [18]
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and resuspended in 30 µl of distilled water and dissolved by adding loading buffer. At lastly, samples were loaded onto 16% tricine-SDS-PAGE for further analysis [19].
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2.5. Purification of HFB1
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Supernatant of the induction medium was collected after 90 hours. Purification was carried out according to one of the following methods: (A) based on the presence of His-tag (B) based on foam formation. We had two approaches in the A-section: indirected and directed methods. The indirected method consisted of 3 steps: Firstly, existing total protein in 50 ml of supernatant was deposited using 90% saturated ammonium sulfate. Secondly, the protein pellets were dissolved in 2 ml of sterile distilled water and desalted using a dialysis membrane with molecular weight cut-off of 3.5 KDa after 24 hours. Lastly, the solution was passed through Ni-NTA-sepharose column according to Invitrogen protocol [20]. The eluted samples were stored at -20 ℃. In the directed method, 30 ml of supernatant which had been collected from the induction medium, was directly passed through Ni-NTA-sepharose column. Furthermore, 30 ml of supernatant related to the culture medium of untransformed cells (untransformed X-33 cells) was also passed through Ni-NTAsepharose column as a control. According to the aforementioned Invitrogen protocol, every 1ml of eluted samples was stored in separated microtubes. In the B-section, two approaches were used for foam production. In the first method, CO2 gas was passed through a column containing supernatant of the culture medium to produce and isolate foam [21]. Then, collected foam was stored at -20 ℃ for further analysis. In the second method, 25 ml of cellfree culture supernatant was poured into 50 ml Falcon tube and shaken manually for 4 minutes to produce foam. The tube was stored in the refrigerator for 2 hours and then the stable foam was collected and rinsed twice with 5 ml of distilled water. Finally, 20µl of each produced sample in all methods was loaded onto 16% tricine-SDS-PAGE for further analysis.
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2.6. Assessment of bubbles’ stability
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10 ml of cell-free culture supernatant without and along with HFB1 were shaken manually in 50 ml Falcon tubes for 4 minutes. At first, the solution containing HFB1 became turbid, subsequently, the bubbles were formed. The height of the formed foam was measured within three weeks. In the other experiment, 50 µl of the solution containing foam was poured on a microscope slide then this slide was dried at room temperature and assessed with a light microscope to analyze micro foam. This slide was evaluated again after 35 days.
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3. Results and discussions
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P. pastoris X-33 strain and pPICZAα vector were selected for extracellular production of HFB1. The sequence of the designed hfb1 gene, without the native signal sequence, was inserted into the pPICZAα plasmid and placed in-frame with the α-mating factor sequence of the vector. The expression of HFB1 in Pichia pastoris was controlled by the methanol-regulated alcohol oxidase (AOX1) promoter. Prior to secretion, the α-factor signal was removed from the N-terminal end of the matures secretory protein by
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yeast proteases. So that, recombinant HFB1 is produced without any additional amino acids in its Nterminus. Double digestion of the pPICZAα-hfb1 vector with Xho1/Not1 together with the production of a DNA fragment of the desired size (278 bp) proved the authenticity of the cloning steps (data not shown). Hfb1 gene was sequenced before transfection to confirm the right direction, size, and sequence of the hfb1 gene. pPICZAα-hfb1 vector was linearized using sac1 restriction enzyme and transformed into the X-33 strain. The growth of yeast cells on YPDS plate including Zeocin indicated that the linearized pPICZAα-hfb1 vector has been integrated into the yeast genome. PCR amplification of yeast genomic DNA was carried out, and the presence of an amplification band of interest approved the proper insertion of the hfb1 gene. Single colonies were grown in YPG medium overnight and then transferred into the YPM medium containing 0.5% (V/V) methanol to induce the alcohol oxidase promoter. In order to determine the suitable time interval for production of protein, 1 ml of the induction medium, which had been collected at various time intervals, was used to assess the protein content of the supernatant and cell lysis. For further analysis, the collected samples were loaded onto 16% tricine -SDS-PAGE. As can be seen in Fig. 2A; HFB1 appeared in the culture medium 42 hours after induction, afterward, its concentration increased over time and received to a maximum level in-90 hours (Fig. 2B), and subsequently decreased. This decline probably is due to the enhancement of P. pastoris proteases into the culture medium [20]. Analysis of the proteins obtained from cell lysis shows that the HFB1 has been produced at a high-level concentration inside the cells. But there were other protein bands around in the area of interest (7.5 KDa). The intensity of these protein bands increased in transformed cells during the expression of recombinant proteins. By considering the fact that these nonspecific bands did not exist in the control sample (untransformed X-33 cell lysis), we