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Production of bioactive recombinant rat soluble receptor for advanced glycation end products (rrsRAGE) in Pichia pastoris
Accepted Manuscript Production of bioactive recombinant rat soluble receptor for advanced glycation end products (rrsRAGE) in Pichia pastoris Peng Xia, Jin Gao, Wen Guan, Jingjing Li, Xiaolan Yu, Fangyuan Wang, Honglin He, Qing Deng, Liang Zhou, Yunsheng Yuan, Wei Han, Yan Yu PII:
S1046-5928(15)30079-6
DOI:
10.1016/j.pep.2015.09.029
Reference:
YPREP 4804
To appear in:
Protein Expression and Purification
Received Date: 12 August 2015 Revised Date:
16 September 2015
Accepted Date: 30 September 2015
Please cite this article as: P. Xia, J. Gao, W. Guan, J. Li, X. Yu, F. Wang, H. He, Q. Deng, L. Zhou, Y. Yuan, W. Han, Y. Yu, Production of bioactive recombinant rat soluble receptor for advanced glycation end products (rrsRAGE) in Pichia pastoris, Protein Expression and Purification (2015), doi: 10.1016/ j.pep.2015.09.029. 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.
ACCEPTED MANUSCRIPT Production of bioactive recombinant rat soluble receptor for advanced glycation end products (rrsRAGE) in Pichia pastoris
Peng Xia1*, Jin Gao2*, Wen Guan1, Jingjing Li2, Xiaolan Yu1, Fangyuan Wang3, Honglin He1, Qing
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Deng1, Liang Zhou1, Yunsheng Yuan1, Wei Han2#, Yan Yu1#
Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and
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Biology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China Laboratory of Regenerative Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai
200240, People’s Republic of China 3
Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated First People’s
Hospital, Shanghai 200080, People’s Republic of China *The authors contributed equally to this work #Corresponding authors: E-mail address: [email protected] (Yan Yu) [email protected] (Wei Han) Tel. and Fax: +86 21 34205833 1
ACCEPTED MANUSCRIPT Abstract Soluble receptor for advanced glycation end products (sRAGE), a natural inhibitor of RAGE, is considered to be a putative therapeutic molecule for a variety of diseases and a biomarker for certain conditions. To further study the function of sRAGE, recombinant rat sRAGE (rrsRAGE) was expressed
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and produced in a eukaryotic system. The open reading frame of rat sRAGE was cloned downstream of the methanol-inducible alcohol oxidase promoter of pPICZαA vector, and Pichia pastoris strain X-33 was used as the host strain. The expression of rrsRAGE was achieved by fermentation in a 15-L bioreactor and the resulting fermentation broth was subjected to purification on a cation exchange
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chromatography column. The purification of rrsRAGE reached 95% after size exclusion chromatography(SEC). The bioactivity of the purified protein was confirmed in a SH-SY5Y cell
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proliferation assay. The biological function of the purified rrsRAGE protein rat CCl4-induced model was then examined. Treatment with rrsRAGE resulted in significantly lower liver fibrosis and lower serum level of ALT, suggesting that sRAGE prevent liver from injury and fibrosis. In conclusion, we achieved high-efficiency production of bioactive rrsRAGE in Pichia pastoris.
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Keywords: Soluble receptor for advanced glycation end products (sRAGE), expression and
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purification, Pichia pastoris
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ACCEPTED MANUSCRIPT Introduction Receptor for advanced glycation end products (RAGE) was first identified as a trans-membrane receptor for final products of glycation and oxidation of proteins and lipids [1] .RAGE can bind several clusters of ligands, including advanced glycation end products (AGEs), high-mobility group box 1
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protein (HMGB1), most S100 family members, and β-amyloid peptides. RAGE plays a central role in diabetes mellitus and exerts a significant effect on diabetic diseases such as diabetic vasculopathy and atherosclerosis [2]. Studies also suggested that expression of RAGE correlated closely to a diverse set of diseases, including types of liver disease [3] renal failure [4] neural degeneration [5] and
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inflammation [6]. Recent clinical research revealed the serum level of soluble RAGE (sRAGE) could act as a biomarker for certain human conditions, such as recurrent pregnancy loss [7] and chronic
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inflammatory disease [8].
Soluble RAGE, a natural inhibitor of RAGE, is widely considered to be a putative therapeutic molecule for a variety of diseases. Although human sRAGE has been expressed by a number of different hosts including Pichia pastoris and Escherichia coli [9, 10] it cannot be used in a rat model because human sRAGE (www.uniprot.org; Q15109 [23–342]) and rat sRAGE (rsRAGE)
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(www.uniprot.org; Q63495 [23–342]) only share 57.01% amino acid sequence homology. This huge difference in the protein sequence of human sRAGE may induce an immunological response in rats. However, the rat bile duct ligand (BDL) model is the most appropriate model in which to study the
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effect of sRAGE on liver protection, therefore another source of sRAGE is required for such studies. The rat sRAGE mRNA encodes 319 amino acids with a calculated molecular weight of 33.86kDa and
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rat sRAGE has an acidic pI value of 6.73. Previously, it was reported that rsRAGE had been produced from insect cells (Sf9 cells) [11] However, because a large amount of sRAGE is required for rat experiments, the insect cell-based expression system cannot meet such requirements because it is difficult and expensive to scale up [12] We therefore proposed P. pastoris or E. coli as vectors for the production of rat sRAGE protein. Given the glycation of the sRAGE protein, the P. pastoris system was selected as being more suitable. Previously, several studies have proved that blockade of RAGE, namely sRAGE administration, could prevent liver from injury and fibrosis in various of animal models. The treatment of sRAGE could attenuate ischemia and reperfusion injury to the liver and acetaminophen-induced hepatotoxicity in mice[3]. Specific siRNA targeting RAGE could also inhibits experimental hepatic fibrosis in rats 3
ACCEPTED MANUSCRIPT [13]. In the other hand, the ligands of RAGE, HMGB1[14], S100A4[15] and AGE, also have been demonstrated capable of promoting the process of liver injury or liver fibrosis. Furthermore, in our previous study, S100A6 has been demonstrated to accelerate liver fibrosis in CCl4-induced animal models. Therefore, we are going to test the in vivo bioactivity of rrsRAGE in CCl4-induced rat models.
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In the present paper, we report a novel, rapid and high-yield purification protocol for recombinant rsRAGE (rrsRAGE) from P. pastoris. The final product was verified to be of high purity and have low endotoxicity, and was shown to be bioactive using cell viability assays. Purified rrsRAGE protein was shown to attenuate liver fibrosis and decrease serum level of alanine aminotransferase(ALT), the
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increase of which could be caused by liver damage.
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Methods Cloning of rsRAGE nucleotide sequences into pPICZαA The
amino
acid
sequence
of
rsRAGE
was
obtained
from
the
Uniprot
website
(www.uniprot.org;Q15109[23-342]) and the coding sequence was optimized according for yeast translation. The sequence with 957 base pairs (bp) was synthesized and cloned into PMD18T-simple
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vector at two restriction sites XhoI and NotI by Genescript Company (Nanjing, China). Then, the rsRAGE-PMD18T-simple vector and the pPICZαA vector were double-digested by XhoI and NotI, the digested products were purified using a DNA extraction kit, and were then ligated using T4-DNA ligase.
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Electrocompetent E. coli strain DH5α cells were prepared and transformed with 50 ng DNA from this ligation reaction mixture and plated on zeocin-resisted Luria Broth (LB) agar. Single colonies were to
identify
the
correct
insert
using
PCR
amplification
with
primers
(S:
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selected
5′-CCAAAGAAGCCAACTCAA-3′; A: 5′-TAAGAACCCTCGGAAACA-3') and transformants were confirmed by sequencing. A single colony with the correct insert was then incubated in LB and the plasmid was prepared and stored for later use.
Transformation and selection of a highly productive single colony of P. pastoris strain X-33 rrsRAGE was transformed into P. pastoris strain X-33. Single colonies were patched on BMGY/BMMY media to check the expression levels [9] The P. pastoris strain X-33-rrsRAGE colonies with higher expression levels were monitored by absorption of the protein to a PVDF membrane and detection by immunohistological techniques. Image J( National Institutes of Health, USA) was used for 4
ACCEPTED MANUSCRIPT expression level quantification. The colony with the highest expression level was selected for the next pilot-scale expression and purification.
