Purification of untagged HIV-1 reverse transcriptase by affinity chromatography
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile...
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Published in | Protein expression and purification Vol. 71; no. 2; pp. 231 - 239 |
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Main Authors | , , , , , , , , , , , |
Format | Journal Article |
Language | English |
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Elsevier Inc
01.06.2010
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Abstract | Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2
mM Mn
2+. The RT retained in the column was eluted after soaking overnight in 10
mM EDTA to retrieve the Mn
2+. In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs. |
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AbstractList | Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2mM Mn(2+). The RT retained in the column was eluted after soaking overnight in 10mM EDTA to retrieve the Mn(2+). In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs. Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2 mM Mn 2+. The RT retained in the column was eluted after soaking overnight in 10 mM EDTA to retrieve the Mn 2+. In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs. Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2 mM Mn super(2+). The RT retained in the column was eluted after soaking overnight in 10 mM EDTA to retrieve the Mn super(2+). In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs. |
Author | Munshi, Vandna Lu, Meiqing Mei, Ye Burlein, Christine Grobler, Jay A. Diamond, Tracy L. Lai, Ming-Tain Ngo, Winnie Miller, Michael D. Hazuda, Daria J. Loughran, Marie H. Williams, Peter D. |
Author_xml | – sequence: 1 givenname: Meiqing surname: Lu fullname: Lu, Meiqing – sequence: 2 givenname: Winnie surname: Ngo fullname: Ngo, Winnie – sequence: 3 givenname: Ye surname: Mei fullname: Mei, Ye – sequence: 4 givenname: Vandna surname: Munshi fullname: Munshi, Vandna – sequence: 5 givenname: Christine surname: Burlein fullname: Burlein, Christine – sequence: 6 givenname: Marie H. surname: Loughran fullname: Loughran, Marie H. – sequence: 7 givenname: Peter D. surname: Williams fullname: Williams, Peter D. – sequence: 8 givenname: Daria J. surname: Hazuda fullname: Hazuda, Daria J. – sequence: 9 givenname: Michael D. surname: Miller fullname: Miller, Michael D. – sequence: 10 givenname: Jay A. surname: Grobler fullname: Grobler, Jay A. – sequence: 11 givenname: Tracy L. surname: Diamond fullname: Diamond, Tracy L. – sequence: 12 givenname: Ming-Tain surname: Lai fullname: Lai, Ming-Tain email: mingtain_lai@merck.com |
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CitedBy_id | crossref_primary_10_1007_s10529_016_2097_0 crossref_primary_10_3390_v8100263 crossref_primary_10_1016_j_isci_2020_101849 crossref_primary_10_1007_s10930_022_10066_5 crossref_primary_10_1007_s11033_024_09583_6 crossref_primary_10_1016_j_tet_2015_01_019 crossref_primary_10_1021_jz300452t crossref_primary_10_1007_s10529_014_1509_2 crossref_primary_10_1093_jac_dkx332 crossref_primary_10_1080_10826068_2024_2317311 |
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Keywords | HIV-1 Affinity chromatography: RNase H Reverse transcriptase |
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SubjectTerms | Affinity chromatography Affinity chromatography: RNase H Antiviral Agents - pharmacology Antiviral Agents - therapeutic use Base Sequence Chromatography, Affinity - methods Genetic Vectors - drug effects HIV Reverse Transcriptase - genetics HIV Reverse Transcriptase - isolation & purification HIV Reverse Transcriptase - metabolism HIV-1 HIV-1 - drug effects HIV-1 - genetics Human immunodeficiency virus 1 Humans Reverse transcriptase Ribonuclease H - genetics |
Title | Purification of untagged HIV-1 reverse transcriptase by affinity chromatography |
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