conclude that the nonspecific bands probably related to intracellular immature HFB1 proteins that have not been processed. Analysis of the total proteins collected from the cell-free culture medium and cell lysis indicated that the purification of HFB1 from the supernatant is more feasible than cell lysis due to lower protein population in the supernatant (Fig. 2A, B). In order to obtain purified HFB1, 30 ml of cell-free culture medium was passed through Ni-NTAsepharose column. HFB1 contains His-tag; thus, it can easily bind to the resin and elute from the column using 5 ml elution buffer. As shown in Fig. 3, the presence of a sharp protein band at 7.5 KDa confirms that purification and isolation of HFB1 were carried out with high purity level. It also means that the secretory signal had been removed from the N-terminus of the recombinant HFB1 by intracellular proteases. The absence of this protein band in the control sample (untransformed X-33 cells) is another evidence of the accuracy of cloning steps and secretory expression of HFB1 in the transformed cells. The concentration of purified HFB1 was calculated to be 300 mg/L based on Bradford assay. In previous studies, Bolyard and Sticklen in 1992 [22], and Penas et al. in 1998 [23] produced hydrophobins ceratoulmin and Fbh1 in E.coli, respectively. But their production yield was below 1 mg/L. In 2010 and 2011, FcHyd5p [24], HGF1 [25], FcHyd3p, and HFB2 [26] hydrophobins were expressed in P. pastoris, without reported production yield. Pedersen et al. produced hydrophobins RodA and RodB in P. pastoris with the concentration of 329 and 262 mg/L, respectively [27]. This result reported on the basis of the fact that they had used fed-batch fermentation to keep pH at 5.0 and monitor the amount of O2 and CO2. After six days, they obtained the concentration of RodA and RodB proteins as mentioned above. But when they prepared these samples for purification, the majority of hydrophobins was lost due to the increase in salt concentration and rise in pH from 5.0 to 7.4. These conditions led to the aggregation of
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hydrophobins. Finally, the concentration of pure proteins decreased until 21 mg/L and 24 mg/L for RodA and RodB, respectively. Despite a decline in the concentration of purified RodA and RodB, these proteins were observed to have high purity [27]. As shown in the Fig. 3, the purity of purified HFB1 was more than 99%; the result is in line with the presented results by Pedersen et al. But the concentration of purified HFB1 was 12.5-fold more than the concentration of purified RodA and RodB. The results of the indirected method (salting out, dialysis, and using affinity column) revealed that at the first step, total protein was concentrated and the presence of 7.5 KDa-band confirmed the existence of recombinant HFB1 (results not shown). The result of dialysis step shown a significant decrease in concentration of HFB1 protein. This finding probably is due to the protein loss in dialysis step, which could be the result of the presence of superficial hydrophobic groups binding to the dialysis tube. In 2010 and 2011, foam fractionation by CO2 gas was invented to solve some problems (gushing phenomena) in the brewery industry [28-29]. Hydrophobic patch plays an important role in the affinity of the protein for CO2 gas. Since CO2 molecules possess lower polarity than the air, HFB1 proteins have a greater affinity for attachment to CO2 molecules in comparison with the air molecules. The foam produced by CO2 gas and then collected. Although this is a simple and cost-effective method for HFB1 purification, this method is not appropriate for purification of HFB1 with high-throughput. In this case, we observed additional weak protein bands on the top of the gel. These protein bands are not interested and related to other yeast proteins that have been secreted into the culture medium (Fig. 4, lane 1). Analysis of collecting foam with shaking method revealed that some nonspecific protein have also been isolated along with HFB1 protein (Fig. 4, lane 2). In 2012, Kottmeier et al. expressed HFB1 in P. pastoris [18], and reported the purity up to 70% for isolated HFB1 using the foam separation method. This result was compatible with our finding of purity of HFB1 by foam separation. By comparing different methods for HFB1 purification, it can be concluded that the directed method has higher performance than the foam separation method. Use of method based on foam formation leads to a decrease in the purity level of purified HFB1 comparison with directed method (the respective values for the purity of proteins were 70% and 99%). According to the obtained results which have been summarized in Table 1, if the high purity of HFB1 is required (for instance stabilization of enzyme and drug delivery), the directed method will be useful for HFB1 purification.
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3.4. Bubbles stability
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Self-assembly at the air/water interface is one of the properties of hydrophobins. So, HFB1 produced stable bubbles in the cell-free culture supernatant. These stable bubbles did not observe in HFB1-free solution. Assessing the height of the formed foam by HFB1 within 3 weeks proved their stability (Fig. 5). Furthermore, a solution containing HFB1 has microscopic bubbles. Analysis of them by microscope shown their differences in size and shape. Re-evaluation of the microscope slide after 35 days confirms that these structures are surprisingly stable, and their shape has remained unchanged.
