人可溶性晚期糖基化终产物受体的毕赤酵母表达及其发酵工艺研究
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摘要
晚期糖基化终产物受体(Receptor for advanced glycation end product, RAGE)是一种跨膜蛋白,RAGE与晚期糖基化终产物(advanced glycation end products, AGEs)的结合可产生多种病理学效应。可溶性晚期糖基化终产物受体(soluble receptor for advanced glycation end product, sRAGE)是RAGE的胞外段,作为RAGE的竞争性拮抗剂,可特异性结合AGEs,从而阻断AGE-RAGE系统通过RAGE胞内段的信号传递而引起的病理学效应,因此具有广阔的应用前景。
目的：
采用巴斯德毕赤酵母表达系统表达人可溶性晚期糖基化终产物受体(human soluble Receptor for advanced glycation end product, hsRAGE)、优化发酵条件并对其体外活性进行研究,为hsRAGE进一步研究和临床应用奠定基础。
方法：
将hsRAGE cDNA插入组成型载体pGAPZaA中,构建重组表达载体pGAPZaA-hsRAGE,并转化到毕赤酵母X-33中,利用含抗生素Zeocin的YPD平板对菌种进行筛选,重组菌株在甘油醛-3-磷酸脱氢酶(GAP)启动子的调控作用下,进行组成型表达。摇瓶发酵初步摸索表达条件,再进一步放大通过发酵罐进行发酵工艺优化。
采用Ni-NTA柱亲和色谱法纯化表达产物。采用ELISA法和Western blot印迹检测hsRAGE与AGE-HSA的结合活性和免疫原性及对内皮细胞NF-κB信号转导途径的影响。
结果：
hsRAGE在pGAPZaA/X-33表达系统中成功表达,获得了相对分子质量约45KDa的重组蛋白。优化后的发酵条件为：发酵温度为30℃、pH6.0,发酵时间60h时蛋白表达水平达到最高值,表达量为58.5mg/L。Western blot显示,重组蛋白能够被相应抗体所识别。纯化后的重组hsRAGE具有与AGE-HSA相互结合的活性,并能够显著的抑制AGE-HSA诱导的人血管内皮细胞系ECV-304细胞NF-κB信号通路中NF-KB/p65基因的表达。
结论：
1、hsRAGE在毕赤酵母组成型表达系统中获得表达；
2、通过发酵工艺研究显著提高了hsRAGE表达产率；
3、毕赤酵母表达的重组hsRAGE具有良好的生物学活性,在治疗RAGE相关疾病上具有良好的应用前景；
4、hsRAGE在毕赤酵母中的成功表达和发酵工艺优化为进一步研究重组hsRAGE的药理药效和大规模生产奠定了良好基础。
Receptor for advanced glycation end product (RAGE) is a transmembrane protein.The association of RAGE and advanced glycation end products (AGEs) can lead to a variety of pathological effects. Soluble receptor for advanced glycation end product (sRAGE) is the extracellular section of RAGE.As a competitive antagonist of RAGE,sRAGE can specifically bind with AGEs and, sequentially,block the pathological effects caused by the cytosol domain of RAGE of AGE-RAGE signaling system.
Objective:
To study the expression of human soluble receptor for advanced glycation end product (hsRAGE) using the Pichia pastoris expression system,optimize fermentation conditions of the recombinant Pichia pastoris engineering strain and study the activity of expressed product in vitro.This work will pave the way for further research and application of hsRAGE.
Methods:
The hsRAGE cDNA was inserted into Pichia pastoris constitutive expression vector, pGAPZaA,the positive colonies were selected by YPD culture plate containing Zeocin.Under the control of the promoter GAP (glyceraldehydes 3 phosphate dehydrogenase),hsRAGE was expressed constitutively.The primary expression conditions experiments were carried out by means of shake flask.Then,the optimization of fermentation technics was carried out in fermentor.
The expressed product was purified by Ni-NTA affinity chromatography and identified by Western blot and enzyme-linked immunosorbent assay (ELISA) blockade experiments in the endothelial cells.The effect of hsRAGE on the NF-κB signal transduction pathway of endothelial cells was detected by Western blot.
Results:
hsRAGE protein was successfully expressed by Pichia Pastroris constitutive expression system, the protein with a molecular weight of about 45kDa.The optimum fermentation of constitutive expression were as follow:30℃,pH6.0,the expression level of hsRAGE reached its peak at 60 hours.The expression level of hsRAGE was 58.5mg/L.This protein could be specifically immunoprecipitated by mouse monoclonal antibodies against human sRAGE. After affinity purification,the protein displays the effect of binding the AGE and blocking AGE-induced nuclear factor NF-κB activation in the endothelial cells.This study could be available for the large-scale industrial production and play an important role in the biological and clinical research of hsRAGE.
Conclusion:
1. The Pichia pastoris constitutive expression system is favourable for expression of hsRAGE.
2. The expression level of hsRAGE was remarkably increase by optimization of fermentation technics.
3. The recombinant hsRAGE expressed by Pichia pastoris has significant biological activity and of brilliant prospect in treat with the RAGE related diseases.