Pilot-scale production of rrsRAGE protein in P. pastoris strain X-33
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Production of rrsRAGE protein in X-33 was controlled by the AOX1 promoter in pPICZαA vector. Four stages were followed: (1) Initializing batch phase: 40 g/L glycerol in basal salts medium was used until the OD600 approached 50–70; (2) fed-batch phase: 50% (w/v) glycerol was pumped at 2 mL/L/h for 4 h; (3) transition phase: 1 mL of 100% methanol was added per 1 L culture to allow for a smooth
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transition into the induction phase; (4) induction phase: methanol alone was fed over a time period of 96 h using a methanol probe to control the methanol concentration. Then, the fermentation broth was
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centrifuged to remove yeast cells and the supernatant was collected for the following purification.
Purification of rrsRAGE
The supernatant, obtained as described above, was filtered through a 0.2-µm membrane, then diluted five-fold with distilled water until the conductivity decreased to below 8 ms/cm. The pH was adjusted
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to 5.0 using acetic acid (HAc) solution. The resulting solution was finally subjected to cation exchange chromatography on a SP Sepharose FF (Amersham Biosciences, Piscataway, NJ, USA) (60 mL) column. After loading, the column was washed with Wash Buffer (20 mM HAc-NaAc, 1 mM EDTA,
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pH 5.0) until the absorption at 280 nm reached the baseline level. The rrsRAGE protein was eluted with a linear gradient of 0–100% in Elution Buffer A (20 mM HAc-NaAc, 1 M NaCl, 1 mM EDTA).
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The elution containing rrsRAGE was loaded on a Superedex 75 (Amersham Biosciences) 90-cm column. The column was directly eluted with Elution Buffer B (20 mM HAc-NaAc, 20 mM NaCl, 1 mM EDTA) and the eluted protein was collected according to the significant increase in absorption at 280 nm. Bradford test kit (Biyotime Company, Wuhan, China) was used to access the concentration of the protein. Western blot analysis was performed on the rrsRAGE using an anti-RAGE antibody (Boster Company, Wuhan, China) and standard procedures. A silver staining test (Novland, Shanghai, China) was conducted to assess the purity of the protein, in which the percentage was analyzed by Fuji LAS-3000 digital imaging workstation and ADVANCED IMAGE DATA ANALYSER(AIDA) software(Fujifilm, Tokyo, Japan). Bioactivity identification of rrsRAGE 5
ACCEPTED MANUSCRIPT The CCK-8 growth inhibition assay was performed to assess the effect of rrsRAGE on SH-SY5Y cells with/without S100A6. SH-SY5Y cells at log phase were diluted to 2×105/mL in cell culture medium(RPMI 1640 medium, Gibco, Shanghai, China) with 10% FBS(Gibco, Shanghai, China) and then incubated in a 96-well plate overnight. After 24 h starvation in RPMI 1640 medium without FBS,
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the medium in each well was replaced with 100 µL medium containing different concentrations of rrsRAGE (0, 10, 30, 50, 150, 200 µg/mL) and S100A6 (50 µg/mL). PBS was used to adjust all wells to the same volume. This test was carried out for 48 h and was repeated six times. After incubation, the medium was replaced with 100 µL of RPMI 1640 medium containing CCK-8(Biyotime Company,
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Wuhan, China) (1:9). The cells were incubated for 1.5 h and the cell density was then measured on a
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plate-reading spectrophotometer (OD450). This process was performed three times.
Identification of the therapeutic effect of rrsRAGE in CCl4-induced rat models All animals were maintained under standard conditions and animal experiments were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University. SD rats, weighing 120-130g, were used and all animals received intraperitoneal injection of 2 ml/kg carbontetrachloride (CCl4)-olive oil
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mixture (1:1) at 1st week and 2nd week and 1ml/kg carbontetrachloride(CCl4)-olive oil mixture (1:1) from 3rd week to the end of 8th week, all twice a week. In addition, another control group (sham group, three rats) with no injection and no treatment was settled for the test of serum levels of ALT. Rats were initially randomly divided into two groups: an experiment group(three rats) who received a
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subcutaneous injection of rrsRAGE at 1.5mg/kg/day from 5th week to the end of 8th week and a
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negative control group(three rats) who received an injection of PBS in the same volume as above. After 8 weeks, all rats were sacrificed and liver samples were fixed and embedded in paraffin, and then the embedded liver samples were cut into 5-µm-thick sections using standard procedures. Masson three-color staining (Jian Cheng, Nanjing, China) was used to stain the sections before microscopic imaging. The stain imparts a blue color to collagen (fibrosis section) against a red background of hepatocytes and other structures. The grade of liver fibrosis was scored following the previous study[16]. Serum samples of all rats were also collected and serum levels of ALT were also tested by ALT test kit (Jian Cheng, Nanjing, China). Results Construction of rrsRAGE expression vectors 6
ACCEPTED MANUSCRIPT To produce rrsRAGE in high yield, specific nucleotide sequences optimized for yeast translation were designed, synthesized, and ligated into the PMD18T-simple vector. The final product (full-length rrsRAGE) was double digested with XhoI and NotI and cloned into the expression vector (pPICZαA vector) to create the recombinant plasmid pPICZαA vector-rrsRAGE (Fig. 1a). The insert was
High expression of rrsRAGE in P. pastoris wild-type strain X-33
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confirmed by PCR amplification (Lane 2 in Fig. S1) and DNA sequencing.
Recombinant plasmids of pPICZαA vector-rrsRAGE were transformed into P. pastoris strain X-33.
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Single clones were analyzed and transformants containing the correct insert were confirmed (Fig. S2). Then, the expression levels of the target protein were monitored using immunochemical techniques and
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detection with a mouse anti-RAGE monoclonal antibody after absorption of the protein onto a PVDF membrane. Figure 1b shows the different expression levels of these single clones and the clone with the highest expression level (indicated by a black circle) was selected for large-scale protein production.
Pilot-scale production of rrsRAGE in yeast fermentation tank
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To obtain large amounts of recombinant rrsRAGE, a simple fermentation procedure in a 30-L fermenter was performed. The OD600 of the culture of the P. pastoris strain harboring pPICZαA vector-rrsRAGE approached 55 after 30 h of fermentation. Then, the fermentation entered the fed-batch phase, when 50%
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(w/v) glycerol was pumped at 2 mL/L/h for 4 h, and the biomass level (wet cell weight) increased from 123 to 145g/L. The subsequent induction of methanol resulted in a rapid increase of yeast cells between
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40-h and 90-h fermentation that plateaued after 90 h. At the end of the fermentation, the wet cell weight reached approximately 323g/L (Fig. 2). SDS-PAGE analysis of the yield in the supernatant showed that the recombinant protein was expressed as a soluble product with a molecular weight of about 34 kDa in accordance with the target protein of sRAGE (Fig. 2).
Purification and identification of rrsRAGE Two peaks, designated P3 and P4, as shown in Fig.3a, were eluted upon cation exchange chromatography and the target protein was determined to be 85% purified by SDS-PAGE after dialysis desalting (Fig. 3b and S3). The products were further purified by Superdex 75 prep grade and the samples in the major peak were collected (Fig. S4). Silver staining confirmed that the purity of the final 7
ACCEPTED MANUSCRIPT product was above 97% and western blotting also indicated this protein was rat sRAGE (Fig. 4). Finally, 200 mg of protein was obtained from the 1-L yeast fermentation. The protein mass was calculated and summarized for each purification step. The final yield of rrsRAGE was 21.8% (Table 1).
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3D structure of rat sRAGE has also been predicted using online software[17](Fig.S5).
