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Expression and purification of non-tagged recombinant mouse SPP1 in E. coli and its biological significance a
a
b
Shunyan Weng , Liang Zhou , Lei Han & Yunsheng Yuan
a
a
Shanghai Key Laboratory of Veterinary Biotechnology; College of Agriculture and Biology; Shanghai Jiao Tong University; Shanghai, P.R. China b
School of Pharmacy; Shanghai Jiao Tong University; Shanghai, P.R. China Published online: 30 Oct 2014.
Click for updates To cite this article: Shunyan Weng, Liang Zhou, Lei Han & Yunsheng Yuan (2014) Expression and purification of non-tagged recombinant mouse SPP1 in E. coli and its biological significance, Bioengineered, 5:6, 405-408, DOI: 10.4161/bioe.34424 To link to this article: http://dx.doi.org/10.4161/bioe.34424
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ADDENDUM Bioengineered 5:6, 405--408; November/December 2014; © 2014 Taylor & Francis Group, LLC
Expression and purification of non-tagged recombinant mouse SPP1 in E. coli and its biological significance Shunyan Weng1, Liang Zhou1, Lei Han2, and Yunsheng Yuan1,* 1
Shanghai Key Laboratory of Veterinary Biotechnology; College of Agriculture and Biology; Shanghai Jiao Tong University; Shanghai, P.R. China;
2
School of Pharmacy; Shanghai Jiao Tong University; Shanghai, P.R. China
S
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ecreted phosphoprotein 1 (SPP1) is a multifunctional protein expressed by cells from a large variety of tissues. It is involved in many physiological and pathological processes, including bone metabolism, inflammation progress, tumor metastasis, injury repair, and hyperoxiainduced injury. Native SPP1 from multiple species have been isolated from the milk and urine, and recombinant SPP1 with different tags have been expressed and purified from bacteria. In our study, DNA fragments corresponding to mouse SPP1 without signal peptide were built into the pET28a(C) vector, and nontagged recombinant mouse SPP1 (rmSPP1) was expressed in Escherichia coli BL21(DE3). rmSPP1 was purified using a novel tri-step procedure, and the product features high purity and low endotoxin level. rmSPP1 can effectively increase hepatocellular carcinoma cell (HCC) proliferation in vitro, demonstrating its biological activity.
Keywords: recombinant SPP1, biological activity, purity, HCC, cell proliferation *Correspondence to: [email protected]
Yunsheng
Yuan;
Email:
Submitted: 07/25/2014 Revised: 08/11/2014 Accepted: 08/11/2014 http://dx.doi.org/10.4161/bioe.34424 Yuan Y, Zhang X, Weng S, Guan W, Xiang D, Gao J, Li J, Han W, Yu Y. Expression and purification of bioactive high-purity recombinant mouse SPP1 in Escherichia coli. Appl Biochem Biotechnol 2014; 173:421-32; PMID:24664233; http://dx.doi. org/10.1007/s12010-014-0849-7
www.landesbioscience.com
Introduction Secreted phosphoprotein 1 (SPP1) is a phosphorylated acidic glycoprotein, and human SPP1 is also named osteopontin (OPN).1,2 It was first identified as a major sialoprotein in bone matrix, expressed by both osteoclast and osteoblast cells. It regulates bone remodeling by inhibiting hydroxyapatite formation. Current studies have shown that SPP1 is expressed in most tissues of the body, including brain, vascular tissues, kidney, dentin, cementum, salivary, gut, mammary, liver, uterus, bone, activated macrophages, and lymphocytes.3 Human SPP1 has 3 isoforms: Bioengineered
OPNa, OPNb, and OPNc. OPNa is the longest variation consisting of 314 amino acids (aa). OPNa mRNA has 7 exons, among which exon5 is absent in OPNb and exon4 is absent in OPNc. Murine SPP1 consists of 294aa, which features 59% amino sequence identity to OPNa.4 SPP1 has several conserved domains across different species that perform crucial biological functions. These domains include an arginine-glycine-aspartic acid (RGD) sequence, a thrombin cleavage site, an aspartate domain, a calcium binding motif, and two heparin binding sites, etc. (Fig. 1).4,5 SPP1 can bind to integrin avb5/(b1, b3) and (a8, a9)b1 via the RGD sequence and regulate cell migration, adhesion, chemotaxis, and so on.1 SPP1 might be a ligand to CD44v receptors as well, and thus it could potentially promote tumor metastasis and cell proliferation.6,7 SPP1 undergoes a series of maturation processes before its secretion, including the excision of its signal peptides (17 aa in human and 16 aa in mouse) at the N-terminus, phosphorylation, and glycosylation. Natural SPP1 has been isolated from milk,8,9 urine,10 and cultured rat smooth muscle cells,11 while recombinant SPP1 with GST or 6 times His have also been expressed in E. coli, and after purification these versions of recombinant SPP1 have been wildly applied to studies on the biological functions of SPP1.12-14 However, these tagged recombinant proteins are unsuitable for investigations on the pharmaceutical potential of SPP1 in animal disease models, because the tags might bring uncertainty to the proteins as drug candidates, while the removal of these tags would greatly decrease the productivity 405
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Figure 1. Functional domains and motifs in mouse SPP1 (modified from refs. 4 and 5).
