Heimbach J C, Garsky V M, Michelson S R, Dixon R A, Sigal I S, Darke P L
Department of Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486.
Biochem Biophys Res Commun. 1989 Nov 15;164(3):955-60. doi: 10.1016/0006-291x(89)91762-2.
An inhibitor of the HIV-1 protease has been employed in the generation of a resin which allows the rapid purification of this enzyme. A peptide substrate analogue, H2N-Ser-Gln-Asn-(Phe-psi[CH2N]-Pro)-Ile-Val-Gln-OH, was coupled to agarose resin. The HIV-1 protease was expressed in E. coli and the supernatant from lysed cells was passed through the affinity resin. Active HIV-1 protease was then eluted with a buffer change to pH 10 and 2 M NaCl. Final purification to a homogeneous preparation, capable of crystallization, was achieved with hydrophobic interaction chromatography. Solutions containing HIV-1 protease bound to competitive inhibitors do not bind to the column.
一种HIV-1蛋白酶抑制剂已被用于生成一种树脂，该树脂可实现对这种酶的快速纯化。一种肽底物类似物H2N-Ser-Gln-Asn-(Phe-ψ[CH2N]-Pro)-Ile-Val-Gln-OH与琼脂糖树脂偶联。HIV-1蛋白酶在大肠杆菌中表达，裂解细胞后的上清液通过亲和树脂。然后通过将缓冲液的pH值变为10并加入2 M NaCl来洗脱活性HIV-1蛋白酶。通过疏水相互作用色谱法最终纯化得到了能够结晶的均一制剂。含有与竞争性抑制剂结合的HIV-1蛋白酶的溶液不会与柱子结合。
