|
|
|
| Cloning and Expression of the dCTP Deaminase Gene from Thermus Phage TSP4 |
| GAO Ting-ting, ZHANG Qi, WEI Yun-lin, JI Xiu-ling, LIN Lian-bing |
| College of Life Science and Engineering, Kunming University of Science and Technology, Kunming 650500, China |
|
|
|
Abstract The gene encoding a dCTP deaminase tspdCD from Thermus phage TSP4 was subcloned into pET-32a, generating a recombinant plasmid pET-32a-tspdCD which was further transformed into Escherichia coli Rosetta(DE3) for expression. Analysis of SDS-PAGE showed that target protein was highly expressed as a soluble form after IPTG induction. The expressed protein was purified using Ni-NTA agarose, which was further used to analyze the enzyme activities including optimum activity temperature, pH and substrate, as well as the effects of metal ions and organic solvents on the enzyme activity. The results showed that the specific activity of the expressed tspdCD was 4.12U/mg. Its optimum activity temperature and pH were 60℃ and 7.5, repectively, and its optimum substrate was dCTP. The enzyme activity was stimulated by Ca2+ and Mg2+ of 2mmol/L, but was inhibited by Ni2+ and Cu2+ of 2 mmol/L. The results also showed that 10%(V/V) ethyl acetate and isopropanol significantly increased the activity of the expressed tspdCD while 10%(V/V) acetone had a significant inhibitory effect for the same enzyme. The successful expression of the phage tspdCD, in E. coli provides a good basis for further functional study of the purified enzyme.
|
|
Received: 17 December 2012
Published: 25 April 2013
|
|
|
|
|
[1] Jones D D,Roberts E L. enzymes of DNA metabolism in clinical diagnosis. 2nd ed. Aberystwyth: Chancery Publications,1986.
[2] 李明,江志红,王超,等. 胞苷脱氨酶的临床应用. 国际检验医学杂志,2010, 31(5): 478-479. Li M,Jiang Z H,Wang C, et al. The clinical application of cytidine deaminase. Int J Lab Med, 2010,31(5): 478-479.
[3] Clifford D Mol, Jonathan M Harris, Evan M McIntosh, et al. Human dUTP pyrophosphatase: uracil recognition by a β hairpin and active sites formed by three separate subunits. Structure, 1996, 4(9), 1077-1092.
[4] Lindahl T. Instability and decay of the primary structure of DNA. Nature, 1993,362:709-715.
[5] Liou J Y, Krishnan P, Hsieh C C, et al. Assessment of the effect of phosphorylated metabolites of anti-human immunodeficiency virus and anti-hepatitis B virus pyrimidine analogs on the behavior of human deoxycytidylate deaminase. Mol Pharmacol, 2003,63:105-110.
[6] Hernandez-Santiago B, Placidi L, Cretton-Scott E, et al. Pharmacology of beta-L-thymidine and beta-L-2’-deoxycytidine in HepG2 cells and primary human hepatocytes: relevance to chemotherapeutic efficacy against hepatitis B virus. Antimicrob Agents Chemother, 2002,46:1728-1733.
[7] Maley F, Maley G F. Mechanisms of enzyme modulation involving deoxycytidylate deaminase and thymidylate synthetase. Adv Enzyme Regul, 1970,8:55-71.
[8] Hai-Feng Hou, Yu-He Liang, Lan-Fen Li, et al. Crystal structures of Streptococcus mutans 2’-deoxycytidylate deaminase and its complex with substrate analog and allosteric regulator dCTP·Mg2+. J Mol Biol, 2008, 377:220-231.
[9] 周敏,戎霞君,高蓓莉,等. 着色性干皮病基因组D和胞苷脱氨酶基因单核苷酸多态性与肺癌易感性的关系. 上海交通大学学报(医学版), 2011,10: 1403-1408. Zhou M, Rong X J, Gao B L, et al. Relationship between single nucleotide polymorphisms of xeroderma pigmentosum complementary group D and cytidine deaminase gene and susceptibility to lung cancer. Journal of Shanghai Jiaotong University, 2011,10:1403-1408.
[10] Kilstrup M, Meng L M,Neuhard J, et al. Genetic evidence for repressor of synthesis of cytosine deaminase and purine biosynthesis enzymes in Escherichia coli. J Bacteriol,1989,171:2124.
[11] Anderson L, Kilstrup M, Neuhard J. Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K 12. Involvement of the glnLG and purR genes in the regulation of codA expression. Arch Microbiol,1989,152:115.
[12] Daniel A, Hamster A, David J, et al. Enzyme/prodrug therapy for head and neck cancer using a catalytically superior cytosine deaminase. Hum Gene Ther,1999,10:1993-2003.
[13] Mullen C A,Kilstrup M, Blaeser R M. Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine:a negative selection system. Proc Natl Acad Sci USA,1992,89(1):33-37.
[14] Huber B E, Austin E A, Good S S, et al. In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer Research, 1993,54: 4619.
[15] Lin L, Hong W, Ji X, et al. Isolation and characterization of an extremely long tail Thermus bacteriophage from Tengchong hot springs in China. J Basic Microbiol, 2010,50(5):452-456.
[16] 张龙翔.生化试验方法和技术.第2版.北京:高等教育出版社,1982.138-140. Zhang L X. Biochemical Test Methods and Techniques. 2nd ed. Beijing: Higher Education Publishing, 1982.138-140.
[17] 肖创清,卢义钦,赵绪忠等. 分光光度法测定血清胞苷脱氨酶活性的研究. 中国医学检验杂志,2001,2(1):25-30. Xiao C Q, Lu Y Q, Zhao X Z, et al. Study on the estimation of serum cytidine deaminase activity by spectrophoto metric method. Chinese Journal of Medical Laboralory Technology, 2001,2(1):25-30.
[18] Signe Smedegaard Helt, Majbritt Thymark, Pernille Harris, et al. Mechanism of dTTP inhibition of the bifunctional dCTP deaminase: dUTPase encoded by Mycobacterium tuberculosis. J Mol Biol, 2008, 376:554-569. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