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4. Conclusions
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According to the results presented in this manuscript, it can be concluded that the hfb1 gene was subcloned into an expression vector (pPICZAα) using Xho1 and Not1 restriction enzymes. Subsequently, HFB1 protein was expressed in P. pastoris X-33 strain and secreted into culture medium. We compared
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different methods for the purification of HFB1 from cell-free culture supernatant and concluded that the HFB1 can be easily purified based on the existence of His-tag at its C-terminus. Our finding showed that directed method has higher efficiency for protein purification in comparison with other methods. Assessment of HFB1 purification based on directed method and the foam separation method revealed 99% and 70% purity level, respectively. In the aforementioned technique (directed method), recombinant HFB1 was purified by Ni-NTA-sepharose column in one step, in the result of, purification has no need of any other pre-purification methods such as ion-exchange chromatography or hydrophobic interaction chromatography. Thus in addition to the saving time, it can also save costs. Considering the fact that HFB1 can be used as a unique protein in some branches of biotechnology, the use of less expensive methods to produce HFB1 is recommended. It seems that the secretory production of the recombinant HFB1 in Pichia pastoris, and subsequently its purification in one step can be accountable for this need. However, it should be considered that we have a long way ahead to achieve these goals. Our analysis also revealed the high potential of HFB1 protein to cover and stabilize microscopic and macroscopic bubbles. HFB1 can form stable foam due to its surface activity and its self-assembling nature while both of these features will require the correct protein structure. It is notable that the production of stable foam is important for food industries such as manufacturing of ice cream and whipped butter. With regard to the points mentioned above, HFB1 can be a good candidate for the production of stable foam in such industries.
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Acknowledgments:
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Financial support of this work is provided by the Research Council of Tarbiat Modares University
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Fig. 1: General schemes of nucleotide and amino acid sequences. (A) The entire coding region of the hfb1 gene (accession no. Z68124) along with its related amino acid sequence. The HFB1 protein contains three parts: signal peptide, prepropeptide, and the hfb1 coding sequence. Initial methionine has been specified by a star (*). (B) Hfb1 nucleotide sequence expressed in P. pastoris. Two primary parts of the native gene have been omitted. Amino acids that have been specified by two stars (**) are cleaved by yeast endoprotease after expression.
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Fig. 2: 16% tricine-SDS-PAGE analyses of HFB1 expression after induction in various times. (A), and (B): Lane M related to protein molecular weight marker. Lanes 1, 3, 5, 7, and 9 total protein content of cell lysis. Lanes 2, 4, 6, 8, and 10 cell-free culture supernatant (TCA precipitated). HFB1 appeared in the culture medium 42 hours after induction.
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Fig. 3: The eluted fractions from the Ni-NTA-sepharose column loaded onto the 16% tricine-SDS-PAGE. Lane 1: The eluted fraction belongs to untransformed cells (X-33 strain). Lane 2-4: HFB1 eluted from the column. The presence of a single band at 7.5 KDa location shows that purification of HFB1 has been carried out more than 99%. Purity level was calculated by gel analyzer software (www.gelanalyzer.com).
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Fig. 4: 16% tricine-SDS-PAGE related to the foam separation methods. Lane M: protein molecular weight marker. Lane 1: The proteins were collected by CO2 gas. Lane 2: The proteins were collected by shaking manually. The presence of a band at 7.5 KDa location indicates the isolation of HFB1, but the existence of the weak protein bands at the top of the gel shows the presence of nonspecific proteins.
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Fig. 5: Photograph of foam stability. (A) Produced foam in the supernatant of the culture medium containing HFB1 on the first day. (B) Re-evaluation of the same tubes after three weeks. In both photographs, positive control consists of recombinant HFB1 has been shown as a left tube, and the right tube is the HFB1-free solution as a negative control sample.
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Table 1: Results of HBF1 purification by different methods Purification methodes† ††
His-tag affinity chromatography Directed Indirected 300 73 300 72 >99 ∽99 57.6 13.8 12.2 12.2 7.4 1.8
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Foam separation Shaken manually Use of CO2 gas 562 160 360 110 <70 70 69.2 21.1 12.2 12.2 8.8 2.7
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Total protein* (mg/L) ** HFB1 protein (mg/L) Purity (%) *** Purification yield (%) Wet cell weight (g) HFB1 per wet cell weight (mg/g) 324 † The initial volume of growth medium was 300 ml for all cases. 325 †† Total protein and HFB1 concentrations obtained 1525 mg/L and 520 mg/L, respectively, before purification. 326 *, ** These values are relevant to steps after HFB1 purification. 327 *** Purification yield was calculated by dividing the HFB1 concentration after purification by the final concentra328 tion of HFB1 before purification.