Recombinant rsRAGE protein inhibits the apoptosis of SH-SY5Y cells induced by S100A6
In a previous study, S100A6 was genetically recombined and shown to induce the apoptosis of SH-SY5Y cells [18]. Soluble RAGE is a natural inhibitor of S100A6, therefore the activity of S100A6
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in SH-SY5Y cells may be inhibited by sRAGE. As shown by cell viability assays, the exposure of serum-starved SH-SY5Y cells to increasing concentrations of rrsRAGE for 48 h resulted in a
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dose-dependent increase in cell viability. While 50 mg/mL of S100A6 significantly decreased cell viability to 55% compared with PBS-treated cells, which was consistent with a previous study [18] 30 mg/mL of rrsRAGE inhibited cell apoptosis resulting in 85% cell viability compared with PBS-treated cells (Fig.5). Thus, the purified rrsRAGE was biologically active and could be used in physiological
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and pathological studies in vitro.
Recombinant rsRAGE attenuates liver fibrosis and decreases serum level of ALT After the treatment of rrsRAGE, the mean grade of liver fibrosis(2.07) was significantly lower(P<0.001)
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than for the PBS group(3.73)(Fig.6a and b). It was also found that lower serum level of ALT appeared in the rrsRAGE treatment group(P<0.01) than in the PBS control group(Fig.6c).These results indicates
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that rrsRAGE can attenuate liver fibrosis and decrease serum level of ALT.
Discussion
As previously shown in many pharmacological studies, sRAGE has a high therapeutic potential for certain diseases including diabetes[19]. In addition, as a natural antagonist of RAGE, inhibiting the binding of ligands such as AGEs, the S100 family, HMGB1, amyloid β peptides, and glycosaminoglycan, it also plays an important role in the ligand-RAGE interaction involved in several diseases including systemic lupus erythematosus [20] subclinical atherosclerosis [21] and Alzheimer’s disease [22]. Recently, sRAGE has been increasingly proposed as a new treatment target for diseases such as polycystic kidney disease [23] obesity, and metabolic syndromes [24]. In our previous study, 8
ACCEPTED MANUSCRIPT we found that some members of the S100 family could accelerate liver fibrosis in CCl4 and BDL models (data not shown). Other ligands of RAGE, such as AGE, S100A4, and HMGB1, have also been shown to activate hepatic stellate cells, the target cells of liver fibrosis [25-27]. To further our studies on sRAGE as a treatment target, it was first necessary to develop a method for the expression and
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purification of recombinant rat sRAGE. The pPICZαA vector, containing a methanol-inducible alcohol oxidase promoter, the strongest and most tightly regulated promoter among the yeast species [28] was employed in the present study. We also designed the optimal nucleotide sequences for protein translation in P. pastoris. Protein
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production from P. pastoris strain X-33 was scalable and to further improve the yield, multiple single clones of the rrsRAGE-X-33 strain were screened and the clone exhibiting the highest expression level
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was selected. The fermentation conditions were optimized and a final yield of 200 mg/L was achieved, with above 97% purification of the protein and confirmed bio-activation. Therefore, we have developed a novel method for the rapid pilot-scale production of high-quality rrsRAGE based on a yeast expression system.
The bioactivity of rrsRAGE was verified both in vitro and in vivo in this study. The rrsRAGE
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could block S100A6-induced inhibition of the proliferation of SH-SY5Y cells, revealing that our rrsRAGE can bind to S100A6 protein in vitro. In the rat CCl4-induced models, it was also observed that rrsRAGE could prevent liver from fibrosis and injury, demonstrating that our rrsRAGE protein can be
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used for medical research.
Compliance with Ethical Standards:
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This study was funded by a Science and Technology Program of Shanghai Grant (NO.12431900600). All Authors declare that he/she has no conflict of interest. All guidelines of the Animal Care and Use Committee of Shanghai Jiao Tong University for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. References
[1] A.M. Schmidt, M. Vianna, M. Gerlach, J. Brett, J. Ryan, J. Kao, C. Esposito, H. Hegarty, W. Hurley, M. Clauss, Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface. J Biol Chem 267 (1992) 14987-14997. [2] J.L. Wautier, C. Zoukourian, O. Chappey, M.P. Wautier, P.J. Guillausseau, R. Cao, O. Hori, D. Stern, A.M. Schmidt, Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats. J Clin Invest 97 9
ACCEPTED MANUSCRIPT (1996) 238-243. [3] S. Zeng, N. Feirt, M. Goldstein, J. Guarrera, N. Ippagunta, U. Ekong, H. Dun, Y. Lu, W. Qu, A.M. Schmidt, J.C. Emond, Blockade of receptor for advanced glycation end product (RAGE) attenuates ischemia and reperfusion injury to the liver in mice. Hepatology 39 (2004) 422-432. [4] T. Miyata, Y. Wada, Z. Cai, Y. Iida, K. Horie, Y. Yasuda, K. Maeda, K. Kurokawa, C. van Ypersele de Strihou, Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure. Kidney Int 51 (1997) 1170-1181.
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[5] V. Meneghini, V. Bortolotto, M.T. Francese, A. Dellarole, L. Carraro, S. Terzieva, M. Grilli, High-mobility group box-1 protein and beta-amyloid oligomers promote neuronal differentiation of adult hippocampal neural progenitors via receptor for advanced glycation end products/nuclear factor-kappaB axis: relevance for Alzheimer's disease. J Neurosci 33 (2013) 6047-6059.
[6] P.R. Reynolds, K.M. Wasley, C.H. Allison, Diesel particulate matter induces receptor for advanced glycation end-products (RAGE) expression in pulmonary epithelial cells, and RAGE signaling influences
SC
NF-kappaB-mediated inflammation. Environ Health Perspect 119 (2011) 332-336.
[7] K. Ota, S. Yamagishi, M. Kim, S. Dambaeva, A. Gilman-Sachs, K. Beaman, J. Kwak-Kim, Elevation of soluble form of receptor for advanced glycation end products (sRAGE) in recurrent pregnancy losses
M AN U
(RPL): possible participation of RAGE in RPL. Fertil Steril 102 (2014) 782-789.
[8] H. Maillard-Lefebvre, E. Boulanger, M. Daroux, C. Gaxatte, B.I. Hudson, M. Lambert, Soluble receptor for advanced glycation end products: a new biomarker in diagnosis and prognosis of chronic inflammatory diseases. Rheumatology (Oxford) 48 (2009) 1190-1196.
[9] T. Ostendorp, M. Weibel, E. Leclerc, P. Kleinert, P.M. Kroneck, C.W. Heizmann, G. Fritz, Expression and purification of the soluble isoform of human receptor for advanced glycation end products (sRAGE) from Pichia pastoris. Biochem Biophys Res Commun 347 (2006) 4-11.
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[10] M.A. Hofmann, S. Drury, C. Fu, W. Qu, A. Taguchi, Y. Lu, C. Avila, N. Kambham, A. Bierhaus, P. Nawroth, M.F. Neurath, T. Slattery, D. Beach, J. McClary, M. Nagashima, J. Morser, D. Stern, A.M. Schmidt, RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 97 (1999) 889-901. [11] C. Renard, O. Chappey, M.P. Wautier, M. Nagashima, E. Lundh, J. Morser, L. Zhao, A.M. Schmidt, Scherrmann, J.L.
Wautier, Recombinant advanced glycation
end product receptor
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J.M.
pharmacokinetics in normal and diabetic rats. Mol Pharmacol 52 (1997) 54-62. [12] H. Asada, T. Uemura, T. Yurugi-Kobayashi, M. Shiroishi, T. Shimamura, H. Tsujimoto, K. Ito, T.
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Sugawara, T. Nakane, N. Nomura, T. Murata, T. Haga, S. Iwata, T. Kobayashi, Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis. Microbial cell factories 10 (2011) 24.
[13] J.R. Xia, N.F. Liu, N.X. Zhu, Specific siRNA targeting the receptor for advanced glycation end products inhibits experimental hepatic fibrosis in rats. International journal of molecular sciences 9 (2008) 638-661.
