An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli  

作　　者：Shi-meng ZHANG Rong FAN Tian-yi YANG Yi SUN Jing-yun LI Qin-zhi XU Ping-kun ZHOU 

机构地区：[1]The Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing100850, China [2]Institute of Microbiology and Epidemiology, Beijing 100071, China

出　　处：《Virologica Sinica》2009年第6期518-528,共11页中国病毒学（英文版）

基　　金：This work was supported by a grant fromthe International Atomic Energy Agency (IAEA) (grantNo: 12510/R1) ;a grant from the Chinese NationalNatural Science Foundation (grant No: 30400120)

摘　　要：Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% （14 of 101） rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 （DE3） due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B （DE3）, which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

关 键 词：HIV tat gene E.COLI Protein expression Codon usage 

分 类 号：Q51[生物学—生物化学] S572[农业科学—烟草工业]