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Highlights
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Recombinant secretory hydrophobin-1 (HFB1) is expressed in Pichia pastoris a simple way for HFB1 Purification with high purity and high concentration (99% and 300 mg/L) is presented We have shown that HFB1 can produce super-stable foam
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S1046-5928(15)30075-9
DOI:
10.1016/j.pep.2015.09.025
Reference:
YPREP 4800
To appear in:
Protein Expression and Purification
Received Date: 27 April 2015 Revised Date:
22 September 2015
Accepted Date: 25 September 2015
Please cite this article as: A. Lohrasbi-Nejad, M. Torkzadeh-Mahani, S. Hosseinkhani, Heterologous expression of a hydrophobin HFB1 and evaluation of its contribution to producing stable foam, Protein Expression and Purification (2015), doi: 10.1016/j.pep.2015.09.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Heterologous expression of a hydrophobin HFB1 and evaluation of its contribution to producing stable foam
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Azadeh Lohrasbi-Nejada, Masoud Torkzadeh-Mahanib, Saman Hosseinkhania* a
Department of Biochemistry, Faculty of Biological Sciences, TarbiatModares University, Tehran, Iran Department of Biotechnology, Institute of Science, High Technology and Environmental Science, Graduate University of Advanced Technology, Kerman, Iran. * Corresponding author. Tel.: +98 21 82884407; fax: +98 21 82884484. E-mail address: [email protected] (S. Hosseinkhani).
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Hydrophobins are small secreted proteins belong to filamentous fungi. These proteins possess a unique ability to self-assemble at air/water interfaces. Hydrophobins have a broad range of biotechnological applications such as stabilizing emulsions and foams, immobilizing proteins on a surface, designing biosensors, affinity tag for protein purification, and drug delivery. We have successfully expressed HFB1 from Trichoderma reesei belonged to class II of hydrophobins in Pichia pastoris. The recombinant gene was under the control of the methanol-inducible AOX1 promoter (alcohol oxidase 1) in the pPICZAα vector. The amount of secreted HFB1 was increased in 90-hours using methanol induction. The recombinant HFB1 was purified based on the presence of His-tag and foam formation. Furthermore, HFB1 was able to produce macro and micro stable air bubbles in the liquid due to the presence of hydrophobic patches on its surface. The liquid medium containing HFB1 becomes turbid after shaking, and then the stable bubbles are formed and remained for three weeks.
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Keywords: Hydrophobin; HFB1; heterologous expression; Pichia pastoris
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Introduction Hydrophobins are small secreted proteins with a surface active, which are produced by filamentous fungi [1]. They consist of about 100 amino acids including 8 cysteine residues in conserved position, which can form 4 intracellular disulfide bonds [2]. Self-assembly in hydrophobic/hydrophilic (such as air/water) interfaces is one of the most important qualities of these proteins [3]. Their role in growth and development of fungi is of utmost importance. Hydrophobins help fungal hyphae to emerge from water by reducing the surface tension of water when secreted into the environment [4-5]. Moreover, they participate in coverage of cell wall fungi and subsequently lead to reverse hydrophobicity. These proteins facilitate appropriate gas exchange, aerial dispersal of spores and connection of hyphae to hydrophobic surfaces by aggregation on the surface of some structures such as fruiting bodies, spores, and hyphae, respectively [6-7]. Based on hydropathy profile, biophysical characteristics, and amino acid sequence similarity, hydrophobins are divided into two groups: class I and class II [8]. All hydrophobins can form amphipathic membranes. Membranes constructed by class I hydrophobins are resistant to dissolution and dissolved in formic acid or trifluoroacetic acid. But membranes created by class II hydrophobins are dissolved in 2% hot SDS or 60% ethanol [9]. Hydrophobins are useful in some branches of biotechnology such as stabilizing enzyme [10-11], biosensor and electrode construction [12], tissue engineering, drug delivery [13], foam stabilizer [14-15], coating techniques [16], and fusion tag for the purification of recombinant proteins [17]. There are many potential practical applications for this protein in technology; however, mass production with low cost is a major obstacle in this regard. Using a proper host such as Pichia pastoris could help surmount this problem. Protein production in Pichia pastoris is preferred among other hosts due to its wide range of post-translational modifications and production of secretory protein [18]. In this study, we have focused on the recombinant production of HFB1 from Trichoderma reesei belong to the class II of hydrophobins. The native form of HFB1 consists of 97 amino acids including 16 amino acids belong to the secretory signal sequence, 6 amino acids belong to the prepropeptide, and 75 amino acids belong to the main structural sequence. The final 7.5 KDa protein is delivered after elimination of the first two parts from N-terminal through intracellular processing. Moreover, we transformed hfb1 gene without a signal and prepropeptide sequences in Pichia pastoris and investigated the secretory expression of HFB1. We have also compared the efficiency of different methods on the yield of purification and finally presented a high-throughput method for purification of HFB1 with high purity and high concentration.