[14] Y.S. Seo, J.H. Kwon, U. Yaqoob, L. Yang, T.M. De Assuncao, D.A. Simonetto, V.K. Verma, V.H. Shah, HMGB1 recruits hepatic stellate cells and liver endothelial cells to sites of ethanol-induced parenchymal cell injury. American journal of physiology Gastrointestinal and liver physiology 305 (2013) G838-848. [15] L. Chen, J. Li, J. Zhang, C. Dai, X. Liu, J. Wang, Z. Gao, H. Guo, R. Wang, S. Lu, F. Wang, H. Zhang, H. Chen, X. Fan, S. Wang, Z. Qin, S100A4 promotes liver fibrosis via activation of hepatic stellate cells. Journal of hepatology 62 (2015) 156-164. 10
ACCEPTED MANUSCRIPT [16] K. Ishak, A. Baptista, L. Bianchi, F. Callea, J. De Groote, F. Gudat, H. Denk, V. Desmet, G. Korb, R.N. MacSween, et al., Histological grading and staging of chronic hepatitis. Journal of hepatology 22 (1995) 696-699. [17] L.A. Kelley, S. Mezulis, C.M. Yates, M.N. Wass, M.J. Sternberg, The Phyre2 web portal for protein modeling, prediction and analysis. Nature protocols 10 (2015) 845-858. [18] H. He, T. Yang, S. Jia, R. Zhang, P. Tu, J. Gao, Y. Yuan, W. Han, Y. Yu, Expression and purification of bioactive high-purity human S100A6 in Escherichia coli. Protein Expr Purif 83 (2012) 98-103.
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[19] J.W. Nin, A. Jorsal, I. Ferreira, C.G. Schalkwijk, M.H. Prins, H.H. Parving, L. Tarnow, P. Rossing, C.D. Stehouwer, Higher plasma soluble Receptor for Advanced Glycation End Products (sRAGE) levels are associated with incident cardiovascular disease and all-cause mortality in type 1 diabetes: a 12-year follow-up study. Diabetes 59 (2010) 2027-2032.
[20] H.A. Martens, H.L. Nienhuis, S. Gross, G. van der Steege, E. Brouwer, J.H. Berden, R.G. de Sevaux, R.H. Derksen, A.E. Voskuyl, S.P. Berger, G.J. Navis, I.M. Nolte, C.G. Kallenberg, M. Bijl, Receptor for
SC
advanced glycation end products (RAGE) polymorphisms are associated with systemic lupus erythematosus and disease severity in lupus nephritis. Lupus 21 (2012) 959-968.
[21] A.W. Souza, K. de Leeuw, M.M. van Timmeren, P.C. Limburg, C.A. Stegeman, M. Bijl, J. Westra, C.G.
M AN U
Kallenberg, Impact of serum high mobility group box 1 and soluble receptor for advanced glycation end-products on subclinical atherosclerosis in patients with granulomatosis with polyangiitis. PLoS One 9 (2014) e96067.
[22] W. Wan, H. Chen, Y. Li, The potential mechanisms of Abeta-receptor for advanced glycation end-products interaction disrupting tight junctions of the blood-brain barrier in Alzheimer's disease. Int J Neurosci 124 (2014) 75-81.
[23] E.Y. Park, B.H. Kim, E.J. Lee, E. Chang, D.W. Kim, S.Y. Choi, J.H. Park, Targeting of receptor for (2014) 9254-9262.
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advanced glycation end products suppresses cyst growth in polycystic kidney disease. J Biol Chem 289 [24] C.T. He, C.H. Lee, C.H. Hsieh, F.C. Hsiao, P. Kuo, N.F. Chu, Y.J. Hung, Soluble Form of Receptor for Advanced Glycation End Products Is Associated with Obesity and Metabolic Syndrome in Adolescents. Int J Endocrinol (2014).
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[25] M. Goodwin, C. Herath, Z. Jia, C. Leung, M.T. Coughlan, J. Forbes, P. Angus, Advanced glycation end products augment experimental hepatic fibrosis. J Gastroenterol Hepatol 28 (2013) 369-376. [26] L. Chen, J. Li, J. Zhang, C. Dai, X. Liu, J. Wang, Z. Gao, H. Guo, R. Wang, S. Lu, F. Wang, H. Zhang, H.
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Chen, X. Fan, S. Wang, Z. Qin, S100A4 promotes liver fibrosis via activation of hepatic stellate cells. J Hepatol (2014).
[27] Y.H. Kao, Y.C. Lin, M.S. Tsai, C.K. Sun, S.S. Yuan, C.Y. Chang, B. Jawan, P.H. Lee, Involvement of the nuclear high mobility group B1 peptides released from injured hepatocytes in murine hepatic fibrogenesis. Biochim Biophys Acta 1842 (2014) 1720-1732. [28] N. Bora, Large-scale production of secreted proteins in Pichia pastoris. Methods Mol Biol 866 (2012) 217-235. Figure legends Fig. 1 Cloning of rat soluble RAGE (rsRAGE) nucleotide sequences into pPICZαA and Detection and selection of P. pastoris transformants containing rsRAGE-pPICZαA (a) The pPICZαA vector with rsRAGE nucleotide sequences. (b) Selection of a highly productive 11
ACCEPTED MANUSCRIPT single colony of P. pastoris wild-type strain X-33 using western blot analysis, the single clone in the black circle was selected.
Fig. 2 Large-scale production of rrsRAGE protein in P. pastoris wild-type strain X-33
expression at 0 and 96 h. Fig. 3 Purification of rrsRAGE from P. pastoris wild-type strain X-33
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The density (OD600) and wet weight of yeast cells per 1 mL and SDS-PAGE analysis of rrsRAGE
(a) Elution profiles of the purified supernatants from X-33/rrsRAGE after column filtration through a
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SP Sepharose FF column. (b) SDS-PAGE analysis of rrsRAGE expression. Lane 1: supernatant from X-33/rrsRAGE; lane 2: washing solution (wash step in picture a); lanes 3–8: elution solution (P1–P6
Fig. 4 Further purification of rrsRAGE
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steps in image a).
(a) Elution profiles from column filtration using Superedex 75 prep grade. (b) SDS-PAGE analysis of rrsRAGE expression. Lane 0: washing buffer (negative control); lanes 1–7: elution of markers (1–7) in
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image (a).
Fig. 5 Detection of the purification of rrsRAGE from the collection of lanes 3–7 in Fig. 5b.
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(a) Silver staining analysis. (b) Western blot analysis.
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Fig.6 Bio-activity analysis of different concentrations of rrsRAGE in the SH-SY5Y cell line after the addition of S100A6
Increasing concentrations of rrsRAGE were added. PBS was used as a negative control. Proliferation was quantified using a CCK-8 kit by measuring the absorbance at 450 nm.(* represent for p<0.05; **
represent for p<0.01; ***represent for p<0.001, compared with 50µg/ml S100A6(+) and no rrsRAGE)
Fig. 7 the treatment of rrsRAGE in CCl4-induced rat model (a) Masson three-color staining of liver tissue, blue section represents the area of the fibrosis. (b) Score of liver fibrosis. (c) Serum levels of ALT. (* represent for p<0.05; ** represent for p<0.01; 12
ACCEPTED MANUSCRIPT ***represent for p<0.001)
Fig.S1 The vectors were checked by PCR amplification: M: marker; lane 1: negative control, pPICZαA vector only; lane 2: pPICZαA vector with rsRAGE nucleotide sequences.
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Fig.S2 Detection of rsRAGE-pPICZαA transformants in P. pastoris by PCR amplification: M: marker; lane 1: negative control (water); lane 2: positive control (plasmid); lanes 3–8: samples from cells with high yield.
Fig.S3 SDS-PAGE analysis of rrsRAGE expression via dialysis desalting. Lane 1: the supernatant from
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X-33/rrsRAGE; lane 2: the elution solution of P3 and P4; lane 3: the eluant after dialysis desalting Fig.S4 Further purification of rrsRAGE
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(a) Elution profiles from column filtration using Superedex 75 prep grade. (b) SDS-PAGE analysis of rrsRAGE expression. Lane 0: washing buffer (negative control); lanes 1–7: elution of markers (1–
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ACCEPTED MANUSCRIPT Table 1 Purification of rrsRAGE using the Pichia pastoris expression system Total protein (mg)
rrsRAGE purification (%)
Total rrsRAGE (mg)
Protein yield (%)
Supernatant
2340.0
39.2
917.3
100.0
SP Sepharose FF
419.7
85.2
357.6
39.0
Superdex 75
205.0
97.4
200.0
21.8
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Purification step
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Highlights: 1. We found a new way to do expression and purification of rat sRAGE protein in Pichia pastoris 2. The sRAGE protein could bind the ligand of RAGE, S100A6, in vitro. 3. The sRAGE protein could prevent rats from liver injury and liver fibrosis in vivo.
