纤维连接蛋白C端肝素结合域多肽在毕赤酵母中的表达、纯化及鉴定
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摘要
为在毕赤酵母中表达纤维连接蛋白C端肝素结合域(Fibronectin C-terminal heparin-binding domainFNCHBD)多肽并研究其功能,通过PCR技术扩增FNCHBD目的基因,将目的基因与T载体连接,经测序正确后,插入pAo815SM酵母表达载体增加基因拷贝数,然后酶切克隆入酵母表达载pPIC9K;将重组质粒Sal I酶切线性化后转化毕赤酵母菌株,筛选工程菌,经甲醇诱导表达,用SDS-PAGE检测发酵上清液,表明有重组蛋白FNCHBD多肽的高表达,表达产物通过离心、超滤、离子交换层析纯化,纯化产物通过SDS-PAGE、Western blotting印迹、质谱及肝素亲和层沉析对表达产物进行鉴定。结果表明利用酵母工程菌成功表达和纯化了FNCHBD多肽,多肽的分子量接近32 kDa,纯化产物的纯度可达95%以上,能被FN多克隆抗体特异识别且具有多肽肝素结合活性,为后续结构及功能的研究奠定基础。
To express and identify fibronectin C-terminal heparin-binding domain(FNCH BD) polypeptides in Pichia pastoris expression system and study its function,the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector.After sequenced,the fragment was inserted into pAo815SM vector,and then cloned into the expression vector pPIC9k.The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115.The positive yeast clone was screened by G418 resistant,and the target protein was induced to express in the medium containing 0.5% methano1.The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography.The purified product was analyzed with mass spectrogram,SDS-PAGE,Western blotting and heparin affinity chromatography.The results showed that the target protein was around 32 kDa and the purity of the product was above 95%.FNCHBD could be specifically recognized by fibronectin polyclonal antibody.These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris,which provides a good strategy to further studies.
To express and identify fibronectin C-terminal heparin-binding domain(FNCH BD) polypeptides in Pichia pastoris expression system and study its function,the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector.After sequenced,the fragment was inserted into pAo815SM vector,and then cloned into the expression vector pPIC9k.The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115.The positive yeast clone was screened by G418 resistant,and the target protein was induced to express in the medium containing 0.5% methano1.The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography.The purified product was analyzed with mass spectrogram,SDS-PAGE,Western blotting and heparin affinity chromatography.The results showed that the target protein was around 32 kDa and the purity of the product was above 95%.FNCHBD could be specifically recognized by fibronectin polyclonal antibody.These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris,which provides a good strategy to further studies.
引文
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[18]Wijelath ES,Rahman S,Namekata M,et al.Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain:enhancement of VEGF biological activity by a singular growth factor matrix protein synergism.Circ Res,2006,99(8):853-860.
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[22]Romanos M.Advances in the use of Pichia pastoris for high level gene expression.Curr Opin Biotechnol,1995,6(5):527-533.
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[24]Maeauley-Patrick S,Fazenda ML,McNeil B,et a1.Heterologous protein production using the Pichia pastoris expression system.Yeast,2005,22(4):249-270.
[2]Hynes RO.Integrins:bidirectional,allostericsignaling machines.Cell,2002,110(6):673-687.
[3]Kang W,Park S,Jang JH.Kinetic and functionalanalysis of the heparin-binding domain offibronectin.Biotechnol Lett,2008,30(1):55-59.
[4]Hynes RO.Fibronectins.New York:Springer-Verlag,1990:333-348.
[5]Sakai T,Johnson KJ,Murozono M,et al.Plasmafibronectin supports neuronal survival and reducesbrain injury following transient focal cerebralischemia but is not essential for skin wound healingand hemostasis.Nat Med,2001,7(3):324-330.
[6]Mao Y,Schwarzbauer JE.Fibronectinfibrillogenesis:a cell-mediated matrix assemblyprocess.Matrix Biol,2005,24(6):389-399.
[7]Yi M,Ruoslahti E.A fibronectin fragment inhibitstumor growth,angiogenesis,and metastasis.ProcNatl Acad Sci USA,2001,98(2):620-624.
[8]Labat-Robert J.Fibronectin in malignancy.SeminCancer Biol,2002,12(3):187-195.
[9]Liu X,Collodi P.Novel form of fibronectin fromzebrafish mediates infectious hematopoieticnecrosis virus infection.J Virol,2002,76(2):492-498.
[10]Adili R,Hong Yang,Guangheng Zhu,et al.Plasmafibronectin depletion enhances platelet aggregationand thrombus formation in mice lacking fibrinogenand vonWillebrand factor.Blood,2009,113(8):1809-1817.
[11]Vaz R,Martins GG,Thorsteinsdottirs S,et al.Fibronectin promotes migration,alignment andfusion in an in vitro myoblast cell model.CellTissue Res,2012,348(3):569-578.
[12]Roumen P,Kenneth MY.Fibronectin at a glance.Cell Science,2002,115(20):3861-3863.
[13]Iwona WP,Jean ES.The ins and outs of fibronectinmatrix assembly.J Cell Sci,2003,116(16):3269-3276.
[14]Zou QY,Guo JR,Chen XF,et al.Preparation of recombinant polypeptide of N-terminal heparin binding domain of fibronectin and its effect on disseminated intravascular coagulation in rats.J Experi Hemaol,2010,18(3):698-703.邹起练,郭江睿,陈小芳,等.纤维连接蛋白N端肝素结合域多肽的制备及其对DIC大鼠的治疗作用.中国实验血液学杂志,2010,18(3):698-703.
[15]Zou QY,Guo JR,Chen XF,et al.Recombinant polypeptide of N-terminal heparin binding domain of fibronectin antogonizes hepatic failure induced by endotoxin in mice.Natl Med J China,2009,89(48):3425-3429.邹起练,郭江睿,陈小芳,等.组纤维连接蛋白N端肝素结合域多肽抗内毒素所致小鼠肝衰竭的作用.中华医学杂志,2009,89(48):3425-3429.
[16]Kim JH,Park SO,Jang HJ,et al.Importance of the heparin-binding domain of fibronectin for enhancing cell adhesion activity of the recombinant fibronectin.Biotechnol Lett,2006,28(17):1409-1413.
[17]Kang W,Park S,Janq JH.Kinetic and functional analysis of the heparin binding domain of fibronectin.Biotechnol Lett,2008,30(1):55-59.
[18]Wijelath ES,Rahman S,Namekata M,et al.Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain:enhancement of VEGF biological activity by a singular growth factor matrix protein synergism.Circ Res,2006,99(8):853-860.
[19]Viji RI,Kumar VB,Kiran MS,et al.Angiogenic response of endothelial cells to heparin-binding domain of fibronectin.Biochem Cell Biol,2008,40(2):215-226.
[20]Kapila YL,Wang S,Dazin P,et al.The heparin-binding domain and V region of fibronectin reglate apoptosis by suppression of p53and c-myc in human primary cells.J Biol Chem,2002,277(10):8482-8491.
[21]Andersson E,Rydengard V,Sonesson A,et al.Antimicrobial activities of heparin-binding peptides.Eur Biochem,2004,271(16):1219-1226.
[22]Romanos M.Advances in the use of Pichia pastoris for high level gene expression.Curr Opin Biotechnol,1995,6(5):527-533.
[23]Faber KN,Hader W,Ab G,et al.Methylotrophic yeasts as factories for the production of foreign proteins.Yeast,1995,11(14):1331-l334.
[24]Maeauley-Patrick S,Fazenda ML,McNeil B,et a1.Heterologous protein production using the Pichia pastoris expression system.Yeast,2005,22(4):249-270.