and increase cost. Recently, we have reported a procedure for expression and purification of biologically active nontagged recombinant mouse SPP1 from E. coli, according to the physical and chemical properties of SPP1.15 In this procedure, the Escherichia coli BL21 (DE3) was used as the host strain for expression of recombinant mouse SPP1 (rmSPP1, L17N294). The procedure of mSPP1 purification could be divided into 3 steps, including process of crude products (isoelectric precipitation and ammonium sulfate precipitation), anion exchange chromatography, and cation exchange chromatography (Fig. 2). rmSPP1 with high purity (97%), low endotoxin level, and affirmative biological activity was produced using this procedure.15 Biological activity is very important to recombinant protein, especially for
products from bacteria. Most eukaryotic proteins have several kinds of post-translation modification, including phosphorylation, glycosylation, acetylation, ubiquitinylation, and sumoylation modification. However, prokaryotic cells lack corresponding enzymes and could not exert some of such post-translational modifications, if not all. In spite of this general disadvantage, recombinant SPP1 from bacteria has been widely used in biological function studies through the past 20 years.16-18 It has been shown that phosphorylation or glycosylation conditions of recombinant SPP1 would not affect its bioactivities except for those related with cell adhesion regulation.2 Interestingly, recombinant SPP1 cleaved by MMP3 has higher bioactivity than fulllength SPP1 in terms of regulating cell migration.12 Current studies have
Figure 2. Flow scheme for recombinant mouse SPP1 expression and purification.
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indicated that recombinant SPP1 could improve hepatocellular carcinoma cell proliferation through binding to CD44 receptor.19 Here, we also described a novel method to test the biological activity of rmSPP1 in vitro. Hepatocellular carcinoma cell lines Huh7 and QGY7701 were employed to test the biological activity of rmSPP1. They were cultured in DMEM with 10% FBS at 5% of CO2, 37 C. For CCK-8 tests, cells were planted to 96-well plates with the density of 3000 cells per well and grown overnight. Cells were then treated with DMEM with 10% FBS containing different concentrations of rmSPP1 (0, 5, 10, 20, 40, and 80 ng/ml) for 4 days, and each group has triple duplicate wells. The experiments were repeated for three times. Cell viability was analyzed with CCK-8 cell counting kit and normalized by control group. Five ng/ml rmSPP1 in cell culture medium effectively increased cell growth rate, as compared with the control group in which PBS was added to cell culture medium instead of rmSPP1. According to previous studies, the highest cell growth ratio would be obtained at 40 ng/ml. (Fig. 3).
Conclusion and Discussion SPP1 in different species share common chemical characters; for example, isoelectric point (PI) of human SPP1 is 4.2, and that of mouse SPP1 is 4.3. Because the separation of proteins using ion exchange chromatography is realized according to certain chemical properties of proteins (e.g., PI), our procedure could be applied to purify human recombinant SPP1 with minor modification, and it could be easily modified for industrial production as well. Producing recombinant protein from bacteria expression system is more economically efficient, and contamination would be less likely to happen, compared with the mammalian cell expression system. Many research works have demonstrated that SPP1 could protect animals against hyperoxia-induced lung injury,20 cardiac ischemia-reperfusion injury,21 alcohol or diethylnitrosamine-induced liver injury,22-24 and brain injury.25,26 So recombinant SPP1 would
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Figure 3. rmSPP1 improves Huh7 and QGY7701 cell proliferation. Cells were seeded to 96-well plates and maintained with DMEM (10%FBS) overnight. Then, the indicated concentration of rmSPP1 was added to medium every 24 h. PBS was used as control. Cell culture was continued for 4 days. Cell viability was analyzed with CCK-8 cell counting kit, and results were normalized by control group (n D 3, § SD). Experiments were repeated 3 times.
