LARGE-SCALE PURIFICATION AND REFOLDING OF HIV-1 PROTEASE FROM ESCHERICHIA-COLI INCLUSION-BODIES
LARGE-SCALE PURIFICATION AND REFOLDING OF HIV-1 PROTEASE FROM ESCHERICHIA-COLI INCLUSION-BODIES
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DOI：
10.1007/bf01028194
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发表时间：
1993-06-01
期刊：
影响因子：
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通讯作者：
HEINRIKSON, RL
中科院分区：
文献类型：
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作者：
HUI, JO;TOMASSELLI, AG;HEINRIKSON, RL
人类免疫缺陷病毒1型(HIV-1)编码的蛋白水解酶是在大肠杆菌中构建的，在该结构中，天然的99个残基多肽之前有一个NH2末端的蛋氨酸启动子。将含有重组HIV-1酶的包涵体溶解在50%的冰醋酸中，然后用Sephadex G-75凝胶过滤。该蛋白质在两个峰中的第二个峰上洗脱，在SDS-PAGE中以M(R)几乎等于-10,000的单一锐带的形式迁移。将纯化的HIV-1蛋白在50%的冰醋酸中稀释，在pH为5.5的条件下，用25体积的缓冲液将纯化的HIV-1蛋白复性为有活性的酶。这种纯化方法也被应用于HIV-2蛋白酶的纯化，提供了一步法生产100毫克量的完全活性的酶。
The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered in Escherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-1 protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band of M(r) almost-equal-to 1 0,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.