S1046-5928(15)30079-6
DOI:
10.1016/j.pep.2015.09.029
Reference:
YPREP 4804
To appear in:
Protein Expression and Purification
Received Date: 12 August 2015 Revised Date:
16 September 2015
Accepted Date: 30 September 2015
Please cite this article as: P. Xia, J. Gao, W. Guan, J. Li, X. Yu, F. Wang, H. He, Q. Deng, L. Zhou, Y. Yuan, W. Han, Y. Yu, Production of bioactive recombinant rat soluble receptor for advanced glycation end products (rrsRAGE) in Pichia pastoris, Protein Expression and Purification (2015), doi: 10.1016/ j.pep.2015.09.029. 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.
ACCEPTED MANUSCRIPT Production of bioactive recombinant rat soluble receptor for advanced glycation end products (rrsRAGE) in Pichia pastoris
Peng Xia1*, Jin Gao2*, Wen Guan1, Jingjing Li2, Xiaolan Yu1, Fangyuan Wang3, Honglin He1, Qing
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Deng1, Liang Zhou1, Yunsheng Yuan1, Wei Han2#, Yan Yu1#
Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and
2
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Biology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China Laboratory of Regenerative Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai
200240, People’s Republic of China 3
Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated First People’s
Hospital, Shanghai 200080, People’s Republic of China *The authors contributed equally to this work #Corresponding authors: E-mail address: [email protected] (Yan Yu) [email protected] (Wei Han) Tel. and Fax: +86 21 34205833 1
ACCEPTED MANUSCRIPT Abstract Soluble receptor for advanced glycation end products (sRAGE), a natural inhibitor of RAGE, is considered to be a putative therapeutic molecule for a variety of diseases and a biomarker for certain conditions. To further study the function of sRAGE, recombinant rat sRAGE (rrsRAGE) was expressed
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and produced in a eukaryotic system. The open reading frame of rat sRAGE was cloned downstream of the methanol-inducible alcohol oxidase promoter of pPICZαA vector, and Pichia pastoris strain X-33 was used as the host strain. The expression of rrsRAGE was achieved by fermentation in a 15-L bioreactor and the resulting fermentation broth was subjected to purification on a cation exchange
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chromatography column. The purification of rrsRAGE reached 95% after size exclusion chromatography(SEC). The bioactivity of the purified protein was confirmed in a SH-SY5Y cell
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proliferation assay. The biological function of the purified rrsRAGE protein rat CCl4-induced model was then examined. Treatment with rrsRAGE resulted in significantly lower liver fibrosis and lower serum level of ALT, suggesting that sRAGE prevent liver from injury and fibrosis. In conclusion, we achieved high-efficiency production of bioactive rrsRAGE in Pichia pastoris.
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Keywords: Soluble receptor for advanced glycation end products (sRAGE), expression and
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purification, Pichia pastoris
2
ACCEPTED MANUSCRIPT Introduction Receptor for advanced glycation end products (RAGE) was first identified as a trans-membrane receptor for final products of glycation and oxidation of proteins and lipids [1] .RAGE can bind several clusters of ligands, including advanced glycation end products (AGEs), high-mobility group box 1
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protein (HMGB1), most S100 family members, and β-amyloid peptides. RAGE plays a central role in diabetes mellitus and exerts a significant effect on diabetic diseases such as diabetic vasculopathy and atherosclerosis [2]. Studies also suggested that expression of RAGE correlated closely to a diverse set of diseases, including types of liver disease [3] renal failure [4] neural degeneration [5] and
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inflammation [6]. Recent clinical research revealed the serum level of soluble RAGE (sRAGE) could act as a biomarker for certain human conditions, such as recurrent pregnancy loss [7] and chronic
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inflammatory disease [8].
Soluble RAGE, a natural inhibitor of RAGE, is widely considered to be a putative therapeutic molecule for a variety of diseases. Although human sRAGE has been expressed by a number of different hosts including Pichia pastoris and Escherichia coli [9, 10] it cannot be used in a rat model because human sRAGE (www.uniprot.org; Q15109 [23–342]) and rat sRAGE (rsRAGE)
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(www.uniprot.org; Q63495 [23–342]) only share 57.01% amino acid sequence homology. This huge difference in the protein sequence of human sRAGE may induce an immunological response in rats. However, the rat bile duct ligand (BDL) model is the most appropriate model in which to study the
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effect of sRAGE on liver protection, therefore another source of sRAGE is required for such studies. The rat sRAGE mRNA encodes 319 amino acids with a calculated molecular weight of 33.86kDa and
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rat sRAGE has an acidic pI value of 6.73. Previously, it was reported that rsRAGE had been produced from insect cells (Sf9 cells) [11] However, because a large amount of sRAGE is required for rat experiments, the insect cell-based expression system cannot meet such requirements because it is difficult and expensive to scale up [12] We therefore proposed P. pastoris or E. coli as vectors for the production of rat sRAGE protein. Given the glycation of the sRAGE protein, the P. pastoris system was selected as being more suitable. Previously, several studies have proved that blockade of RAGE, namely sRAGE administration, could prevent liver from injury and fibrosis in various of animal models. The treatment of sRAGE could attenuate ischemia and reperfusion injury to the liver and acetaminophen-induced hepatotoxicity in mice[3]. Specific siRNA targeting RAGE could also inhibits experimental hepatic fibrosis in rats 3
ACCEPTED MANUSCRIPT [13]. In the other hand, the ligands of RAGE, HMGB1[14], S100A4[15] and AGE, also have been demonstrated capable of promoting the process of liver injury or liver fibrosis. Furthermore, in our previous study, S100A6 has been demonstrated to accelerate liver fibrosis in CCl4-induced animal models. Therefore, we are going to test the in vivo bioactivity of rrsRAGE in CCl4-induced rat models.
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In the present paper, we report a novel, rapid and high-yield purification protocol for recombinant rsRAGE (rrsRAGE) from P. pastoris. The final product was verified to be of high purity and have low endotoxicity, and was shown to be bioactive using cell viability assays. Purified rrsRAGE protein was shown to attenuate liver fibrosis and decrease serum level of alanine aminotransferase(ALT), the
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increase of which could be caused by liver damage.
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Methods Cloning of rsRAGE nucleotide sequences into pPICZαA The
amino
acid
sequence
of
rsRAGE
was
obtained
from
the
Uniprot
website
(www.uniprot.org;Q15109[23-342]) and the coding sequence was optimized according for yeast translation. The sequence with 957 base pairs (bp) was synthesized and cloned into PMD18T-simple
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vector at two restriction sites XhoI and NotI by Genescript Company (Nanjing, China). Then, the rsRAGE-PMD18T-simple vector and the pPICZαA vector were double-digested by XhoI and NotI, the digested products were purified using a DNA extraction kit, and were then ligated using T4-DNA ligase.
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Electrocompetent E. coli strain DH5α cells were prepared and transformed with 50 ng DNA from this ligation reaction mixture and plated on zeocin-resisted Luria Broth (LB) agar. Single colonies were to
identify
the
correct
insert
using
PCR
amplification
with
primers
(S:
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selected
5′-CCAAAGAAGCCAACTCAA-3′; A: 5′-TAAGAACCCTCGGAAACA-3') and transformants were confirmed by sequencing. A single colony with the correct insert was then incubated in LB and the plasmid was prepared and stored for later use.
Transformation and selection of a highly productive single colony of P. pastoris strain X-33 rrsRAGE was transformed into P. pastoris strain X-33. Single colonies were patched on BMGY/BMMY media to check the expression levels [9] The P. pastoris strain X-33-rrsRAGE colonies with higher expression levels were monitored by absorption of the protein to a PVDF membrane and detection by immunohistological techniques. Image J( National Institutes of Health, USA) was used for 4
ACCEPTED MANUSCRIPT expression level quantification. The colony with the highest expression level was selected for the next pilot-scale expression and purification.