be a potential drug candidate in the future. rmSPP1 can be applied to rodent disease models for studying pharmaceutical mechanisms prior to preclinical studies. On the other side, under certain biological circumstances, SPP1 could promote tumor cell migration and proliferation in both paracrine and autocrine manners.7,27 Neutralization of endogenous SPP1 with anti-SPP1 antibody may be an effective method to inhibit tumor metastasis. Native recombinant SPP1 with high purity and biological activity is more appropriate than synthesized peptide in terms of raising polyclonal antibodies, and it is also more appropriate than tagged SPP1 in terms of raising monoclonal antibodies. In summary, we have provided a new strategy to express and purify native recombinant mouse SPP1 which has significant biological activity in vitro. Disclosure of Potential Conflicts of Interest No potential conflict of interest was disclosed. Acknowledgments
We thank Dr Yan Yu from Shanghai Jiao Tong University for reading and correcting the manuscript.
www.landesbioscience.com
Funding
This work was supported by the National Natural and Science Foundation of China (81302825 Y.S.Y.) and Key Laboratory of Urban Agriculture (South) Ministry of Agriculture (10UA002 Y.S.Y.). References 1. Mazzali M, Kipari T, Ophascharoensuk V, Wesson JA, Johnson R, Hughes J. Osteopontin–a molecule for all seasons. QJM 2002; 95:3-13; PMID:11834767; http:// dx.doi.org/10.1093/qjmed/95.1.3 2. Rangaswami H, Bulbule A, Kundu GC. Osteopontin: role in cell signaling and cancer progression. Trends Cell Biol 2006; 16:79-87; PMID:16406521; http://dx. doi.org/10.1016/j.tcb.2005.12.005 3. Sodek J, Ganss B, McKee MD. Osteopontin. Crit Rev Oral Biol Med 2000; 11:279-303; PMID:11021631; http://dx.doi.org/10.1177/10454411000110030101 4. Yuan Y, Xie M, Qian Z, Zhang X, Yan D, Yu Y. Osteopontin: an important molecule in liver diseases. World Chin J Digestology 2011; 19:814. 5. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Berman JS. Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 2001; 107:1055-61; PMID:11342566; http://dx.doi.org/ 10.1172/JCI12980 6. Gao C, Guo H, Downey L, Marroquin C, Wei J, Kuo PC. Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells. Carcinogenesis 2003; 24:1871-8; PMID:12949055; http://dx.doi.org/ 10.1093/carcin/bgg139 7. Lee JL, Wang MJ, Sudhir PR, Chen GD, Chi CW, Chen JY. Osteopontin promotes integrin activation through outside-in and inside-out mechanisms: OPNCD44V interaction enhances survival in gastrointestinal cancer cells. Cancer Res 2007; 67:2089-97; PMID:17332338; http://dx.doi.org/10.1158/00085472.CAN-06-3625
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8. Sørensen S, Justesen SJ, Johnsen AH. Purification and characterization of osteopontin from human milk. Protein Expr Purif 2003; 30:238-45; PMID:12880773; http://dx.doi.org/10.1016/S1046-5928(03)00102-5 9. Bayless KJ, Davis GE, Meininger GA. Isolation and biological properties of osteopontin from bovine milk. Protein Expr Purif 1997; 9:309-14; PMID:9126601; http://dx.doi.org/10.1006/prep.1996.0699 10. Christensen B, Petersen TE, Sørensen ES. Post-translational modification and proteolytic processing of urinary osteopontin. Biochem J 2008; 411:53-61; PMID: 18072945; http://dx.doi.org/10.1042/BJ20071021 11. Liaw L, Almeida M, Hart CE, Schwartz SM, Giachelli CM. Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro. Circ Res 1994; 74:214-24; PMID:8293561; http://dx.doi.org/10.1161/01.RES.74.2.214 12. Gao YA, Agnihotri R, Vary CP, Liaw L. Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments. Matrix Biol 2004; 23:457-66; PMID: 15579312; http://dx.doi.org/10.1016/j.matbio.2004. 09.003 13. Ashkar S, Teplow DB, Glimcher MJ, Saavedra RA. In vitro phosphorylation of mouse osteopontin expressed in E. coli. Biochem Biophys Res Commun 1993; 191:126-33; PMID:8447818; http://dx.doi.org/ 10.1006/bbrc.1993.1193 14. Jang JH, Kim JH. Improved cellular response of osteoblast cells using recombinant human osteopontin protein produced by Escherichia coli. Biotechnol Lett 2005; 27:1767-70; PMID:16314968; http://dx.doi. org/10.1007/s10529-005-3551-6 15. Yuan Y, Zhang X, Weng S, Guan W, Xiang D, Gao J, Li J, Han W, Yu Y. Expression and purification of bioactive high-purity recombinant mouse SPP1 in Escherichia coli. Appl Biochem Biotechnol 2014; 173:421-32; PMID:24664233; http://dx.doi.org/10.1007/s12010014-0849-7 16. Agnihotri R, Crawford HC, Haro H, Matrisian LM, Havrda MC, Liaw L. Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin). J Biol Chem 2001; 276:28261-7; PMID:11375993; http://dx.doi.org/ 10.1074/jbc.M103608200 17. Saad FA, Salih E, Glimcher MJ. Identification of osteopontin phosphorylation sites involved in bone remodeling and inhibition of pathological calcification. J Cell Biochem 2008; 103:852-6; PMID:17615552; http:// dx.doi.org/10.1002/jcb.21453 18. Mitchell EA, Chaffey BT, McCaskie AW, Lakey JH, Birch MA. Controlled spatial and conformational display of immobilised bone morphogenetic protein-2 and osteopontin signalling motifs regulates osteoblast adhesion and differentiation in vitro. BMC Biol 2010; 8:57; PMID:20459712; http://dx.doi.org/10.1186/17417007-8-57 19. Phillips RJ, Helbig KJ, Van der Hoek KH, Seth D, Beard MR. Osteopontin increases hepatocellular carcinoma cell growth in a CD44 dependant manner. World J Gastroenterol 2012; 18:3389-99; PMID:22807608; http://dx.doi. org/10.3748/wjg.v18.i26.3389 20. Zhang XF, Liu S, Zhou YJ, Zhu GF, Foda HD. Osteopontin protects against hyperoxia-induced lung injury by inhibiting nitric oxide synthases. Chin Med J (Engl) 2010; 123:929-35; PMID:20497690 21. Wang Y, Chen B, Shen D, Xue S. Osteopontin protects against cardiac ischemia-reperfusion injury through late preconditioning. Heart Vessels 2009; 24:116-23; PMID:19337795; http://dx.doi.org/10.1007/s00380008-1094-1 22. Ge X, Leung TM, Arriazu E, Lu Y, Urtasun R, Christensen B, Fiel MI, Mochida S, Sørensen ES, Nieto N. Osteopontin binding to lipopolysaccharide lowers tumor necrosis factor-a and prevents early alcoholinduced liver injury in mice. Hepatology 2014; 59:1600-16; PMID:24214181; http://dx.doi.org/ 10.1002/hep.26931
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25. Suzuki H, Hasegawa Y, Kanamaru K, Zhang JH. Mechanisms of osteopontin-induced stabilization of blood-brain barrier disruption after subarachnoid hemorrhage in rats. Stroke 2010; 41:1783-90; PMID:20616319; http://dx.doi.org/10.1161/ STROKEAHA.110.586537 26. Topkoru BC, Altay O, Duris K, Krafft PR, Yan J, Zhang JH. Nasal administration of recombinant osteopontin attenuates early brain injury after subarachnoid hemorrhage. Stroke 2013; 44:3189-94;
