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

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1 VIROLOGICA SINICA March 2006 HIV-1 * 1,3 1, ,** ( ; ; ) Expression and Purification of HIV-1 Protease and the Establishment of a Method for Protease Inhibitor Screening WANG Yun-hua 1,3, WANG Rui-rui 1,3, YANG Liu-meng 1, Li Jian-feng 2 XU Wei-ming 2 ZHENG Yong-tang 1, (1. Laboratory of Immunopharmacology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan ,China; 2. Institute of Medical Biology, Chinese Academy of Medical Sciences, Kunming, Yunnan China ; 3. Graduate School of Chinese Academy of Sciences, Beijing , China ) AbstractHIV-protease coding sequence was amplified from HIV-1 IIIB 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. Key wordshiv-1; protease; expression; purification; inhibitors : HIV-1 IIIB RNA RT-PCR HIV-1 pet28a HIV-1 E. coli BL21 DE3 IPTG 40% Triton X-100 8M sephacyl s-200 H.R 90% indinavir HIV-1 : Q786 : A (2006) HIV 4000 HAART (Highly active antiretroviral therapy) 1998 HIV , * (2003AA219142)(2004BA719A14)(KSCX2-SW-216; KSCX1-SW-11)(2002C0066M)(2004NG12) (1974-) HIV ** (1962-) Corresponding author. Tel/Fax: ; zhengyt@mail.kiz.ac.cn

2 ,. 127 HIV [1,2] HIV HIV HIV [3] HPLC [4] (Fluorescence resonance energy transfer, FRET) [5,6] HIV pet28a E. coli. DH5BL-21DE3 LA-Taq RT-PCR Indinavir Merk HIV DABCYL-γ-Abu-Ser- Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS AnaSpec ÄKTA explore sephcryl S-200 H.R. Pharmacia Viva flow200 Vivascience AG Fluoroskan Ascent FL Thermo Electron Sigma 1.2 HIV-1 IIIB RNA 5 -GCCCATGGCC T CAGGTCACTCTTTGG-3 5 -GTGGATCCTTAAAAATTTAAAAGTGCACC A-3 RT-PCR HIV min95 5min, 941 min 521 min721 min30 722min 1.5% PCR pet28a Nco I BamH I E. coli. DH5 PCR DNA 1.3 BL-21 DE3 37 OD 600nm g/mL IPTG 8h A(50 mmol/l PB ph7.0, 50 mmol/l NaCl5 mmol/l DTT) B(50 mmol/l PB ph7.0, 50 mmol/l NaCl5 mmol/l DTT 1% Triton X-100) 3 C (50 mmol/l PBpH mmol/l NaCl5 mmol/l DTT8 mol/l ) 3 h r/min 20 min C sephacyl s-200 H.R C 40mL 100 4L D (50 mmol/l PBpH7.050 mmol/l NaCl5 mmol/l DTT10%) 4 24 h 20 ml12000 r/min 20min 50% ph 50 mmol/l HEPES, 125 mmol/l 1 mmol/l EDTA,1 mmol/l DTT, 0.5 mg/ml BSA A ph B A NaCl 1 mol/l L 4.75 ng/l, 2 mol/l 60min Anaspec EDANS (5-[ (2-aminoethyl)amino]naphthalen e-1-sulfonic acid 96 EDNAS nmol/l, 100L 355/485

3 L 4.75 ng/ L, 2 mol/l 20s Indinavir indinavir 5 10 mol/l2 mol/l400 nmol/l80 nmol/l16 nmol/l3.2 nmol/l0.64 nmol/l0.128 nmol/l 25 15min 50L 60min indinavir EC RT-PCR pet28a pet28a-pr PCR DNA 2.2 HIV-1 BL-21 DE3 IPTG 40 % Triton X-100 sephacyl s-200 H.R SDS-PAGE 90.5 % 1-158g/mL 2 2 HIV-1 Fig. 2 SDS-PAGE analysis of purified and refolded HIV-1 protease 1 LMW protein molecular weight standard 2HIV-1 protease recovered from refolded diluent. 2.3 ph / 3pH ph5.0, 1 mol/l NaCl 3 Fig. 3 Optimization of reaction buffer 4 EDANS Fig.4 Standard curve of EDANS 1 HIV-1 Fig. 1 expression and purification of HIV-1 protease 1 LMW protein molecular weight standard; 2 lysate of pet28a-pr/bl-21de3 cell before induction ; 3lysate of cell induced by IPTG for 8 h; 4inclusion body protein washed by Triton X-100; 5HIV-1 protease purified by sephacyl s-200 H.R. 2.4 EDANS 4-

4 , mol /(L.min.mg) 5 Fig. 5 5 HIV-1 Analysis of recombinant HIV-1 protease activity 2.5 Indinavir indinavir, indinavir EC nmol/l 6 Indinavir HIV-1 Fig. 6 Inhibition of indinavir on HIV-1 protease activity 3 HIV HIV HIV HIV-1 IIIB HIV [7] [6,8,9,10] 40% 90% 42.82mol /(L.min.mg) [8,9] ph ph mol/L NaCl ( 3) Leuthardt [8] HIV HIV HIV HIV HIV-1 indinavir indinavir EC 50 ( 6) HIV-1 HIV HIV References [1] Daar E S,Richman D D. Confronting the emergence of drug-resistant HIV type 1 : impact of antiretroviral therapy on individual and population resistance [J]. AIDS Res Hum Retroviruses, (5): [2] Clavel F, Hance A J. HIV drug resistance [J]. N Engl J Med, 2004, 35010: [3] Zheng Y T (). In vitro screening and assays for anti-hiv agents [J]. Chin J New Drugs (), 2002, 11 (8): [4] Tomasselli A G, Heinrikson R L. Targeting the HIV protease in AIDS therapy : a current clinical perspective [J ]. Biochem Biophys

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