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2. Materials and methods
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2.1. Strains, vectors, and reagents
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Pichia pastoris strain X-33, Escherichia coli strain DH5α was obtained from our laboratory. All restriction enzymes, T4 DNA ligase, and alkaline phosphatase were procured from Thermo Fisher Scientific, Inc. DNA extraction and plasmid purification kits were also supplied by Bioneer, Inc. The entire coding region of hfb1 gene was directly synthesized and cloned into the pUC57 vector by BioBasic, Inc. It was subsequently subcloned into the pPICZAα vector that was purchased from Iranian Gene Bank (Pasteur Insti-
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tute of Iran), and Ni-NTA-sepharose resin was purchased from Invitrogen, Inc. Other chemicals were of analytical grade and obtained from Merck & Co., Inc.
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2.2. Vector construction
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The entire coding region of the hfb1 gene (278 bp) was directly synthesized and cloned into the pUC57 vector. Its coding sequence was synthesized according to Genbank sequence (accession no. Z68124) without sequences related to secretory signal and prepropeptide (Fig. 1). Two nucleotide fragments were inserted into the 5’ and 3’ ends of the hfb1 gene. These fragments were possessed restriction enzyme cleavage sites and added to both ends of the gene. The upstream segment contains the kex2 protease recognition sequence which was located after the Xho1 cleavage site (5’ end of the gene) and followed by sequencing related to alanine and glutamate. The downstream fragment includes a nucleotide sequence encoding the 6 His-tag previous the Not1 recognition site (3’ end of the gene). The pPICZAα vector which contains α-mating factor signal sequence was selected to produce secretory HFB1 protein. The gene of interest, after digestion with Xho1 and Not1 endonuclease, was ligated into Xho1/Not1 linearized pPICZAα plasmid. So that, hfb1 gene placed in a frame with α-factor secretion signal to ensure the efficient secretion of HFB1.
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The pPICZAα-hfb1 plasmid was linearized with sac1 restriction enzyme and transformed into the X-33 strain of the P. pastoris by electroporation. Transformed plasmids were cultured on a YPDS agar medium containing Zeocin (100 mg/ml) for 5 days. 11 Zeocin-resistant clones were selected, and then integration of the hfb1 gene into the host’s genome was approved by PCR.
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2.4. Selection and Expression of hfb1
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6 clones of transformed cells containing the recombinant gene and a single colony of untransformed cells (X-33 strain) were grown in 5 ml of YPG medium at 30 ℃ with agitation at 250 rpm until the cell density reached an OD600 of 2 to 6. The cells were harvested by centrifugation at 3000 g for 5 minutes at room temperature. After removing the supernatant, the cell pellet was resuspended in 50 ml of YPM medium and cultured at 28 ℃, 250 rpm. In order to maintain induction, methanol was added to the medium every 24 hours at a final concentration of 0.5% (V/V). To determine the optimum time interval for the high-level production of protein, 1 ml of the expression medium was removed and transferred into a microtube at various time intervals. After centrifugation of microtubes at 21000 g, the primary supernatant and cell pellets were separated from each other. Afterward, the pellets were resuspended in 1x PBS and lysed by bead beating (0.25–0.5 mm diameter). All lysate samples were centrifuged to separate the secondary supernatant from the cell debris, and finally 20 µl of these samples were assessed by SDSPAGE. In order to precipitate of the protein content of the primary supernatant, 23 µl of 100% trichloroacetic acid (TCA) was added to 200 µl of each sample and then stored at -20 ℃. Thereafter, the samples were centrifuged at 21000 g for 1 hour at 4 ℃. The supernatant was removed and the protein pellet was rinsed twice with 70% acetone. Then these pellets were dried on a heat block at 95 ℃ for 5 minutes [18]
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and resuspended in 30 µl of distilled water and dissolved by adding loading buffer. At lastly, samples were loaded onto 16% tricine-SDS-PAGE for further analysis [19].
