基于PHA颗粒-PhaP标签和内含肽元件的极端嗜盐古菌蛋白的表达纯化系统

Development of PhaP-tagged protein expression and purification systems for extremely halophilic archaea

摘要:

摘要:【目的】建立一个基于PHA颗粒-PhaP标签的简便实用的极端嗜盐古菌蛋白表达纯化系统，并探讨嗜盐古菌内含肽在该系统优化中的应用。【方法】以嗜盐古菌-大肠杆菌穿梭载体pWL502为基本骨架，构建带有嗜盐古菌强启动子和PhaP 融合标签的表达载体;在地中海富盐菌phaP缺陷株(ΔphaP)中融合表达目的基因，通过蔗糖密度梯度离心分离纯化结合于PHA颗粒上的PhaP融合蛋白;在phaP基因与多克隆位点之间引入特定的嗜盐古菌内含肽元件，尝试通过定点突变改变该内含肽的剪切活性。【结果】成功构建了以 PhaP作为N端融合标签的表达载体pPM以及作为C端融合标签的载体pIP;在phaP基因簇强启动子控制下，二者均实现了目标蛋白的高效表达;通过PHA颗粒介导的蛋白分离纯化策略，实现了以PhaP为融合标签的目标蛋白的分离纯化;发现内含肽序列Hbt21在地中海富盐菌中保持了高效的剪接活性，通过定点突变其C端末位氨基酸天冬酰胺(N182)及邻位的丝氨酸(S183)失活了该内含肽的C端剪接活性。【结论】首次建立了一个基于PHA颗粒-PhaP标签的简便节约的极端嗜盐古菌蛋白表达纯化系统，并确定了嗜盐古菌型内含肽C 端剪接的活性位点，为该内含肽将来应用于PhaP 融合蛋白的标签去除奠定了基础。

Abstract:

Abstract:［Objective］ To establish a convenient halophilic protein expression and purification system based on the haloarchaeal-type PhaP and polyhydroxyalkanoate (PHA) granule．［Methods］We cloned a strong haloarchaeal promoter and the phaP-tag into the haloarchaea- Escherichia coli shuttle vector pWL502，and then used the constructed vector to express the PhaP-tagged haloarchaeal proteins in the phaP-deleted strain Haloferax mediterranei ΔphaP．We purified the PhaP-fusion proteins，which were associated with PHA granules，by sucrose density gradient centrifugation．We also inserted a haloarchaeal intein-containing fragment between phaP and multiple cloning sites，and modulated the intein splicing activity by site-directed mutagenesis.［Results］We successfully constructed two expression vectors，pPM and pIP，in which PhaP was used as N-terminal and C-terminal fusion tag，respectively．The haloarchaeal proteins were effectively expressed by both vectors． The PhaP-tagged proteins were easily purified through the strategy of PHA granulemediated protein purification．In addition，we found that the intein-containing fragment Hbt21 from Halobacterium sp．NRC-1 had maintained splicing activity in H． mediterranei，and its C-terminal cleavage could be blocked or attenuated by mutating the conserved asparagine (N182) or serine (S183)，respectively．［Conclusion］ We have established a convenient and economical halophilic protein expression and purification system． We have also identified the splicing active sites of a haloarchaeal intein，which showed potential for removing the PhaP-tag from the purified proteins．