Pilot-scale production of rrsRAGE protein in P. pastoris strain X-33
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Production of rrsRAGE protein in X-33 was controlled by the AOX1 promoter in pPICZαA vector. Four stages were followed: (1) Initializing batch phase: 40 g/L glycerol in basal salts medium was used until the OD600 approached 50–70; (2) fed-batch phase: 50% (w/v) glycerol was pumped at 2 mL/L/h for 4 h; (3) transition phase: 1 mL of 100% methanol was added per 1 L culture to allow for a smooth
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transition into the induction phase; (4) induction phase: methanol alone was fed over a time period of 96 h using a methanol probe to control the methanol concentration. Then, the fermentation broth was
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centrifuged to remove yeast cells and the supernatant was collected for the following purification.
Purification of rrsRAGE
The supernatant, obtained as described above, was filtered through a 0.2-µm membrane, then diluted five-fold with distilled water until the conductivity decreased to below 8 ms/cm. The pH was adjusted
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to 5.0 using acetic acid (HAc) solution. The resulting solution was finally subjected to cation exchange chromatography on a SP Sepharose FF (Amersham Biosciences, Piscataway, NJ, USA) (60 mL) column. After loading, the column was washed with Wash Buffer (20 mM HAc-NaAc, 1 mM EDTA,
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pH 5.0) until the absorption at 280 nm reached the baseline level. The rrsRAGE protein was eluted with a linear gradient of 0–100% in Elution Buffer A (20 mM HAc-NaAc, 1 M NaCl, 1 mM EDTA).
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The elution containing rrsRAGE was loaded on a Superedex 75 (Amersham Biosciences) 90-cm column. The column was directly eluted with Elution Buffer B (20 mM HAc-NaAc, 20 mM NaCl, 1 mM EDTA) and the eluted protein was collected according to the significant increase in absorption at 280 nm. Bradford test kit (Biyotime Company, Wuhan, China) was used to access the concentration of the protein. Western blot analysis was performed on the rrsRAGE using an anti-RAGE antibody (Boster Company, Wuhan, China) and standard procedures. A silver staining test (Novland, Shanghai, China) was conducted to assess the purity of the protein, in which the percentage was analyzed by Fuji LAS-3000 digital imaging workstation and ADVANCED IMAGE DATA ANALYSER(AIDA) software(Fujifilm, Tokyo, Japan). Bioactivity identification of rrsRAGE 5
ACCEPTED MANUSCRIPT The CCK-8 growth inhibition assay was performed to assess the effect of rrsRAGE on SH-SY5Y cells with/without S100A6. SH-SY5Y cells at log phase were diluted to 2×105/mL in cell culture medium(RPMI 1640 medium, Gibco, Shanghai, China) with 10% FBS(Gibco, Shanghai, China) and then incubated in a 96-well plate overnight. After 24 h starvation in RPMI 1640 medium without FBS,
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the medium in each well was replaced with 100 µL medium containing different concentrations of rrsRAGE (0, 10, 30, 50, 150, 200 µg/mL) and S100A6 (50 µg/mL). PBS was used to adjust all wells to the same volume. This test was carried out for 48 h and was repeated six times. After incubation, the medium was replaced with 100 µL of RPMI 1640 medium containing CCK-8(Biyotime Company,
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Wuhan, China) (1:9). The cells were incubated for 1.5 h and the cell density was then measured on a
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plate-reading spectrophotometer (OD450). This process was performed three times.
Identification of the therapeutic effect of rrsRAGE in CCl4-induced rat models All animals were maintained under standard conditions and animal experiments were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University. SD rats, weighing 120-130g, were used and all animals received intraperitoneal injection of 2 ml/kg carbontetrachloride (CCl4)-olive oil
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mixture (1:1) at 1st week and 2nd week and 1ml/kg carbontetrachloride(CCl4)-olive oil mixture (1:1) from 3rd week to the end of 8th week, all twice a week. In addition, another control group (sham group, three rats) with no injection and no treatment was settled for the test of serum levels of ALT. Rats were initially randomly divided into two groups: an experiment group(three rats) who received a
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subcutaneous injection of rrsRAGE at 1.5mg/kg/day from 5th week to the end of 8th week and a
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negative control group(three rats) who received an injection of PBS in the same volume as above. After 8 weeks, all rats were sacrificed and liver samples were fixed and embedded in paraffin, and then the embedded liver samples were cut into 5-µm-thick sections using standard procedures. Masson three-color staining (Jian Cheng, Nanjing, China) was used to stain the sections before microscopic imaging. The stain imparts a blue color to collagen (fibrosis section) against a red background of hepatocytes and other structures. The grade of liver fibrosis was scored following the previous study[16]. Serum samples of all rats were also collected and serum levels of ALT were also tested by ALT test kit (Jian Cheng, Nanjing, China). Results Construction of rrsRAGE expression vectors 6
ACCEPTED MANUSCRIPT To produce rrsRAGE in high yield, specific nucleotide sequences optimized for yeast translation were designed, synthesized, and ligated into the PMD18T-simple vector. The final product (full-length rrsRAGE) was double digested with XhoI and NotI and cloned into the expression vector (pPICZαA vector) to create the recombinant plasmid pPICZαA vector-rrsRAGE (Fig. 1a). The insert was
High expression of rrsRAGE in P. pastoris wild-type strain X-33
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confirmed by PCR amplification (Lane 2 in Fig. S1) and DNA sequencing.
Recombinant plasmids of pPICZαA vector-rrsRAGE were transformed into P. pastoris strain X-33.
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Single clones were analyzed and transformants containing the correct insert were confirmed (Fig. S2). Then, the expression levels of the target protein were monitored using immunochemical techniques and
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detection with a mouse anti-RAGE monoclonal antibody after absorption of the protein onto a PVDF membrane. Figure 1b shows the different expression levels of these single clones and the clone with the highest expression level (indicated by a black circle) was selected for large-scale protein production.
Pilot-scale production of rrsRAGE in yeast fermentation tank
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To obtain large amounts of recombinant rrsRAGE, a simple fermentation procedure in a 30-L fermenter was performed. The OD600 of the culture of the P. pastoris strain harboring pPICZαA vector-rrsRAGE approached 55 after 30 h of fermentation. Then, the fermentation entered the fed-batch phase, when 50%
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(w/v) glycerol was pumped at 2 mL/L/h for 4 h, and the biomass level (wet cell weight) increased from 123 to 145g/L. The subsequent induction of methanol resulted in a rapid increase of yeast cells between
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40-h and 90-h fermentation that plateaued after 90 h. At the end of the fermentation, the wet cell weight reached approximately 323g/L (Fig. 2). SDS-PAGE analysis of the yield in the supernatant showed that the recombinant protein was expressed as a soluble product with a molecular weight of about 34 kDa in accordance with the target protein of sRAGE (Fig. 2).
Purification and identification of rrsRAGE Two peaks, designated P3 and P4, as shown in Fig.3a, were eluted upon cation exchange chromatography and the target protein was determined to be 85% purified by SDS-PAGE after dialysis desalting (Fig. 3b and S3). The products were further purified by Superdex 75 prep grade and the samples in the major peak were collected (Fig. S4). Silver staining confirmed that the purity of the final 7
ACCEPTED MANUSCRIPT product was above 97% and western blotting also indicated this protein was rat sRAGE (Fig. 4). Finally, 200 mg of protein was obtained from the 1-L yeast fermentation. The protein mass was calculated and summarized for each purification step. The final yield of rrsRAGE was 21.8% (Table 1).
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3D structure of rat sRAGE has also been predicted using online software[17](Fig.S5).
