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KMID : 0578319940040010079
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Molecules and Cells
1994 Volume.4 No. 1 p.79 ~ p.84
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Expression and Ourification of HIV-1 Protease Utikizing a Maltose Binding Protein
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Kim, Do Hyung
Lee, Ki Jeong/Sung, Young Chul/Choi, kwan Yong
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Abstract
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Human immunodeficiency virus type 1 (HIV-1) protease is essential for the replication of the virus and it is therefore an attractive target for antiviral drugs of HIV-1. The gene coding for this enzyme was cloned in pMALrcRI and expressed as a MBP-pol fusion protein in E. soli XLI-blue. The expressed protease was found to be toxic to F_ soli. The activity assay with the undecapeptide His-Lys-Ala-Arg-Val-Leu-(p-nitro)-Phe-Glu-Ala-Nle-Ser-amide as a substrate showed the specific protease activity. The protease exhibited autoprocessing as proved and by Western blotting with polyclonal antibody against maltose binding protein. The expressed protease was purified to the homogeniety by a series of chromatographies using DEAE, SE cellulose, Superose 12 gel filtraton and Mono S HPLC columns. The purified protease migrated on SDS-PAGE as expected and had a Km (9.8 pM) with the above undecapeptide substrate.
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KEYWORD
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