PMID:24008574; http://dx.doi.org/10.1161/ STROKEAHA.113.001574 27. Medico E, Gentile A, Lo Celso C, Williams TA, Gambarotta G, Trusolino L, Comoglio PM. Osteopontin is an autocrine mediator of hepatocyte growth factorinduced invasive growth. Cancer Res 2001; 61:5861-8; PMID:11479227
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23. Ge X, Lu Y, Leung TM, Sørensen ES, Nieto N. Milk osteopontin, a nutritional approach to prevent alcoholinduced liver injury. Am J Physiol Gastrointest Liver Physiol 2013; 304:G929-39; PMID:23518682; http:// dx.doi.org/10.1152/ajpgi.00014.2013 24. He C, Fan X, Chen R, Liang B, Cao L, Guo Y, Zhao J. Osteopontin is involved in estrogen-mediated protection against diethylnitrosamine-induced liver injury in mice. Food Chem Toxicol 2012; 50:2878-85; PMID: 22609492; http://dx.doi.org/10.1016/j.fct.2012.05.012
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Volume 5 Issue 6
Bioengineered Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kbie20
Expression and purification of non-tagged recombinant mouse SPP1 in E. coli and its biological significance a
a
b
Shunyan Weng , Liang Zhou , Lei Han & Yunsheng Yuan
a
a
Shanghai Key Laboratory of Veterinary Biotechnology; College of Agriculture and Biology; Shanghai Jiao Tong University; Shanghai, P.R. China b
School of Pharmacy; Shanghai Jiao Tong University; Shanghai, P.R. China Published online: 30 Oct 2014.
Click for updates To cite this article: Shunyan Weng, Liang Zhou, Lei Han & Yunsheng Yuan (2014) Expression and purification of non-tagged recombinant mouse SPP1 in E. coli and its biological significance, Bioengineered, 5:6, 405-408, DOI: 10.4161/bioe.34424 To link to this article: http://dx.doi.org/10.4161/bioe.34424
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ADDENDUM Bioengineered 5:6, 405--408; November/December 2014; © 2014 Taylor & Francis Group, LLC
Expression and purification of non-tagged recombinant mouse SPP1 in E. coli and its biological significance Shunyan Weng1, Liang Zhou1, Lei Han2, and Yunsheng Yuan1,* 1
Shanghai Key Laboratory of Veterinary Biotechnology; College of Agriculture and Biology; Shanghai Jiao Tong University; Shanghai, P.R. China;
2
School of Pharmacy; Shanghai Jiao Tong University; Shanghai, P.R. China
S
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ecreted phosphoprotein 1 (SPP1) is a multifunctional protein expressed by cells from a large variety of tissues. It is involved in many physiological and pathological processes, including bone metabolism, inflammation progress, tumor metastasis, injury repair, and hyperoxiainduced injury. Native SPP1 from multiple species have been isolated from the milk and urine, and recombinant SPP1 with different tags have been expressed and purified from bacteria. In our study, DNA fragments corresponding to mouse SPP1 without signal peptide were built into the pET28a(C) vector, and nontagged recombinant mouse SPP1 (rmSPP1) was expressed in Escherichia coli BL21(DE3). rmSPP1 was purified using a novel tri-step procedure, and the product features high purity and low endotoxin level. rmSPP1 can effectively increase hepatocellular carcinoma cell (HCC) proliferation in vitro, demonstrating its biological activity.
