Expression and Purification of HIV-1 Protease and the Establishment of a Method for Protease Inhibitor Screening

WANG Yun-hua; WANG Rui-rui; YANG Liu-meng; Li Jian-feng; XU Wei-ming; ZHENG Yong-tang

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April 2006

WANG Yun-hua, WANG Rui-rui, YANG Liu-meng, Li Jian-feng, XU Wei-ming and ZHENG Yong-tang. Expression and Purification of HIV-1 Protease and the Establishment of a Method for Protease Inhibitor Screening[J]. Virologica Sinica, 2006, 21(2): 126-130.

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.

HIV-1蛋白酶的表达、纯化及其抑制剂体外筛选方法的建立

王云华 ^1,3 ,
王睿睿 ^1,3 ,
杨柳萌 ¹ ,
李健峰 ² ,
徐维明 ² ,
郑永唐 ^1,,

1.
1中国科学院昆明动物研究所分子免疫药理学实验室，云南昆明 650223
2.
中国医学科学院医学生物学研究所，云南昆明650231
3.
国科学院研究生院，北京 100039

通讯作者： 郑永唐,

摘要

从HIV-1 IIIB 病毒RNA经RT-PCR得到HIV-1蛋白酶编码序列，克隆到pet28a 质粒中构建HIV-1蛋白酶表达载体。阳性克隆转染E. coli BL21 DE3，经IPTG诱导，蛋白酶以包涵体的形式表达，表达量占菌体总蛋白量的40%。包涵体经Triton X-100洗涤后溶解于8M尿素，溶解后的蛋白溶液经sephacyl s-200 H.R分子筛柱纯化后纯度达到90%以上，收集蛋白酶峰稀释复性并通过超滤进行浓缩。经检测，纯化的蛋白酶具有较高的活性。用荧光标记的蛋白酶底物检测不同浓度indinavir对蛋白酶活性的影响，表明该方法可以用于蛋白酶抑制剂的筛选。
- HIV-1
- , 蛋白酶
- , 表达
- , 纯化
- , 抑制剂

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,

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.
- HIV-1
- , protease
- , expression
- , purification
- , inhibitors

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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.