Prokaryotic Expression of Dendrorhynchus zhejiangensis Actin Gene and Self-assembly of Recombinant Protein in vitro

Abstract

Abstract: Actin is a major component of cytoskeleton. In cells, actin associates with actin binding proteins（ABPs）to form highly ordered polymerization structure on the basis of F-actin, which plays a variety of important physiological functions. According to the full length cDNA of Dendrorhynchus zhejiangensis actin, the prokaryotic expression plasmid for actin（pET-28a-A）was constructed and transformed into E.coli BL21(DE3). After IPTG induction, the recombinant proteins were highly expressed and contained in inclusion bodies. The recombinant proteins from inclusion bodies were purified by Nickel nitrilotriacetic（Ni-NTA）agarose affinity chromatography, and further dialyzed into a refolding buffer. The results of sodium dodecyl sulfate polycrylamide gel（SDS-PAGE）showed that the relative molecular weight of r-actin was 43 kD. In in vitro polymerization condition, the atomic force microscope（AFM）was used to observe and analyze the large-scale aggregate structure of r-actin during self-assembly. Our results showed that in vitro assembly of r-actin polymerized into complicated discrete structures, like filopodia-like fiber bundles, random coiled actin clusters, in addition to form disorganized protein aggregates. These data implicate that in vitro assembly of r-actin reflects its inherent thermodynamic properties of polymerization；further study would help to explore the regulatory function of actin binding proteins（ABPs）in the assembly of actin supramolecular aggregate structures.

Key words: Dendrorhynchus zhejiangensis, Actin Prokaryotic expression, Atomic force microscope, Self-assembly

Li Ye, Chen Lei, Zhou Jun, Li Chenghua, Su Xiurong, Li Taiwu. Prokaryotic Expression of Dendrorhynchus zhejiangensis Actin Gene and Self-assembly of Recombinant Protein in vitro[J]. Biotechnology Bulletin, 2013, 0(8): 113-118.

References

[1] 吕慈仙, 李太武, 江锦波, 等. 浙江沿海最新发现一种枝吻类纽虫[J]. 水产科学, 2008, 27（12）：652-654.
[2] 王孟前, 李太武, 江锦坡, 等. 浙江沿海一新纽虫的18S rDNA序列研究[J].科技通报, 2010, 26（1）：67-71.
[3] 尹左芬, 曾棻. 纵沟纽虫科（Lineidae）、枝吻纽虫属(Dendror-hynchus)的一新种—湛江枝吻纽虫(D.zhanjianggensis)的研究[J]. 海洋通报, 1984, 3（6）：51-58.
[4] 尹左芬, 曾棻.异纽类纵沟纽虫科一新属新种—中华枝吻纽虫[J]. 海洋与湖沼, 1985, 16（4）：323-336.
[5] 尹左芬, 曾棻. 纵沟纽虫科（Lineidae）具分枝吻纽虫的一新属新种—疣多枝吻纽虫(Polydendrorhyn chus papillaris)[J]. 山东海洋学院学报, 1986, 16（4）：1-9.
[6] 孙世春. 中国单针类纽虫一新记录[J]. 青岛海洋大学学报, 1994, 24（3）：436-438.
[7] Strand M, Sundberg P. Genus Tetrastemma Ehrenberg, 1831（Phylu-mNemertea）—anatural group? Phylogenetic relationships inferred from partial 18S rDNA sequences[J]. Molecular Phylogenetics and Evolution, 2005, 37：144-152.
[8] Sundberg P, Strand M. Genetics do not reflect habitat differences in Riseriellus occultus（Heteronemertea, Nemertea）from Spain and Wales[J]. Marine Biology Research, 2007, 3：117-122.
[9] Chen HX, Sundberg P, Jon L, et al. The complete mitochondrial genome of Cephalothrix simula（Iwata）（Nemertea：Palaeoneme-rtea）[J]. Gene, 2009, 442：8-12.
[10] Simon JA, Melanie AH, Bruce AM. Mechanism of actin polymeriza-tion in cellular ATP depletion[J]. JBC, 2003, 11：1-30.
[11] Christophe LC, Dominique P, Marie FC. ATP hydrolysis on actin-related protein 2/3 complex causes debranching of dendritic actin arrays[J]. PNAS, 2003, 100：6337-6342.
[12] Hason J, Lowy J. The structure of F-Actin and of Actin filaments isolated from muscle[J]. J Mol Biol, 1963, 6：46.
[13] Poliard TD, Cooper JA. Actin and actin-binding proteins：A critical evaluation of mechanisms and functions[J]. Annu Rev Biochem, 1986, 55：987-1035.
[14] 秦玉明, 苏秀榕, 李晔, 等. 缢蛏(Sinonovacula constricta)cDNA文库的构建及肌动蛋白基因的研究[J]. 海洋与湖沼, 2010, 41（1）：54-59.
[15] Coluccio LM, Bretscher A. Reassociation of microvillar core proteins：making a microvillar core in vitro[J]. J Cell Biol, 1989, 108：495-502.
[16] Wei MY, Leon LJ, Lee Y. Selective attachment of F-actin with controlled length for developing an intelligent nanodevice[J]. Journal of Colloid and Interface Science, 2011, 356：182-189.
[17] Quamann BM, Kessels M, Kelly RB. Molecular links between Endocysis and the actin cytoskeleton[J]. The Journal of Cell Biology, 2000, 150：111.
[18] Vidal IL, Mckenna ST, Hepler PK. Actin polymerization is essential for pollen tube growth[J]. Molbiol Cell, 2001, 12：2534-2545.
[19] 陈蕾, 李晔, 苏秀榕, 等. 浙江枝吻纽虫(Dendrorhynchus zhejiangensis)凝溶胶蛋白和肌动蛋白基因的克隆与序列分析[J]. 海洋与湖沼, 2011, 42（2）：1-8.
[20] 杨晓仪, 林键, 吴文言. 重组蛋白包涵体的复性研究[J]. 生命科学研究, 2004, 8（2）：100-105.
[21] Maeda Y, Ueda A, Imoto T. Effective renaturation of denatured and reduced immunoglobulin G in vitro without assistance of chaperone[J]. Protein Eng, 1996, 9（1）：95-100.
[22] Hwang HS, Chung HS. Preparation of active recombinant cathepsin K expressed in bacteria as inclusion body[J]. Protein Expr Purif, 2002, 25（3）：541-546.
[23] Estes J, Selden L, Kinosian H, et al. Tightly bound divalent cations of actin[J]. J Muscle Res Cell Motil, 1992, 13：272-284.
[24] Lehto T, Miaczynska M, Zerialb M, et al. Observing the growth of individual actin filaments in cell extracts by time - lap se atomic force microscopy[J]. FEBS Letters, 2003, 551：25-28.
[25] 张军, 王远亮, 程超, 等.利用激光AFM和TEM探索肌动蛋白体外自装配聚合结构[J]. 激光生物学报, 2008, 17（1）：1-6.
[26] Chaudhurio, Parekh SH, Fletcher DA. Reversible stress softening of actin networks[J]. Nature, 2007, 445（7125）：295-298.
[27] Kawabata K, Sado Y, Nagayama M, et al. Visuation of dynamic organization of cytoskeleton gels in living cells by hybrid-SPM[J]. Chinese Journal of Polymer Science, 2003, 21：169.
[28] Tan XW, Promoda A, Perera P, et al. Comparison of candidate materials for a synthetic osteo-odonto keratoprosthesis device[J]. IOVS, 2011, 52（1）：21-29.