Keywords: recombinant SPP1, biological activity, purity, HCC, cell proliferation *Correspondence to: [email protected]
Yunsheng
Yuan;
Email:
Submitted: 07/25/2014 Revised: 08/11/2014 Accepted: 08/11/2014 http://dx.doi.org/10.4161/bioe.34424 Yuan Y, Zhang X, Weng S, Guan W, Xiang D, Gao J, Li J, Han W, Yu Y. Expression and purification of bioactive high-purity recombinant mouse SPP1 in Escherichia coli. Appl Biochem Biotechnol 2014; 173:421-32; PMID:24664233; http://dx.doi. org/10.1007/s12010-014-0849-7
www.landesbioscience.com
Introduction Secreted phosphoprotein 1 (SPP1) is a phosphorylated acidic glycoprotein, and human SPP1 is also named osteopontin (OPN).1,2 It was first identified as a major sialoprotein in bone matrix, expressed by both osteoclast and osteoblast cells. It regulates bone remodeling by inhibiting hydroxyapatite formation. Current studies have shown that SPP1 is expressed in most tissues of the body, including brain, vascular tissues, kidney, dentin, cementum, salivary, gut, mammary, liver, uterus, bone, activated macrophages, and lymphocytes.3 Human SPP1 has 3 isoforms: Bioengineered
OPNa, OPNb, and OPNc. OPNa is the longest variation consisting of 314 amino acids (aa). OPNa mRNA has 7 exons, among which exon5 is absent in OPNb and exon4 is absent in OPNc. Murine SPP1 consists of 294aa, which features 59% amino sequence identity to OPNa.4 SPP1 has several conserved domains across different species that perform crucial biological functions. These domains include an arginine-glycine-aspartic acid (RGD) sequence, a thrombin cleavage site, an aspartate domain, a calcium binding motif, and two heparin binding sites, etc. (Fig. 1).4,5 SPP1 can bind to integrin avb5/(b1, b3) and (a8, a9)b1 via the RGD sequence and regulate cell migration, adhesion, chemotaxis, and so on.1 SPP1 might be a ligand to CD44v receptors as well, and thus it could potentially promote tumor metastasis and cell proliferation.6,7 SPP1 undergoes a series of maturation processes before its secretion, including the excision of its signal peptides (17 aa in human and 16 aa in mouse) at the N-terminus, phosphorylation, and glycosylation. Natural SPP1 has been isolated from milk,8,9 urine,10 and cultured rat smooth muscle cells,11 while recombinant SPP1 with GST or 6 times His have also been expressed in E. coli, and after purification these versions of recombinant SPP1 have been wildly applied to studies on the biological functions of SPP1.12-14 However, these tagged recombinant proteins are unsuitable for investigations on the pharmaceutical potential of SPP1 in animal disease models, because the tags might bring uncertainty to the proteins as drug candidates, while the removal of these tags would greatly decrease the productivity 405
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Figure 1. Functional domains and motifs in mouse SPP1 (modified from refs. 4 and 5).
and increase cost. Recently, we have reported a procedure for expression and purification of biologically active nontagged recombinant mouse SPP1 from E. coli, according to the physical and chemical properties of SPP1.15 In this procedure, the Escherichia coli BL21 (DE3) was used as the host strain for expression of recombinant mouse SPP1 (rmSPP1, L17N294). The procedure of mSPP1 purification could be divided into 3 steps, including process of crude products (isoelectric precipitation and ammonium sulfate precipitation), anion exchange chromatography, and cation exchange chromatography (Fig. 2). rmSPP1 with high purity (97%), low endotoxin level, and affirmative biological activity was produced using this procedure.15 Biological activity is very important to recombinant protein, especially for
products from bacteria. Most eukaryotic proteins have several kinds of post-translation modification, including phosphorylation, glycosylation, acetylation, ubiquitinylation, and sumoylation modification. However, prokaryotic cells lack corresponding enzymes and could not exert some of such post-translational modifications, if not all. In spite of this general disadvantage, recombinant SPP1 from bacteria has been widely used in biological function studies through the past 20 years.16-18 It has been shown that phosphorylation or glycosylation conditions of recombinant SPP1 would not affect its bioactivities except for those related with cell adhesion regulation.2 Interestingly, recombinant SPP1 cleaved by MMP3 has higher bioactivity than fulllength SPP1 in terms of regulating cell migration.12 Current studies have
Figure 2. Flow scheme for recombinant mouse SPP1 expression and purification.
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indicated that recombinant SPP1 could improve hepatocellular carcinoma cell proliferation through binding to CD44 receptor.19 Here, we also described a novel method to test the biological activity of rmSPP1 in vitro. Hepatocellular carcinoma cell lines Huh7 and QGY7701 were employed to test the biological activity of rmSPP1. They were cultured in DMEM with 10% FBS at 5% of CO2, 37 C. For CCK-8 tests, cells were planted to 96-well plates with the density of 3000 cells per well and grown overnight. Cells were then treated with DMEM with 10% FBS containing different concentrations of rmSPP1 (0, 5, 10, 20, 40, and 80 ng/ml) for 4 days, and each group has triple duplicate wells. The experiments were repeated for three times. Cell viability was analyzed with CCK-8 cell counting kit and normalized by control group. Five ng/ml rmSPP1 in cell culture medium effectively increased cell growth rate, as compared with the control group in which PBS was added to cell culture medium instead of rmSPP1. According to previous studies, the highest cell growth ratio would be obtained at 40 ng/ml. (Fig. 3).