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2.5. Purification of HFB1
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Supernatant of the induction medium was collected after 90 hours. Purification was carried out according to one of the following methods: (A) based on the presence of His-tag (B) based on foam formation. We had two approaches in the A-section: indirected and directed methods. The indirected method consisted of 3 steps: Firstly, existing total protein in 50 ml of supernatant was deposited using 90% saturated ammonium sulfate. Secondly, the protein pellets were dissolved in 2 ml of sterile distilled water and desalted using a dialysis membrane with molecular weight cut-off of 3.5 KDa after 24 hours. Lastly, the solution was passed through Ni-NTA-sepharose column according to Invitrogen protocol [20]. The eluted samples were stored at -20 ℃. In the directed method, 30 ml of supernatant which had been collected from the induction medium, was directly passed through Ni-NTA-sepharose column. Furthermore, 30 ml of supernatant related to the culture medium of untransformed cells (untransformed X-33 cells) was also passed through Ni-NTAsepharose column as a control. According to the aforementioned Invitrogen protocol, every 1ml of eluted samples was stored in separated microtubes. In the B-section, two approaches were used for foam production. In the first method, CO2 gas was passed through a column containing supernatant of the culture medium to produce and isolate foam [21]. Then, collected foam was stored at -20 ℃ for further analysis. In the second method, 25 ml of cellfree culture supernatant was poured into 50 ml Falcon tube and shaken manually for 4 minutes to produce foam. The tube was stored in the refrigerator for 2 hours and then the stable foam was collected and rinsed twice with 5 ml of distilled water. Finally, 20µl of each produced sample in all methods was loaded onto 16% tricine-SDS-PAGE for further analysis.
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2.6. Assessment of bubbles’ stability
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10 ml of cell-free culture supernatant without and along with HFB1 were shaken manually in 50 ml Falcon tubes for 4 minutes. At first, the solution containing HFB1 became turbid, subsequently, the bubbles were formed. The height of the formed foam was measured within three weeks. In the other experiment, 50 µl of the solution containing foam was poured on a microscope slide then this slide was dried at room temperature and assessed with a light microscope to analyze micro foam. This slide was evaluated again after 35 days.
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3. Results and discussions
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P. pastoris X-33 strain and pPICZAα vector were selected for extracellular production of HFB1. The sequence of the designed hfb1 gene, without the native signal sequence, was inserted into the pPICZAα plasmid and placed in-frame with the α-mating factor sequence of the vector. The expression of HFB1 in Pichia pastoris was controlled by the methanol-regulated alcohol oxidase (AOX1) promoter. Prior to secretion, the α-factor signal was removed from the N-terminal end of the matures secretory protein by
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yeast proteases. So that, recombinant HFB1 is produced without any additional amino acids in its Nterminus. Double digestion of the pPICZAα-hfb1 vector with Xho1/Not1 together with the production of a DNA fragment of the desired size (278 bp) proved the authenticity of the cloning steps (data not shown). Hfb1 gene was sequenced before transfection to confirm the right direction, size, and sequence of the hfb1 gene. pPICZAα-hfb1 vector was linearized using sac1 restriction enzyme and transformed into the X-33 strain. The growth of yeast cells on YPDS plate including Zeocin indicated that the linearized pPICZAα-hfb1 vector has been integrated into the yeast genome. PCR amplification of yeast genomic DNA was carried out, and the presence of an amplification band of interest approved the proper insertion of the hfb1 gene. Single colonies were grown in YPG medium overnight and then transferred into the YPM medium containing 0.5% (V/V) methanol to induce the alcohol oxidase promoter. In order to determine the suitable time interval for production of protein, 1 ml of the induction medium, which had been collected at various time intervals, was used to assess the protein content of the supernatant and cell lysis. For further analysis, the collected samples were loaded onto 16% tricine -SDS-PAGE. As can be seen in Fig. 2A; HFB1 appeared in the culture medium 42 hours after induction, afterward, its concentration increased over time and received to a maximum level in-90 hours (Fig. 2B), and subsequently decreased. This decline probably is due to the enhancement of P. pastoris proteases into the culture medium [20]. Analysis of the proteins obtained from cell lysis shows that the HFB1 has been produced at a high-level concentration inside the cells. But there were other protein bands around in the area of interest (7.5 KDa). The intensity of these protein bands increased in transformed cells during the expression of recombinant proteins. By considering the fact that these nonspecific bands did not exist in the control sample (untransformed X-33 cell lysis), we conclude that the nonspecific bands probably related to intracellular