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Volume 24 Issue 6
December 2009
    Citation: Shi-meng ZHANG, Rong FAN, Tian-yi YANG, Yi1 SUN, Jing-yun LI, Qin-zhi XU, Ping-kun ZHOU. An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli .VIROLOGICA SINICA, 2009, 24(6) : 518-528.  http://dx.doi.org/10.1007/s12250-009-3068-6
    shu

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

    cstr: 32224.14.s12250-009-3068-6
    • 1.

      The Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing 100850, China

    • 2.

      Institute of Microbiology and Epidemiology, Beijing 100071, China

    • Corresponding author: Ping-kun ZHOU, zhoupk@nic.bmi.ac.cn
    • Received Date: 05 May 2009
      Accepted Date: 16 August 2009
      Available online: 01 December 2009

      Fund Project: a grant from the International Atomic Energy Agency (IAEA) 12510/R1a grant from the Chinese NationalNatural Science Foundation 30400120

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

        Corresponding author: Ping-kun ZHOU, zhoupk@nic.bmi.ac.cn
      • 1. The Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing 100850, China
      • 2. Institute of Microbiology and Epidemiology, Beijing 100071, China
      • Received Date: 05 May 2009
      • Accepted Date: 16 August 2009
      Fund Project:  a grant from the International Atomic Energy Agency (IAEA) 12510/R1a grant from the Chinese NationalNatural Science Foundation 30400120

      Abstract: 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.

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