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Volume 21 Issue 2
April 2006
    Citation: WANG Yun-hua, WANG Rui-rui, YANG Liu-meng, Li Jian-feng, XU Wei-ming, ZHENG Yong-tang. Expression and Purification of HIV-1 Protease and the Establishment of a Method for Protease Inhibitor Screening .VIROLOGICA SINICA, 2006, 21(2) : 126-130.
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    Expression and Purification of HIV-1 Protease and the Establishment of a Method for Protease Inhibitor Screening

    • 1.

      1. Laboratory of Immunopharmacology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223,China

    • 2.

      Institute of Medical Biology, Chinese Academy of Medical Sciences, Kunming, Yunnan 650231，China

    • 3.

      Graduate School of Chinese Academy of Sciences, Beijing 100039, China

    • Corresponding author: ZHENG Yong-tang, 
    • Available online: 20 March 2006
    • HIV-protease coding sequence was amplified from HIV-1IIIB RNA by one- step RT-PCR. The PCR product was inserted into the pet28a expression vector with an additional ATG start codon before and TAA stop codon after the coding sequence. Positive clone was selected and transformed into E. coli BL21 DE3. Cells were cultured in LB medium and induced by IPTG. The recombinant protease was expressed in the form of inclusion body and was up to 40% of total bacterial protein. The inclusion body was washed by Triton X-100 before dissolved in 8M urea and then applied on sephacyl s-200 H.R column. The purity of the protease reached 90% after purification. Protease fraction was collected and diluted in refolding buffer and left at 4℃ for 24 h. The diluent was concentrated by ultrafiltration. The activity of recombinant protease and the inhibiting effect of indinavir were analyzed by FRET with synthesized fluorescence-labeled peptide. The results above indicates that the method can be used to screen inhibitor of HIV-1 protease.
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      Expression and Purification of HIV-1 Protease and the Establishment of a Method for Protease Inhibitor Screening

        Corresponding author: ZHENG Yong-tang,
      • 1. 1. Laboratory of Immunopharmacology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223,China
      • 2. Institute of Medical Biology, Chinese Academy of Medical Sciences, Kunming, Yunnan 650231，China
      • 3. Graduate School of Chinese Academy of Sciences, Beijing 100039, China

      Abstract: HIV-protease coding sequence was amplified from HIV-1IIIB RNA by one- step RT-PCR. The PCR product was inserted into the pet28a expression vector with an additional ATG start codon before and TAA stop codon after the coding sequence. Positive clone was selected and transformed into E. coli BL21 DE3. Cells were cultured in LB medium and induced by IPTG. The recombinant protease was expressed in the form of inclusion body and was up to 40% of total bacterial protein. The inclusion body was washed by Triton X-100 before dissolved in 8M urea and then applied on sephacyl s-200 H.R column. The purity of the protease reached 90% after purification. Protease fraction was collected and diluted in refolding buffer and left at 4℃ for 24 h. The diluent was concentrated by ultrafiltration. The activity of recombinant protease and the inhibiting effect of indinavir were analyzed by FRET with synthesized fluorescence-labeled peptide. The results above indicates that the method can be used to screen inhibitor of HIV-1 protease.

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