GST-融合蛋白的医.docVIP

GST融合蛋白纯化--筛选表达株(英文） 日期：2012-06-27来源：互联网作者：青岚点击： 197次 相关专题 蛋白表达技术全攻略 Purification of GST fusion proteins in E.coli GST Sugden lab,McArdle Laboratory for Cancer Research,University of Wisconsin-Madison Medical School Screen of GST-Fusion Protein Expression (pGEX system by Amersham: for check clones for expression of the desired fusion protein prior to large-scale purification) Pick several colonies of E.coli transformed with the pGEX recombinants into separate tubes containing 2 ml of 2 x YTA medium. -Note: For comparison,it is advisable to inoculate a control tube with bacteria transformed with the parental pGEX plasmid. Grow liquid cultures to an A600 of 0.6-0.8 (3~5 h)with vigorous agitation at 20~37℃. Incubate fusion protein expression by adding 2 μl of 100 mM IPTG (final concentration 0.1 mM). Continue incubation for an additional 1~2 h Transfer 1.5 ml of the liquid cultures to labeled 1.5 ml microcentrifuge tubes. Centrifuge in a microcentrifuge for 5 sec and discard the supernatant. Resuspend each pellet in 300 μl of ice-cold 1 x PBS,remove 10 μl of these resuspend cells into labeled tubes (for later use in SDSanalysis). -Note: Except where noted,keep all samples and tubes on ice. Lyse the cells using the sonicator equipped with an appropriate probe. -Note: Lysis is complete when the cloudy cell suspension becomes translucent.The frequency and intensity of sonication should be adjusted such that complete lysis occurs in 10 sec,without frothing (it may denature proteins). -Note: Crude sonicates can be screened for the relative level of expression of GST fugion proteins using the GST substrates CDNB (1-chloro-2,4-dinitrobenzene). Centrifuge in a microcentrifuge for 5 min to remove insoluble materials.Save a10 μl aliquots of the insoluble material for analysis by SDS.Transfer the supernatants to fresh tubes, Add 20 μl of a 50% slurry of Glutathione Sepharose 4B (prepared as described above)to each supernatant and mix gently for 5 min at r.t.. Add 100 μl of 1 x PB