Recombinant rsRAGE protein inhibits the apoptosis of SH-SY5Y cells induced by S100A6
In a previous study, S100A6 was genetically recombined and shown to induce the apoptosis of SH-SY5Y cells [18]. Soluble RAGE is a natural inhibitor of S100A6, therefore the activity of S100A6
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in SH-SY5Y cells may be inhibited by sRAGE. As shown by cell viability assays, the exposure of serum-starved SH-SY5Y cells to increasing concentrations of rrsRAGE for 48 h resulted in a
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dose-dependent increase in cell viability. While 50 mg/mL of S100A6 significantly decreased cell viability to 55% compared with PBS-treated cells, which was consistent with a previous study [18] 30 mg/mL of rrsRAGE inhibited cell apoptosis resulting in 85% cell viability compared with PBS-treated cells (Fig.5). Thus, the purified rrsRAGE was biologically active and could be used in physiological
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and pathological studies in vitro.
Recombinant rsRAGE attenuates liver fibrosis and decreases serum level of ALT After the treatment of rrsRAGE, the mean grade of liver fibrosis(2.07) was significantly lower(P<0.001)
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than for the PBS group(3.73)(Fig.6a and b). It was also found that lower serum level of ALT appeared in the rrsRAGE treatment group(P<0.01) than in the PBS control group(Fig.6c).These results indicates
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that rrsRAGE can attenuate liver fibrosis and decrease serum level of ALT.
Discussion
As previously shown in many pharmacological studies, sRAGE has a high therapeutic potential for certain diseases including diabetes[19]. In addition, as a natural antagonist of RAGE, inhibiting the binding of ligands such as AGEs, the S100 family, HMGB1, amyloid β peptides, and glycosaminoglycan, it also plays an important role in the ligand-RAGE interaction involved in several diseases including systemic lupus erythematosus [20] subclinical atherosclerosis [21] and Alzheimer’s disease [22]. Recently, sRAGE has been increasingly proposed as a new treatment target for diseases such as polycystic kidney disease [23] obesity, and metabolic syndromes [24]. In our previous study, 8
ACCEPTED MANUSCRIPT we found that some members of the S100 family could accelerate liver fibrosis in CCl4 and BDL models (data not shown). Other ligands of RAGE, such as AGE, S100A4, and HMGB1, have also been shown to activate hepatic stellate cells, the target cells of liver fibrosis [25-27]. To further our studies on sRAGE as a treatment target, it was first necessary to develop a method for the expression and
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purification of recombinant rat sRAGE. The pPICZαA vector, containing a methanol-inducible alcohol oxidase promoter, the strongest and most tightly regulated promoter among the yeast species [28] was employed in the present study. We also designed the optimal nucleotide sequences for protein translation in P. pastoris. Protein
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production from P. pastoris strain X-33 was scalable and to further improve the yield, multiple single clones of the rrsRAGE-X-33 strain were screened and the clone exhibiting the highest expression level
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was selected. The fermentation conditions were optimized and a final yield of 200 mg/L was achieved, with above 97% purification of the protein and confirmed bio-activation. Therefore, we have developed a novel method for the rapid pilot-scale production of high-quality rrsRAGE based on a yeast expression system.
The bioactivity of rrsRAGE was verified both in vitro and in vivo in this study. The rrsRAGE
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could block S100A6-induced inhibition of the proliferation of SH-SY5Y cells, revealing that our rrsRAGE can bind to S100A6 protein in vitro. In the rat CCl4-induced models, it was also observed that rrsRAGE could prevent liver from fibrosis and injury, demonstrating that our rrsRAGE protein can be
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used for medical research.
Compliance with Ethical Standards:
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This study was funded by a Science and Technology Program of Shanghai Grant (NO.12431900600). All Authors declare that he/she has no conflict of interest. All guidelines of the Animal Care and Use Committee of Shanghai Jiao Tong University for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. References
[1] A.M. Schmidt, M. Vianna, M. Gerlach, J. Brett, J. Ryan, J. Kao, C. Esposito, H. Hegarty, W. Hurley, M. Clauss, Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface. J Biol Chem 267 (1992) 14987-14997. [2] J.L. Wautier, C. Zoukourian, O. Chappey, M.P. Wautier, P.J. Guillausseau, R. Cao, O. Hori, D. Stern, A.M. Schmidt, Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats. J Clin Invest 97 9
ACCEPTED MANUSCRIPT (1996) 238-243. [3] S. Zeng, N. Feirt, M. Goldstein, J. Guarrera, N. Ippagunta, U. Ekong, H. Dun, Y. Lu, W. Qu, A.M. Schmidt, J.C. Emond, Blockade of receptor for advanced glycation end product (RAGE) attenuates ischemia and reperfusion injury to the liver in mice. Hepatology 39 (2004) 422-432. [4] T. Miyata, Y. Wada, Z. Cai, Y. Iida, K. Horie, Y. Yasuda, K. Maeda, K. Kurokawa, C. van Ypersele de Strihou, Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure. Kidney Int 51 (1997) 1170-1181.
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[5] V. Meneghini, V. Bortolotto, M.T. Francese, A. Dellarole, L. Carraro, S. Terzieva, M. Grilli, High-mobility group box-1 protein and beta-amyloid oligomers promote neuronal differentiation of adult hippocampal neural progenitors via receptor for advanced glycation end products/nuclear factor-kappaB axis: relevance for Alzheimer's disease. J Neurosci 33 (2013) 6047-6059.
[6] P.R. Reynolds, K.M. Wasley, C.H. Allison, Diesel particulate matter induces receptor for advanced glycation end-products (RAGE) expression in pulmonary epithelial cells, and RAGE signaling influences
SC
NF-kappaB-mediated inflammation. Environ Health Perspect 119 (2011) 332-336.
[7] K. Ota, S. Yamagishi, M. Kim, S. Dambaeva, A. Gilman-Sachs, K. Beaman, J. Kwak-Kim, Elevation of soluble form of receptor for advanced glycation end products (sRAGE) in recurrent pregnancy losses
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(RPL): possible participation of RAGE in RPL. Fertil Steril 102 (2014) 782-789.
[8] H. Maillard-Lefebvre, E. Boulanger, M. Daroux, C. Gaxatte, B.I. Hudson, M. Lambert, Soluble receptor for advanced glycation end products: a new biomarker in diagnosis and prognosis of chronic inflammatory diseases. Rheumatology (Oxford) 48 (2009) 1190-1196.
[9] T. Ostendorp, M. Weibel, E. Leclerc, P. Kleinert, P.M. Kroneck, C.W. Heizmann, G. Fritz, Expression and purification of the soluble isoform of human receptor for advanced glycation end products (sRAGE) from Pichia pastoris. Biochem Biophys Res Commun 347 (2006) 4-11.
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[10] M.A. Hofmann, S. Drury, C. Fu, W. Qu, A. Taguchi, Y. Lu, C. Avila, N. Kambham, A. Bierhaus, P. Nawroth, M.F. Neurath, T. Slattery, D. Beach, J. McClary, M. Nagashima, J. Morser, D. Stern, A.M. Schmidt, RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 97 (1999) 889-901. [11] C. Renard, O. Chappey, M.P. Wautier, M. Nagashima, E. Lundh, J. Morser, L. Zhao, A.M. Schmidt, Scherrmann, J.L.
Wautier, Recombinant advanced glycation
end product receptor
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J.M.
pharmacokinetics in normal and diabetic rats. Mol Pharmacol 52 (1997) 54-62. [12] H. Asada, T. Uemura, T. Yurugi-Kobayashi, M. Shiroishi, T. Shimamura, H. Tsujimoto, K. Ito, T.
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Sugawara, T. Nakane, N. Nomura, T. Murata, T. Haga, S. Iwata, T. Kobayashi, Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis. Microbial cell factories 10 (2011) 24.
[13] J.R. Xia, N.F. Liu, N.X. Zhu, Specific siRNA targeting the receptor for advanced glycation end products inhibits experimental hepatic fibrosis in rats. International journal of molecular sciences 9 (2008) 638-661.
