提取拟南芥总RNA,反转录获得cDNA后PCR扩增获得1 212 bp目的基因AtSTK。将该基因片段构建到PET-32a表达载体,转化大肠杆菌Rosetta菌,用IPTG进行诱导表达。该基因表达的最佳诱导条件为IPTG终浓度1mmol/L,诱导时间为4 h,此时融合蛋白表达量最高。该融合蛋白存在于包含体中,经包含体溶解、复性,最终纯化获得了AtSTK融合蛋白,为AtSTK蛋白的理化性质研究奠定了基础。
Total RNA was extracted from Arabidopsis leaves. 1212 bp AtSTKgene was amplified by RT-PCR and constructed into the expression vector PET-32a. Then the recombinant plasmid was transformed into E. coli Rosetta. The optimal induction conditions for AtSTKgene expression were 1 mmol/L IPTG and 4 hours induction. Under these conditions,the expression level of the fusion protein was highest. It was found that the form of the fusion protein was inclusion body. So the inclusion body was treated by dissolving and renaturation. Finally,the purified At-STK fusion protein was obtained. This work laid the foundation for the next study on the physical and chemical prop-erties of AtSTK protein.
[1] Chenk P W,Snaar J B E. Signal perception and transduc-tion:the role of protein kinases[J]. Biochim Biophys Ac-ta,1999,1449(1):1-24.
[2] Ferreira Pg. The Arabidopsis functional homolog of the P34cdc2protein kinase[J]. Plant Cell,1991,3(5):531-540.
[3] Lee J H,Montagu M V,Verbruggen N. A highly conserved kinase is an essential component for sress tolerance in yeast and plant cells[J]. Proc Natl Acad Sci USA,1999,9(6):5873-5877.
[4] Robinson J J,Cobb M H. Mitogen-activated protein kinase pathway[J]. Curr Opin Cell Biol,1997,9:180-186.
[5] Pardo J M,Reddy M P,Yang S. Stress signaling through Ca2 +/calmodulin-dependent protein phosphatase calci-neurin mediates salt adaptation in plants[J]. Proc Natl Acad Sci USA,1998,95:9681-9686.
[6] Zhu J K.genetic Analysis of plant salt tolerance using Ar-abidopsis[J]. Plant Physiol,2000,124:941-948.
[7] Zhu J K. Plant salt tolerance review[J]. Trends Plant Sci-ence,2001,6(2):66-71.
[8] Zhu J K. Regulation of ion homeostasis under salt stress review[J]. Current Opinion Plant Biology,2003,6(5): 441-445.
[9] Rudrabhatla P,Rajasekharan R. Developmentally regula-ted dual-specificity kinase that is induced by abiotic stresses[J]. Plant Physiol,2002,130(1):380-390.
[10] Rudrabhatla P,Reddy M M,Rajasekharan R.genome-wide analysis and experimentation of plant serine/threo-nine/tyrosine-specific protein kinases[J]. Plant Mol Bi-ol,2006,60(2):293-319.
[11] Chen W,Wang Y Y,Zhou M J,et al. Prokaryotic expres-sion,purification,and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyl transferase 14[J]. Protein Expression and Purification,2007,56:1-7.
[12] Han X Q,Lin X M,Chen H J,et al. The prokaryotic ex-pression and bioactivity of the recombinant red fire ant venom allergen soli4[J]. Agricultural Sciences in Chi-na,2009,8(2):182-187.
[13] 邓毛子,石春薇,王芳,等.结核分枝杆菌Ag85A蛋白的原核表达、纯化和免疫反应性[J]. 基础医学与 临床,2010,30(2):118-119.
[14] Rao C N,Reeder D J,Stack S M,et al. Prokaryotic ex-pression,purification,and reconstitution of biological ac-tivities(antiprotease,antitumor,and heparin-binding) for tissue factor pathway inhibitor-2[J]. Biochemical and Biophysical Research Communications,2000,3(276): 1286-1293.
[15] 胡晓梅,黄建军,饶贤才,等. 分泌型重组铜绿假单胞菌外毒素A基因工程菌的发酵研究[J]. 医学研究生 学报,2004,17(5):388-390.
[16] Rudolph H,Bohmg,Lilie H,et al. Folding Proteins
[M]. 2nd ed. New York:IRL Press,1997.
[17] Clark E D B. Refolding of recombinant proteins[J]. Curr Opin Biotechnol,1998,9(2):157-163.
[18] West S M,Chaudhui J B,Howell J A. Improved protein refolding using hollow-fibre membrane dialysis[J]. Bio-technol Bioeng,1998,57(5):590-599.
[19] Suttnar J E,Dyr E,Hamsikova J,et al. Procedure forre-folding and purification of recombinant proteins fromE-sche-richia coliinclusion bodies using a strong anion ex-changer[J]. Chromatography B,1994,656(1):123-126.
[20] Wermer M H,Cloreg M,Gronenborn A M,et al. Refold-ing proteins bygel filtration chromatography[J]. FEBS Lett,1994,345(2-3):125-130.