4. The successful expression of hsRAGE in Pichia Pastoris and optimization of fermentation technics lay the foundations for the further study and large-scale production of hsRAGE.
目的：
采用巴斯德毕赤酵母表达系统表达人可溶性晚期糖基化终产物受体(human soluble Receptor for advanced glycation end product, hsRAGE)、优化发酵条件并对其体外活性进行研究,为hsRAGE进一步研究和临床应用奠定基础。
方法：
将hsRAGE cDNA插入组成型载体pGAPZaA中,构建重组表达载体pGAPZaA-hsRAGE,并转化到毕赤酵母X-33中,利用含抗生素Zeocin的YPD平板对菌种进行筛选,重组菌株在甘油醛-3-磷酸脱氢酶(GAP)启动子的调控作用下,进行组成型表达。摇瓶发酵初步摸索表达条件,再进一步放大通过发酵罐进行发酵工艺优化。
采用Ni-NTA柱亲和色谱法纯化表达产物。采用ELISA法和Western blot印迹检测hsRAGE与AGE-HSA的结合活性和免疫原性及对内皮细胞NF-κB信号转导途径的影响。
结果：
hsRAGE在pGAPZaA/X-33表达系统中成功表达,获得了相对分子质量约45KDa的重组蛋白。优化后的发酵条件为：发酵温度为30℃、pH6.0,发酵时间60h时蛋白表达水平达到最高值,表达量为58.5mg/L。Western blot显示,重组蛋白能够被相应抗体所识别。纯化后的重组hsRAGE具有与AGE-HSA相互结合的活性,并能够显著的抑制AGE-HSA诱导的人血管内皮细胞系ECV-304细胞NF-κB信号通路中NF-KB/p65基因的表达。
结论：
1、hsRAGE在毕赤酵母组成型表达系统中获得表达；
2、通过发酵工艺研究显著提高了hsRAGE表达产率；
3、毕赤酵母表达的重组hsRAGE具有良好的生物学活性,在治疗RAGE相关疾病上具有良好的应用前景；
4、hsRAGE在毕赤酵母中的成功表达和发酵工艺优化为进一步研究重组hsRAGE的药理药效和大规模生产奠定了良好基础。
Receptor for advanced glycation end product (RAGE) is a transmembrane protein.The association of RAGE and advanced glycation end products (AGEs) can lead to a variety of pathological effects. Soluble receptor for advanced glycation end product (sRAGE) is the extracellular section of RAGE.As a competitive antagonist of RAGE,sRAGE can specifically bind with AGEs and, sequentially,block the pathological effects caused by the cytosol domain of RAGE of AGE-RAGE signaling system.
Objective:
To study the expression of human soluble receptor for advanced glycation end product (hsRAGE) using the Pichia pastoris expression system,optimize fermentation conditions of the recombinant Pichia pastoris engineering strain and study the activity of expressed product in vitro.This work will pave the way for further research and application of hsRAGE.
Methods:
The hsRAGE cDNA was inserted into Pichia pastoris constitutive expression vector, pGAPZaA,the positive colonies were selected by YPD culture plate containing Zeocin.Under the control of the promoter GAP (glyceraldehydes 3 phosphate dehydrogenase),hsRAGE was expressed constitutively.The primary expression conditions experiments were carried out by means of shake flask.Then,the optimization of fermentation technics was carried out in fermentor.
The expressed product was purified by Ni-NTA affinity chromatography and identified by Western blot and enzyme-linked immunosorbent assay (ELISA) blockade experiments in the endothelial cells.The effect of hsRAGE on the NF-κB signal transduction pathway of endothelial cells was detected by Western blot.
Results:
hsRAGE protein was successfully expressed by Pichia Pastroris constitutive expression system, the protein with a molecular weight of about 45kDa.The optimum fermentation of constitutive expression were as follow:30℃,pH6.0,the expression level of hsRAGE reached its peak at 60 hours.The expression level of hsRAGE was 58.5mg/L.This protein could be specifically immunoprecipitated by mouse monoclonal antibodies against human sRAGE. After affinity purification,the protein displays the effect of binding the AGE and blocking AGE-induced nuclear factor NF-κB activation in the endothelial cells.This study could be available for the large-scale industrial production and play an important role in the biological and clinical research of hsRAGE.
Conclusion:
1. The Pichia pastoris constitutive expression system is favourable for expression of hsRAGE.
2. The expression level of hsRAGE was remarkably increase by optimization of fermentation technics.
3. The recombinant hsRAGE expressed by Pichia pastoris has significant biological activity and of brilliant prospect in treat with the RAGE related diseases.
4. The successful expression of hsRAGE in Pichia Pastoris and optimization of fermentation technics lay the foundations for the further study and large-scale production of hsRAGE.
引文
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[5]郑文华,许旭.美拉德反应的研究进展[J].化学进展,2005,17(1).
