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Research of the Improvement of Soluble Expression of HIV-1 Integrase Induced by T66I Mutation in E.coli |
YANG Dong, LIU Bin,HE Hong-qiu,ZHANG Xiao-yi,ZHANG Hai-feng,XU Xian-jin,CHEN Wei-zu,WANG Cun-xin |
Life Science and Bioengineering College of Beijing University of Technology , Beijing 100124 ,China |
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Abstract During research of the reported drug-resistance mutation T66I, it was found that this mutation can also improve the solubility of integrase. IN1–288/T66I and WT were expressed at the same time, 10μl of the supernatant was extracted for SDSPAGE analysis and His-tagged protein staining. Result shows that the solubility of IN1–288/T66I is 2~3 fold of WT. 4.72mg protein was obtained from 600ml of cells expressing IN1–288/T66I. The strand transfer activity of IN1–288/T66I and IN1-288/F185K /C280S were analyzed by advanced ELISA, which shows that two proteins are almost equal in catalytic activity.Another method, being different from F185K /C280S mutation, to improve the soluble expression of integrase has been provided. IN1–288/T66I can also be used to screen integrase inhibitor, which can avoid the drug-resistance brought by T66I mutation.
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Received: 17 March 2010
Published: 25 August 2010
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[1] Sherman P A, Fyfe J A. Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity. Proc Natl Acad Sci USA,1990, 87 (13) : 51995123.
[2] Bushman F D, Craigie R. Activities of human immunodeficiency virus ( HIV ) integration protein in vitro: specific cleavage and integration of HIV DNA. Proc Natl Acad Sci USA, 1991, 88 (4) : 13391343.
[3] Chiu T K, Davies D R. Structure and function of HIV1 integrase. Curr Top Med Chem, 2004, 4(9): 965977.
[4] Pommier Y, Johnson A A, Marchand C. Integrase inhibitors to treat HIV/Aids. Nat Rev Drug Discov, 2005, 4 ( 3 ) : 236248.
[5] Pluymers W, de Clercq E, Debyser Z. HIV1 integration as a target for antiretroviral therapy: a review. Curr Drug Targets Infect Disord, 2001, 1 (2) : 133149.
[6] Summa V, Petrocchi A, et al. Discovery of Raltegravir, a potent, selective orally bioavailable HIVintegrase inhibitor for the treatment of HIVAIDS infection. J. Med. Chem. 2008, 51:58435855.
[7] Evering T H, Markowitz M. Raltegravir: an integrase inhibitor for HIV1. Expert opinion on investigational drugs. 2008, 17(13):413422.
[8] Jenkins T M, Hickman A B, Dyda F, et al. Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues. Proc Natl Acad Sci USA, 1995, 92(13): 60576061.
[9] Jenkins T M, Engelman A, Ghirlando R, et al. A soluble active mutant of HIV1 integrase: involvement of both the core and carboxylterminal domains in multimerization. J Biol Chem, 1996,271 (13): 77127718.
[10] Lataillade M, Chiarella J, Kozal J M. Natural polymorphism of the HIV1 integrase gene and mutations associated with integrase inhibitor resistance. Antivir Ther, 2007, 12(4): 563570.
[11] Jessica Marinello, Christophe Marchand. Comparison of raltegravir and elvitegravir on HIV1 integrase catalytic reactions and on a series of drugresistant integrase mutants. Biochemistry, 2008, 47 (36), 93459354.
[12] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem, 1976, 72: 248254.
[13] He H Q, Ma X H, Liu B, et al. A highthroughput format assay for HIV1 integrase strand transfer reaction using magnetic beads. Acta Pharmacol Sin, 2008, 29 (3): 397404.
[14] 何红秋,胡建平,刘斌,等.HIV1整合酶核心区野生型和F185K突变型活性和溶解性的比较及分子动力学模拟分析.生物化学与生物物理进展,2009, 36(9): 11461153. He H Q, Hu J P, Liu B, et al.Progress in Biochemistry and Biophysics,2009,36(9):11461153.
[15] Carrio M M, Villaverde A. Construction and deconstruction of bacterial inclusion bodies. J Biotechnol, 2002, 96(1): 312.
[16] Sotriffer C A, Ni H, Mc Cammon J A. Active site binding modes of HIV1 integrase inhibitors. J Med Chem, 2000, 43:41094117.
[17] Lee D J, Robinson W E J. Preliminary mapping of a putativeinhibitorbinding pocket for human immunodeficiency virustype 1 integrase inhibitors. Antimicrob Agents Chemother, 2006, 50:134142.
[18] Wilkinson D L, Harrison R G. Predicting the solubility of recombinant proteins in Escherichia coli. Biotechnology (N Y), 1991, 9(5):443448.
[19] Bjellqvist B, Hughes G J. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis, 1993, 14: 10231031. |
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