人源fidgetin蛋白的表达纯化和生化活性研究
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摘要
Fidgetin是2000年发现的AAA蛋白家族的一个新成员,同源序列分析表明fidgetin与spastin、katanin属于AAA蛋白家族的同一亚家族,有研究表明fidgetin也同样具有ATP依赖的微管切割活性。本研究尝试使用昆虫杆状病毒表达体系表达纯化人源fidgetin蛋白及其截短片段。通过对不同表达纯化条件的摸索,得到了fidgetin的截短蛋白,并对其ATP酶活性、微管结合能力、多聚体状态进行了分析,这对进一步研究人源fidgetin的作用机制及功能提供了很好的依据。
Fidgetin is a new member of the AAA protein family which has been found in 2000,and homologous sequence analysis shows that it belongs to the same subfamily of the AAA family as spastin and katanin,and it also has ATP-dependent microtubule-severing activity.In this paper,we try to express and purify human fidgetin and its short fragment with insect baculovirus expression system.Through the exploration of different expression conditions,we purified the short fidgetin,then analyzed its ATPase activity,microtubule binding ability and multimeric state.These results provide a good basis for further studies of the mechanism and function of human fidgetin.
Fidgetin is a new member of the AAA protein family which has been found in 2000,and homologous sequence analysis shows that it belongs to the same subfamily of the AAA family as spastin and katanin,and it also has ATP-dependent microtubule-severing activity.In this paper,we try to express and purify human fidgetin and its short fragment with insect baculovirus expression system.Through the exploration of different expression conditions,we purified the short fidgetin,then analyzed its ATPase activity,microtubule binding ability and multimeric state.These results provide a good basis for further studies of the mechanism and function of human fidgetin.
引文
[1]MILLER J M,ENEMARK E J,Fundamental characteristics of AAA+protein family structure and function[J].Archaea,2016,2016(1):1-12.
[2]MUKHERJEE S,VALENCIA J D D,STEWMAN S,et al.Human fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis[J].Cell Cycle,2012,11(12):2359-2366.
[3]LEO L,YU W,D’ROZARIO M,et al.,Vertebrate fidgetin restrains axonal growth by severing labile domains of microtubules[J].Cell Reports,2015,12(11):1723-1730.
[4]YAKUSHIJI Y,YAMANAKA K,OGURA T.Identification of a cysteine residue important for the ATPase activity of C.elegans fidgetin homologue[J].FEBSLetters,2004,578(1/2):191-197.
[5]YAKUSHIJI Y,NISHIKORI S,YAMANAKA K,et al.Mutational analysis of the functional motifs in the ATPase domain of Caenorhabditis elegans fidgetin homologue FIGL-1:firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA AT-Pases[J].Journal of Structural Biology,2006,156(1):93-100.
[6]PENG W,LIN Z,LI W,et al.,Structural insights into the unusually strong ATPase activity of the AAA domain of the Caenorhabditis elegans fidgetin-like-1(FIGL-1)protein[J].Journal of Biological Chemistry,2013,288(41):29305-29312.
[7]ZHAO X,JIN M,WANG M,et al.,Fidgetin-like 1is a ciliogenesis-inhibitory centrosome protein[J].Cell Cycle,2016,15(17):2367-2375.
[8]YANG Y,MAHAFFEY C L,BRUBN,et al.Interaction between fidgetin and protein kinase A-anchoring protein AKAP95is critical for palatogenesis in the mouse[J].Journal of Biological Chemistry,2006,281(31):22352-22359.
[9]LEO L,YU W,D’ROZARIO M,et al.Vertebrate fidgetin restrains axonal growth by severing labile domains of microtubules[J].Cell Reports,2015,12(11):1723-1730.
[2]MUKHERJEE S,VALENCIA J D D,STEWMAN S,et al.Human fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis[J].Cell Cycle,2012,11(12):2359-2366.
[3]LEO L,YU W,D’ROZARIO M,et al.,Vertebrate fidgetin restrains axonal growth by severing labile domains of microtubules[J].Cell Reports,2015,12(11):1723-1730.
[4]YAKUSHIJI Y,YAMANAKA K,OGURA T.Identification of a cysteine residue important for the ATPase activity of C.elegans fidgetin homologue[J].FEBSLetters,2004,578(1/2):191-197.
[5]YAKUSHIJI Y,NISHIKORI S,YAMANAKA K,et al.Mutational analysis of the functional motifs in the ATPase domain of Caenorhabditis elegans fidgetin homologue FIGL-1:firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA AT-Pases[J].Journal of Structural Biology,2006,156(1):93-100.
[6]PENG W,LIN Z,LI W,et al.,Structural insights into the unusually strong ATPase activity of the AAA domain of the Caenorhabditis elegans fidgetin-like-1(FIGL-1)protein[J].Journal of Biological Chemistry,2013,288(41):29305-29312.
[7]ZHAO X,JIN M,WANG M,et al.,Fidgetin-like 1is a ciliogenesis-inhibitory centrosome protein[J].Cell Cycle,2016,15(17):2367-2375.
[8]YANG Y,MAHAFFEY C L,BRUBN,et al.Interaction between fidgetin and protein kinase A-anchoring protein AKAP95is critical for palatogenesis in the mouse[J].Journal of Biological Chemistry,2006,281(31):22352-22359.
[9]LEO L,YU W,D’ROZARIO M,et al.Vertebrate fidgetin restrains axonal growth by severing labile domains of microtubules[J].Cell Reports,2015,12(11):1723-1730.