immature HFB1 proteins that have not been processed. Analysis of the total proteins collected from the cell-free culture medium and cell lysis indicated that the purification of HFB1 from the supernatant is more feasible than cell lysis due to lower protein population in the supernatant (Fig. 2A, B). In order to obtain purified HFB1, 30 ml of cell-free culture medium was passed through Ni-NTAsepharose column. HFB1 contains His-tag; thus, it can easily bind to the resin and elute from the column using 5 ml elution buffer. As shown in Fig. 3, the presence of a sharp protein band at 7.5 KDa confirms that purification and isolation of HFB1 were carried out with high purity level. It also means that the secretory signal had been removed from the N-terminus of the recombinant HFB1 by intracellular proteases. The absence of this protein band in the control sample (untransformed X-33 cells) is another evidence of the accuracy of cloning steps and secretory expression of HFB1 in the transformed cells. The concentration of purified HFB1 was calculated to be 300 mg/L based on Bradford assay. In previous studies, Bolyard and Sticklen in 1992 [22], and Penas et al. in 1998 [23] produced hydrophobins ceratoulmin and Fbh1 in E.coli, respectively. But their production yield was below 1 mg/L. In 2010 and 2011, FcHyd5p [24], HGF1 [25], FcHyd3p, and HFB2 [26] hydrophobins were expressed in P. pastoris, without reported production yield. Pedersen et al. produced hydrophobins RodA and RodB in P. pastoris with the concentration of 329 and 262 mg/L, respectively [27]. This result reported on the basis of the fact that they had used fed-batch fermentation to keep pH at 5.0 and monitor the amount of O2 and CO2. After six days, they obtained the concentration of RodA and RodB proteins as mentioned above. But when they prepared these samples for purification, the majority of hydrophobins was lost due to the increase in salt concentration and rise in pH from 5.0 to 7.4. These conditions led to the aggregation of
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hydrophobins. Finally, the concentration of pure proteins decreased until 21 mg/L and 24 mg/L for RodA and RodB, respectively. Despite a decline in the concentration of purified RodA and RodB, these proteins were observed to have high purity [27]. As shown in the Fig. 3, the purity of purified HFB1 was more than 99%; the result is in line with the presented results by Pedersen et al. But the concentration of purified HFB1 was 12.5-fold more than the concentration of purified RodA and RodB. The results of the indirected method (salting out, dialysis, and using affinity column) revealed that at the first step, total protein was concentrated and the presence of 7.5 KDa-band confirmed the existence of recombinant HFB1 (results not shown). The result of dialysis step shown a significant decrease in concentration of HFB1 protein. This finding probably is due to the protein loss in dialysis step, which could be the result of the presence of superficial hydrophobic groups binding to the dialysis tube. In 2010 and 2011, foam fractionation by CO2 gas was invented to solve some problems (gushing phenomena) in the brewery industry [28-29]. Hydrophobic patch plays an important role in the affinity of the protein for CO2 gas. Since CO2 molecules possess lower polarity than the air, HFB1 proteins have a greater affinity for attachment to CO2 molecules in comparison with the air molecules. The foam produced by CO2 gas and then collected. Although this is a simple and cost-effective method for HFB1 purification, this method is not appropriate for purification of HFB1 with high-throughput. In this case, we observed additional weak protein bands on the top of the gel. These protein bands are not interested and related to other yeast proteins that have been secreted into the culture medium (Fig. 4, lane 1). Analysis of collecting foam with shaking method revealed that some nonspecific protein have also been isolated along with HFB1 protein (Fig. 4, lane 2). In 2012, Kottmeier et al. expressed HFB1 in P. pastoris [18], and reported the purity up to 70% for isolated HFB1 using the foam separation method. This result was compatible with our finding of purity of HFB1 by foam separation. By comparing different methods for HFB1 purification, it can be concluded that the directed method has higher performance than the foam separation method. Use of method based on foam formation leads to a decrease in the purity level of purified HFB1 comparison with directed method (the respective values for the purity of proteins were 70% and 99%). According to the obtained results which have been summarized in Table 1, if the high purity of HFB1 is required (for instance stabilization of enzyme and drug delivery), the directed method will be useful for HFB1 purification.
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Self-assembly at the air/water interface is one of the properties of hydrophobins. So, HFB1 produced stable bubbles in the cell-free culture supernatant. These stable bubbles did not observe in HFB1-free solution. Assessing the height of the formed foam by HFB1 within 3 weeks proved their stability (Fig. 5). Furthermore, a solution containing HFB1 has microscopic bubbles. Analysis of them by microscope shown their differences in size and shape. Re-evaluation of the microscope slide after 35 days confirms that these structures are surprisingly stable, and their shape has remained unchanged.