[14] Y.S. Seo, J.H. Kwon, U. Yaqoob, L. Yang, T.M. De Assuncao, D.A. Simonetto, V.K. Verma, V.H. Shah, HMGB1 recruits hepatic stellate cells and liver endothelial cells to sites of ethanol-induced parenchymal cell injury. American journal of physiology Gastrointestinal and liver physiology 305 (2013) G838-848. [15] L. Chen, J. Li, J. Zhang, C. Dai, X. Liu, J. Wang, Z. Gao, H. Guo, R. Wang, S. Lu, F. Wang, H. Zhang, H. Chen, X. Fan, S. Wang, Z. Qin, S100A4 promotes liver fibrosis via activation of hepatic stellate cells. Journal of hepatology 62 (2015) 156-164. 10
ACCEPTED MANUSCRIPT [16] K. Ishak, A. Baptista, L. Bianchi, F. Callea, J. De Groote, F. Gudat, H. Denk, V. Desmet, G. Korb, R.N. MacSween, et al., Histological grading and staging of chronic hepatitis. Journal of hepatology 22 (1995) 696-699. [17] L.A. Kelley, S. Mezulis, C.M. Yates, M.N. Wass, M.J. Sternberg, The Phyre2 web portal for protein modeling, prediction and analysis. Nature protocols 10 (2015) 845-858. [18] H. He, T. Yang, S. Jia, R. Zhang, P. Tu, J. Gao, Y. Yuan, W. Han, Y. Yu, Expression and purification of bioactive high-purity human S100A6 in Escherichia coli. Protein Expr Purif 83 (2012) 98-103.
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[19] J.W. Nin, A. Jorsal, I. Ferreira, C.G. Schalkwijk, M.H. Prins, H.H. Parving, L. Tarnow, P. Rossing, C.D. Stehouwer, Higher plasma soluble Receptor for Advanced Glycation End Products (sRAGE) levels are associated with incident cardiovascular disease and all-cause mortality in type 1 diabetes: a 12-year follow-up study. Diabetes 59 (2010) 2027-2032.
[20] H.A. Martens, H.L. Nienhuis, S. Gross, G. van der Steege, E. Brouwer, J.H. Berden, R.G. de Sevaux, R.H. Derksen, A.E. Voskuyl, S.P. Berger, G.J. Navis, I.M. Nolte, C.G. Kallenberg, M. Bijl, Receptor for
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advanced glycation end products (RAGE) polymorphisms are associated with systemic lupus erythematosus and disease severity in lupus nephritis. Lupus 21 (2012) 959-968.
[21] A.W. Souza, K. de Leeuw, M.M. van Timmeren, P.C. Limburg, C.A. Stegeman, M. Bijl, J. Westra, C.G.
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Kallenberg, Impact of serum high mobility group box 1 and soluble receptor for advanced glycation end-products on subclinical atherosclerosis in patients with granulomatosis with polyangiitis. PLoS One 9 (2014) e96067.
[22] W. Wan, H. Chen, Y. Li, The potential mechanisms of Abeta-receptor for advanced glycation end-products interaction disrupting tight junctions of the blood-brain barrier in Alzheimer's disease. Int J Neurosci 124 (2014) 75-81.
[23] E.Y. Park, B.H. Kim, E.J. Lee, E. Chang, D.W. Kim, S.Y. Choi, J.H. Park, Targeting of receptor for (2014) 9254-9262.
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advanced glycation end products suppresses cyst growth in polycystic kidney disease. J Biol Chem 289 [24] C.T. He, C.H. Lee, C.H. Hsieh, F.C. Hsiao, P. Kuo, N.F. Chu, Y.J. Hung, Soluble Form of Receptor for Advanced Glycation End Products Is Associated with Obesity and Metabolic Syndrome in Adolescents. Int J Endocrinol (2014).
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[25] M. Goodwin, C. Herath, Z. Jia, C. Leung, M.T. Coughlan, J. Forbes, P. Angus, Advanced glycation end products augment experimental hepatic fibrosis. J Gastroenterol Hepatol 28 (2013) 369-376. [26] L. Chen, J. Li, J. Zhang, C. Dai, X. Liu, J. Wang, Z. Gao, H. Guo, R. Wang, S. Lu, F. Wang, H. Zhang, H.
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Chen, X. Fan, S. Wang, Z. Qin, S100A4 promotes liver fibrosis via activation of hepatic stellate cells. J Hepatol (2014).
[27] Y.H. Kao, Y.C. Lin, M.S. Tsai, C.K. Sun, S.S. Yuan, C.Y. Chang, B. Jawan, P.H. Lee, Involvement of the nuclear high mobility group B1 peptides released from injured hepatocytes in murine hepatic fibrogenesis. Biochim Biophys Acta 1842 (2014) 1720-1732. [28] N. Bora, Large-scale production of secreted proteins in Pichia pastoris. Methods Mol Biol 866 (2012) 217-235. Figure legends Fig. 1 Cloning of rat soluble RAGE (rsRAGE) nucleotide sequences into pPICZαA and Detection and selection of P. pastoris transformants containing rsRAGE-pPICZαA (a) The pPICZαA vector with rsRAGE nucleotide sequences. (b) Selection of a highly productive 11
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Fig. 2 Large-scale production of rrsRAGE protein in P. pastoris wild-type strain X-33
expression at 0 and 96 h. Fig. 3 Purification of rrsRAGE from P. pastoris wild-type strain X-33
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The density (OD600) and wet weight of yeast cells per 1 mL and SDS-PAGE analysis of rrsRAGE
(a) Elution profiles of the purified supernatants from X-33/rrsRAGE after column filtration through a
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SP Sepharose FF column. (b) SDS-PAGE analysis of rrsRAGE expression. Lane 1: supernatant from X-33/rrsRAGE; lane 2: washing solution (wash step in picture a); lanes 3–8: elution solution (P1–P6
Fig. 4 Further purification of rrsRAGE
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steps in image a).
(a) Elution profiles from column filtration using Superedex 75 prep grade. (b) SDS-PAGE analysis of rrsRAGE expression. Lane 0: washing buffer (negative control); lanes 1–7: elution of markers (1–7) in
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image (a).
Fig. 5 Detection of the purification of rrsRAGE from the collection of lanes 3–7 in Fig. 5b.
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(a) Silver staining analysis. (b) Western blot analysis.
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Fig.6 Bio-activity analysis of different concentrations of rrsRAGE in the SH-SY5Y cell line after the addition of S100A6
Increasing concentrations of rrsRAGE were added. PBS was used as a negative control. Proliferation was quantified using a CCK-8 kit by measuring the absorbance at 450 nm.(* represent for p<0.05; **
represent for p<0.01; ***represent for p<0.001, compared with 50µg/ml S100A6(+) and no rrsRAGE)
Fig. 7 the treatment of rrsRAGE in CCl4-induced rat model (a) Masson three-color staining of liver tissue, blue section represents the area of the fibrosis. (b) Score of liver fibrosis. (c) Serum levels of ALT. (* represent for p<0.05; ** represent for p<0.01; 12
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Fig.S1 The vectors were checked by PCR amplification: M: marker; lane 1: negative control, pPICZαA vector only; lane 2: pPICZαA vector with rsRAGE nucleotide sequences.
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Fig.S2 Detection of rsRAGE-pPICZαA transformants in P. pastoris by PCR amplification: M: marker; lane 1: negative control (water); lane 2: positive control (plasmid); lanes 3–8: samples from cells with high yield.
Fig.S3 SDS-PAGE analysis of rrsRAGE expression via dialysis desalting. Lane 1: the supernatant from
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X-33/rrsRAGE; lane 2: the elution solution of P3 and P4; lane 3: the eluant after dialysis desalting Fig.S4 Further purification of rrsRAGE
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(a) Elution profiles from column filtration using Superedex 75 prep grade. (b) SDS-PAGE analysis of rrsRAGE expression. Lane 0: washing buffer (negative control); lanes 1–7: elution of markers (1–
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rrsRAGE purification (%)
Total rrsRAGE (mg)
Protein yield (%)
Supernatant
2340.0
39.2
917.3
100.0
SP Sepharose FF
419.7
85.2
357.6
39.0
Superdex 75
205.0
97.4
200.0
21.8
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Highlights: 1. We found a new way to do expression and purification of rat sRAGE protein in Pichia pastoris 2. The sRAGE protein could bind the ligand of RAGE, S100A6, in vitro. 3. The sRAGE protein could prevent rats from liver injury and liver fibrosis in vivo.