Conclusion and Discussion SPP1 in different species share common chemical characters; for example, isoelectric point (PI) of human SPP1 is 4.2, and that of mouse SPP1 is 4.3. Because the separation of proteins using ion exchange chromatography is realized according to certain chemical properties of proteins (e.g., PI), our procedure could be applied to purify human recombinant SPP1 with minor modification, and it could be easily modified for industrial production as well. Producing recombinant protein from bacteria expression system is more economically efficient, and contamination would be less likely to happen, compared with the mammalian cell expression system. Many research works have demonstrated that SPP1 could protect animals against hyperoxia-induced lung injury,20 cardiac ischemia-reperfusion injury,21 alcohol or diethylnitrosamine-induced liver injury,22-24 and brain injury.25,26 So recombinant SPP1 would
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Figure 3. rmSPP1 improves Huh7 and QGY7701 cell proliferation. Cells were seeded to 96-well plates and maintained with DMEM (10%FBS) overnight. Then, the indicated concentration of rmSPP1 was added to medium every 24 h. PBS was used as control. Cell culture was continued for 4 days. Cell viability was analyzed with CCK-8 cell counting kit, and results were normalized by control group (n D 3, § SD). Experiments were repeated 3 times.
be a potential drug candidate in the future. rmSPP1 can be applied to rodent disease models for studying pharmaceutical mechanisms prior to preclinical studies. On the other side, under certain biological circumstances, SPP1 could promote tumor cell migration and proliferation in both paracrine and autocrine manners.7,27 Neutralization of endogenous SPP1 with anti-SPP1 antibody may be an effective method to inhibit tumor metastasis. Native recombinant SPP1 with high purity and biological activity is more appropriate than synthesized peptide in terms of raising polyclonal antibodies, and it is also more appropriate than tagged SPP1 in terms of raising monoclonal antibodies. In summary, we have provided a new strategy to express and purify native recombinant mouse SPP1 which has significant biological activity in vitro. Disclosure of Potential Conflicts of Interest No potential conflict of interest was disclosed. Acknowledgments
We thank Dr Yan Yu from Shanghai Jiao Tong University for reading and correcting the manuscript.
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Funding
This work was supported by the National Natural and Science Foundation of China (81302825 Y.S.Y.) and Key Laboratory of Urban Agriculture (South) Ministry of Agriculture (10UA002 Y.S.Y.). References 1. Mazzali M, Kipari T, Ophascharoensuk V, Wesson JA, Johnson R, Hughes J. Osteopontin–a molecule for all seasons. QJM 2002; 95:3-13; PMID:11834767; http:// dx.doi.org/10.1093/qjmed/95.1.3 2. Rangaswami H, Bulbule A, Kundu GC. Osteopontin: role in cell signaling and cancer progression. Trends Cell Biol 2006; 16:79-87; PMID:16406521; http://dx. doi.org/10.1016/j.tcb.2005.12.005 3. Sodek J, Ganss B, McKee MD. Osteopontin. Crit Rev Oral Biol Med 2000; 11:279-303; PMID:11021631; http://dx.doi.org/10.1177/10454411000110030101 4. Yuan Y, Xie M, Qian Z, Zhang X, Yan D, Yu Y. Osteopontin: an important molecule in liver diseases. World Chin J Digestology 2011; 19:814. 5. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Berman JS. Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 2001; 107:1055-61; PMID:11342566; http://dx.doi.org/ 10.1172/JCI12980 6. Gao C, Guo H, Downey L, Marroquin C, Wei J, Kuo PC. Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells. Carcinogenesis 2003; 24:1871-8; PMID:12949055; http://dx.doi.org/ 10.1093/carcin/bgg139 7. Lee JL, Wang MJ, Sudhir PR, Chen GD, Chi CW, Chen JY. Osteopontin promotes integrin activation through outside-in and inside-out mechanisms: OPNCD44V interaction enhances survival in gastrointestinal cancer cells. Cancer Res 2007; 67:2089-97; PMID:17332338; http://dx.doi.org/10.1158/00085472.CAN-06-3625
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Volume 5 Issue 6