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According to the results presented in this manuscript, it can be concluded that the hfb1 gene was subcloned into an expression vector (pPICZAα) using Xho1 and Not1 restriction enzymes. Subsequently, HFB1 protein was expressed in P. pastoris X-33 strain and secreted into culture medium. We compared
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different methods for the purification of HFB1 from cell-free culture supernatant and concluded that the HFB1 can be easily purified based on the existence of His-tag at its C-terminus. Our finding showed that directed method has higher efficiency for protein purification in comparison with other methods. Assessment of HFB1 purification based on directed method and the foam separation method revealed 99% and 70% purity level, respectively. In the aforementioned technique (directed method), recombinant HFB1 was purified by Ni-NTA-sepharose column in one step, in the result of, purification has no need of any other pre-purification methods such as ion-exchange chromatography or hydrophobic interaction chromatography. Thus in addition to the saving time, it can also save costs. Considering the fact that HFB1 can be used as a unique protein in some branches of biotechnology, the use of less expensive methods to produce HFB1 is recommended. It seems that the secretory production of the recombinant HFB1 in Pichia pastoris, and subsequently its purification in one step can be accountable for this need. However, it should be considered that we have a long way ahead to achieve these goals. Our analysis also revealed the high potential of HFB1 protein to cover and stabilize microscopic and macroscopic bubbles. HFB1 can form stable foam due to its surface activity and its self-assembling nature while both of these features will require the correct protein structure. It is notable that the production of stable foam is important for food industries such as manufacturing of ice cream and whipped butter. With regard to the points mentioned above, HFB1 can be a good candidate for the production of stable foam in such industries.
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Acknowledgments:
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Financial support of this work is provided by the Research Council of Tarbiat Modares University
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Fig. 1: General schemes of nucleotide and amino acid sequences. (A) The entire coding region of the hfb1 gene (accession no. Z68124) along with its related amino acid sequence. The HFB1 protein contains three parts: signal peptide, prepropeptide, and the hfb1 coding sequence. Initial methionine has been specified by a star (*). (B) Hfb1 nucleotide sequence expressed in P. pastoris. Two primary parts of the native gene have been omitted. Amino acids that have been specified by two stars (**) are cleaved by yeast endoprotease after expression.
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Fig. 2: 16% tricine-SDS-PAGE analyses of HFB1 expression after induction in various times. (A), and (B): Lane M related to protein molecular weight marker. Lanes 1, 3, 5, 7, and 9 total protein content of cell lysis. Lanes 2, 4, 6, 8, and 10 cell-free culture supernatant (TCA precipitated). HFB1 appeared in the culture medium 42 hours after induction.
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Fig. 3: The eluted fractions from the Ni-NTA-sepharose column loaded onto the 16% tricine-SDS-PAGE. Lane 1: The eluted fraction belongs to untransformed cells (X-33 strain). Lane 2-4: HFB1 eluted from the column. The presence of a single band at 7.5 KDa location shows that purification of HFB1 has been carried out more than 99%. Purity level was calculated by gel analyzer software (www.gelanalyzer.com).
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Fig. 4: 16% tricine-SDS-PAGE related to the foam separation methods. Lane M: protein molecular weight marker. Lane 1: The proteins were collected by CO2 gas. Lane 2: The proteins were collected by shaking manually. The presence of a band at 7.5 KDa location indicates the isolation of HFB1, but the existence of the weak protein bands at the top of the gel shows the presence of nonspecific proteins.
AC C
EP
TE D
307 308 309 310 311 312 313
314 315 316 317 318 319 320 321 322
Fig. 5: Photograph of foam stability. (A) Produced foam in the supernatant of the culture medium containing HFB1 on the first day. (B) Re-evaluation of the same tubes after three weeks. In both photographs, positive control consists of recombinant HFB1 has been shown as a left tube, and the right tube is the HFB1-free solution as a negative control sample.
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Table 1: Results of HBF1 purification by different methods Purification methodes† ††
His-tag affinity chromatography Directed Indirected 300 73 300 72 >99 ∽99 57.6 13.8 12.2 12.2 7.4 1.8
††
Foam separation Shaken manually Use of CO2 gas 562 160 360 110 <70 70 69.2 21.1 12.2 12.2 8.8 2.7
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SC
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Total protein* (mg/L) ** HFB1 protein (mg/L) Purity (%) *** Purification yield (%) Wet cell weight (g) HFB1 per wet cell weight (mg/g) 324 † The initial volume of growth medium was 300 ml for all cases. 325 †† Total protein and HFB1 concentrations obtained 1525 mg/L and 520 mg/L, respectively, before purification. 326 *, ** These values are relevant to steps after HFB1 purification. 327 *** Purification yield was calculated by dividing the HFB1 concentration after purification by the final concentra328 tion of HFB1 before purification.
329 330 331 332
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EP
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333
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Highlights
RI PT SC M AN U TE D EP
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Recombinant secretory hydrophobin-1 (HFB1) is expressed in Pichia pastoris a simple way for HFB1 Purification with high purity and high concentration (99% and 300 mg/L) is presented We have shown that HFB1 can produce super-stable foam
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