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植物研究  2015 

黑莓果实UGT78H2重组蛋白诱导的响应面优化与蛋白纯化

DOI: 10.7525/j.issn.1673-5102.2015.05.014, PP. 724-729

Keywords: 黑莓,UGT78H2,原核表达,响应面分析,重组蛋白

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Abstract:

?UGT78H2是从黑莓果实中新发现的一个植物糖基转移酶家族成员,获得重组蛋白是后续深入研究该基因功能的基础。本研究通过构建原核表达载体,并应用响应面分析方法对重组蛋白的诱导条件(如诱导温度、IPTG浓度、菌液浓度和诱导时间)进行了优化,结果表明:(1)构建了pET32a-UGT78H2原核表达载体,并成功导入到BL21(DE3)pLysS细菌中;(2)经响应面优化,在29.5℃培养工程菌至菌液OD600=0.51,加入终浓度为0.4mmol·L-1的IPTG诱导7.4h,可获得最大量重组蛋白166.4μg·mL-1(占总蛋白41.6%);(3)诱导时间和培养温度极显著地影响重组蛋白表达量,诱导剂IPTG和诱导前菌液浓度之间的交互效应显著影响重组蛋白的表达;(4)获得的带S-tag和His-tag的UGT78H2重组蛋白分子量为67.9kDa,主要以包涵体形式存在,通过Ni-NTA柱纯化,成功获得了重组蛋白。以上结果为进一步采用酶学方法进行UGT78H2蛋白功能鉴定提供了基础资料。

References

[1]  2.Reyes-Carmona J,Yousef G G,Mart Nez-Peniche R A,et al.Antioxidant capacity of fruit extracts of blackberry( Rubus sp.) produced in different climatic regions[J].Journal of food science,2005,70(7):497-503.
[2]  3.Bowen-Forbes C S,Zhang Y,Nair M G.Anthocyanin content,antioxidant,anti-inflammatory and anticancer properties of blackberry and raspberry fruits[J].Journal of food composition and analysis,2010,23(6):554-560.
[3]  4.王丽玲.树莓,黑莓引种驯化及组培快繁技术的研究[D].杨凌:西北农林科技大学,2004.
[4]  5.伍贤进,傅家瑞.黑莓在湖南的引种及果实的生理特性[J].植物资源与环境,1999,8(2):49-52.
[5]  6.李维林,吴文龙,闾连飞.黑莓品种宝森在江苏南京的表现[J].中国果树,2007,(4):19-21.
[6]  7.Siriwoharn T,Wrolstad R E,Finn C E,et al.Influence of cultivar,maturity,and sampling on blackberry( Rubus L.Hybrids) anthocyanins,polyphenolics,and antioxidant properties[J].Journal of Agricultural and Food Chemistry,2004,52(26):8021-8030.
[7]  8.Chen Q,Yu H W,Tang H R,et al.Identification and expression analysis of genes involved in anthocyanin and proanthocyanidin biosynthesis in the fruit of blackberry[J].Scientia Horticulturae,2012,141:61-68.
[8]  9.Lewers K S,Saski C A,Cuthbertson B J,et al.A blackberry( Rubus L.) expressed sequence tag library for the development of simple sequence repeat markers[J].BMC plant biology,2008,8(1):69.
[9]  10.Lairson L,Henrissat B,Davies G,et al.Glycosyltransferases:structures,functions,and mechanisms[J].Annual Review of Biochemistry,2008,77:521-555.
[10]  11.Caputi L,Malnoy M,Goremykin V,et al.A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyltransferases revealed the expansion of the family during the adaptation of plants to life on land[J].The Plant Journal,2012,69(6):1030-1042.
[11]  12.Breton C,?najdrov L,Jeanneau C,et al.Structures and mechanisms of glycosyltransferases[J].Glycobiology,2006,16(2):29R-37R.
[12]  13.Chen Q,Yu H,Wang X,et al.An alternative cetyltrimethylammonium bromide-based protocol for RNA isolation from blackberry( Rubus L.)[J].Genetics and molecular research:GMR,2011,11(2):1773-1782.
[13]  14.Breton C,Fournel-Gigleux S,Palcic M M.Recent structures,evolution and mechanisms of glycosyltransferases[J].Current opinion in structural biology,2012,22(5):540-549.
[14]  16.Nogu R C,Larsson A M,Guyot J C,et al.Fractional factorial approach combining 4 Escherichia coli strains,3 culture media,3 expression temperatures and 5 N-terminal fusion tags for screening the soluble expression of recombinant proteins[J].Protein expression and purification,2012,84(2):204-213.
[15]  17.田生和,全家妩.人TRAIL细胞外段基因在大肠杆菌中的表达及其包涵体复性[J].中国生物制品学杂志,2005,18(2):114-116.
[16]  18.Li C,Zhang R,Chen B,et al.Purification of recombinant histidine-tagged catalytic domain of MMP-13 in one-step using affinity column and renaturation of it with histidine-tag[J].Journal of liquid chromatogrphy and related technologies,2014,37:2118-2130.
[17]  19.Einem S V,Schwarz E,Rudolph R.A novel two-step renaturation procedure for efficient production of recombinant BMP-2[J].Protein expression and purification,2010,73:65-69.
[18]  20.高志民,于小清,郑波,等.毛竹PsbS基因的克隆及其原核表达特征研究[J].热带亚热带作物学报,2011,19(6):513-518.
[19]  21.王江,罗冬娇,孙爱华,等.基于响应面分析方法的钩端螺旋体三联属特异性蛋白抗原原核表达条件的优化[J].微生物学报,2008,48(7):893-899.
[20]  22.邹运明,马武龙,高慎阳,等.IPTG诱导浓度对重组李氏溶血素表达的影响[J].东北农业大学学报,2006,37(2):199-201.
[21]  15.Schein C H.Production of soluble recombinant proteins in bacteria[J].Nature Biotechnology,1989,7(11):1141-1149.

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