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[7]李慎军.羰基化终末产物对阿尔茨海默病的影响[J].国外医学-老年医学分册,2006,27(5)：217-219.
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[23]Tagachi A, Blood DC, del Toro G. Blockade of RAGE-amphoterin signaling suppresses tumor growth and metastases[J]. Nature,2000,405(6784):354-360
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[2]Brownlee M, Lilly L. Glycation and diabetic complications[J]. Diabetes,1994,43: 8362841.
[3]Thornalley PJ. Glutathione-dependent detoxification of alpha-oxoaldehydes by the glyoxalase syste:involvement in disease mechanisms and antiproliferative activity of glyoxalase I inhibitors [J]. Chem Biol Interact,1998:111-112,137-51.
[4]侯凡凡,张讯.慢性肾功能衰竭时晚期糖基化终产物的病理生理[J].Chin J Nephrol,1997,13(5):312-314.
[5]郑文华,许旭.美拉德反应的研究进展[J].化学进展,2005,17(1).
[6]R Singh, A Barden, T Mori, et al. Advanced glycation end-products:a review[J]. Diabetologia,2001(44):129-146
[7]李慎军.羰基化终末产物对阿尔茨海默病的影响[J].国外医学-老年医学分册,2006,27(5)：217-219.
[8]Olsen BS, Johannesen J, Sjolie AK, et al. Metabolic control and prevalence of microvascular complications in young Danish patients with Type 1 diabetes mellitus. Danish Study Group of Diabetes in Childhood[J]. Diabet Med,1999, 16(1):79-85.
[9]Brownlee M. Biochemistry and molecular cell biology of diabetic complications[J]. Nature,2001(414):813-820
[10]蒲里津.晚期糖基化终产物致动脉粥样硬化的机制[J].国际心血管病杂志,2006,33(2)：98-100.
[11]Basta G, Schmid AM, de-Caterina R. Advanced glycation end products and vascular inflammation:implications for accelerated atherosclerosis in diabetes[J]. Cardiovasc Res,2004,63(4):582-592.
[12]孟丹,刘乃丰.糖基化终产物与糖尿病血管并发症关系的研究进展[J].中国病理生理杂志Chinese Journal of Pathophysiology,2000,16(11):1244-1248.
[13]刘乃丰,黄晓明,金晖.糖基化终产物对大鼠主动脉平滑肌细胞sis基因表达的作用[J].中国糖尿病杂志,1997,5：237.
[14]Hattori Y, Suzuki M, Hattori S, et al. Vascular smooth muscule cell activation by glycated albumin(Amadori adducts)[J]. Hypertension,2002,39(1):22-28.
[15]Vlassara H, Bucala R, Striker L. Pathogenic effects of advanced glycoslylation:Biochemical, biologic, and clinical implications for diabetes and aging[J]. Lab Invest,1994,70(2):138-51.
[16]Brett J, Schmidt AM, Yan SD, et al. Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues[J]. Am J Pathol,1993,143(6):1699-712.
[17]Yamamoto Y, Kato I, Doi T, et al. Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice[J]. J Clin Invest,2001, 108(2):261-8.
[18]Oldfield MD, Bach LA, Forbes JM, et al. Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products(RAGE) [J]. J Clin Invest,2001,108(12):1853-63.
[19]Skolnik EY, Yang Z, Makita Z, et al. Human and rat mesangial cell receptors for glucose-modified proteins:potential role in kidney tissue remodeling and diabetic nephropathy [J]. J Exp Med,1991,174(4):931-9.
[20]Sugaya K, Fukagawa T, Matsumoto Kl, et al. Three genes in the human MHC class III region near the junction with the class Ⅱ:gene for receptor of advanced glycosylation end products,PBX2 homeobox gene and a notch homolog,human counterpart of mouse mammary tumor geneint-3[J]. Genomics, 1994,23(2):408-19.
[21]Neeper M, Schmidt AM, Brett J, et al. Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins [J]. J Biol Chem, 1992 Jul 25,267(21):14998-5004.
[22]Claus W. Heizmann. The mechanism by which dietary AGEs are a risk to human health is via their interaction with RAGE:Arguing against the motion[J]. Mol Nutr Food Res,2007,51,1116-1119.
[23]Tagachi A, Blood DC, del Toro G. Blockade of RAGE-amphoterin signaling suppresses tumor growth and metastases[J]. Nature,2000,405(6784):354-360
[24]Bierhaus A,Schiekofer S,Schwa ninger M,et al.Diabetes,2001,50(12):2792-2808
[25]Bierhans A, schickofer S, Schwaninger M, et al. Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB[J]. Diabetes,2001, 50(12):2792-2808.
[26]Peter P. Nawroth, David M. Stern. Soluble forms of RAGE:An index of vascular stress? A commentary on "Soluble RAGE in type 2 diabetes:Association with oxidative stress" [J]. Free Radical Biology & Medicine,2007,43:506-10.
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